Embed Size (px)
Citation preview
i
Accelerating the generation of homozygosity and genome fixation in pea (Pisum sativum L.)
Federico Ribalta
This thesis is presented for the degree of Doctor of Philosophy at
The University of Western Australia
2014
Centre for Legumes in Mediterranean Agriculture
School of Plant Biology
Faculty of Science
The University of Western Australia
iii
Abstract
Pea cultivars are nearly homozygous and thus homogeneous when they are
released. The traditional method of selfing is slow and inefficient, taking up to ten
generations of inbreeding following a cross to achieve a high level of homozygosity.
Current single-seed-descent (SSD) methodologies enable a maximum of three
generations per year to be developed in pea. Doubled haploidy and an in vitro based
modified SSD technology have been utilised in many important crops for the rapid
achievement of homozygosity, and thus acceleration of the breeding process. In pea,
due to the lack of robust protocols, neither of these technologies is routinely used in a
breeding program. The aim of this study was to accelerate the breeding process in pea
by developing in vitro techniques to more rapidly achieve a high level of homozygosity
and to gain a better understanding of the fundamental mechanisms involved in these
processes. These techniques include: 1) haploidisation from cultured anthers in
selected genotypes of varying backgrounds; and 2) in vitro flowering and seed-set for
use in SSD breeding strategies. The development of robust genotype-independent in
vitro protocols will be of great value to accelerate the breeding process in pea.
In this research a number of key factors in the development of a robust pea
anther culture protocol were identified and optimised. The combined application of
multiple stress treatments, including the novel stress agent sonication, and the
optimisation of key culture factors led to the development of an efficient protocol for
the routine induction of androgenesis and embryo production from extracted anthers
of various pea genotypes. A flow cytometry study was undertaken to further
understand the effect of individual and combined stress treatments on androgenesis
elicitation. Analysis of the flow cytometry results revealed clear differences in the
relative nuclear DNA content of microspores within anthers after stress treatments
iv
and enabled prediction of whether a combination of stresses were elicitors or
enhancers of androgenesis. Flow cytometry is thus proposed as a method for the
quick assessment of the effect of individual and combined stress treatments, based on
the relative nuclear DNA content.
An optimised in vitro based SSD system was developed which enabled rapid in
vitro flowering and seed-set across a range of pea genotypes including, for the first
time, mid to late flowering types. In this protocol, the antigibberellin Flurprimidol was
used to control in vitro plant size, and plants with the meristem removed and excised
shoot tip explants were cultured into glass tubes under white fluorescent light. The
involvement of antigibberellin and light quality on plant growth response is discussed.
Using this strategy more than five generations per year can be obtained with mid to
late flowering genotypes and over six generations per year for early to mid flowering
genotypes.
The results presented in this research form a solid basis for further efforts
designed to enhance androgenic response in pea and extend double haploid
technology to other legumes. However, further research is required before this
technology will be routinely available within a pea breeding program. In the absence
of a robust DH protocol, the in vitro based SSD system reported herein will offer a
valuable alternative method for the rapid achievement of homozygosity by shortening
each generation cycle.
v
Acknowledgements
I would like to thank the Grains Research and Development Corporation (GRDC)
for providing a full PhD scholarship with living allowance and research support; and
the Graduate Research School Office (GRS), the School of Plant Biology (UWA) and the
Centre for Legumes in Mediterranean Agriculture (CLIMA) for their financial
assistance. Also, I would like to thank the French National Institute for Agricultural
Research (INRA), and in particular, Dr Sergio Ochatt and the UMRLEG team for hosting
me in their laboratory during my three month training period in Dijon.
I am particularly grateful to my PhD supervisors Janine Croser, Willie Erskine,
Patrick Finnegan and Sergio Ochatt, for their great input, support and patience during
the period of my research. I would like to acknowledge Rob Creasy and Bill Piasini for
their glasshouse expertise, and Dr Kathy Heel (CMCA) for her flow cytometry
expertise. I would also like to thank fellow colleagues and friends who have supported
me during this project.
Finally, a very special thanks to my parents Horacio and Marisabel, my brothers
Javier and Nacho, and to my wife Kylie and our baby boy Diego, for their help,
encouragement and support during the entire project.
vi
Statement of candidate contribution
This thesis contains no material which has been submitted for the award of any
other degree or diploma in any other University and to the best of my knowledge it
contains no copy or paraphrase of material previously published by any other person,
except where due reference is made. All the glasshouse and laboratory work related
to this thesis was carried out solely by Federico Ribalta. The publications pertaining to
this thesis were written by Federico Ribalta and the co-authors were involved in the
experimental planning phase, discussion of results, structure of the papers and
editorial comments to finalise them.
vii
Publications pertaining to this thesis
International journal publications:
Ribalta FM, Croser JS, Ochatt SJ (2012). Flow cytometry enables identification
of sporophytic eliciting stress treatments in gametic cells. Journal of Plant
Physiology; Vol 169, Issue 1, pp. 104-110.
Ribalta FM, Croser JS, Erskine W, Finnegan PM, Lülsdorf MM, Ochatt SJ
(2014). Antigibberellin-induced reduction of internode length favors in vitro
flowering and seed-set in different pea genotypes. Biologia Plantarum; Vol 58,
Issue 1, pp. 39-43.
International conference proceedings:
Ribalta F, Croser J, Ochatt S (2010). Doubled haploid production in field pea
via anther culture. In: Proceedings of the 5th International Food Legumes
Research Conference (IFLRC V) and 7th European Conference on Grain Legumes
(AEP VII), Antalya, Turkey. This poster was awarded as the ‘Best early career
researcher poster’ by the Conference Organising Committee.
Ribalta F, Ochatt S, Erskine W, Finnegan P, Croser J (2013). Accelerating the
generation of homozygosity and genome fixation in pea. In: Proceedings of the
PBA Inaugural Pulse Conference, Adelaide, Australia. This poster was awarded
as the ‘Best PhD student poster’ by the Conference Organising Committee.
Edwards K, Munday C, Castello MC, Ribalta F, Nelson K, Lülsdorf M, Erskine
W, Croser J (2013). Six generations in one year? Accelerating generation
turnover in the grain legumes. In: Proceedings of the PBA Inaugural Pulse
Conference, Adelaide, Australia.
viii
Abbreviations: ABA - abscisic acid; BAP - 6-benzylaminopurine; DAPI - 4',6-diamidino-
2-phenylindole dihydrochloride; DH - doubled haploid; FDA - fluorescein diacetate; GA
- gibberellic acid; GFP - green fluorescent protein; GMO - genetically modified
organism; HSP - heat shock protein; IAA-asp - indol-3 acetic acid-asparagine; MS -
Murashige & Skoog; NAA - a-naphthalene acetic acid; RIL - recombinant inbred lines;
SSD - single seed descent; 2,4-D - 2,4-dichlorophenoxyacetic acid.
ix
Table of contents
Thesis abstract . iii
Acknowledgements v
Statement of candidate contribution vi
Publications pertaining to this thesis . vii
Abbreviations viii
Table of contents . ix
List of figures . xii
List of tables xv
Chapter 1: Introduction and literature review .1
1.1 Introduction . .1
1.2 Literature review . 4
1.2.1 Pea background . 4
1.2.1.1 Classification 4 1.2.1.2 Origin of cultivated pea . 5 1.2.1.3 Distribution and production . . 5
1.2.2 Genetic improvement in pea . 7 1.2.2.1 Pea yield and breeding constraints . 7
1.2.2.2 Genetic improvement techniques . 9 1.2.2.3 Double haploidy . 10
1.2.2.3.1 Introduction . 10 1.2.2.3.2 Production of doubled haploids (DHs) via
androgenesis . 12 1.2.2.3.3 Role of abiotic stress treatments on androgenesis 14
elicitation 1.2.2.3.4 Application of haploids and DHs in plant breeding,
genetics and functional genomics . 18 1.2.2.3.5 Pea doubled haploid research status . . 20 1.2.2.3.6 Application of flow cytometry in plant breeding . 24
1.2.2.4 In vitro based single-seed-descent (SSD) . 26 1.2.2.4.1 Introduction . 26
1.2.2.4.2 In vitro flowering in pea 27 1.2.2.4.3 Factors affecting flowering in pea . 28 1.2.2.4.4 Genetics of flowering in pea . 31
1.2.2.5 Doubled haploidy vs. Single-Seed-Descent . .32 1.2.2.6 Benefits of pea research to the Australian industry sector . 34
1.3 Research objectives and thesis structure . . 35 1.4 References .. 36
x
Chapter 2: The application of multiple stress treatments and the optimisation of culture conditions lead to the routine induction of androgenesis from intact anthers of pea (Pisum sativum L.) .47
2.1 Introduction .47
2.2 Materials and methods . . .52 2.2.1 Plant material .52 2.2.2 Donor plants growth conditions . 52 2.2.3 Harvest of buds and determination of microspore developmental stage . 53 2.2.4 Effect of bud harvest number on microspore viability . 53
2.2.5 Bud sterilisation . 54 2.2.6 Culture media . 54
2.2.7 Anther extraction procedure 54 2.2.8 Stress treatments 55
2.2.8.1 Cold pre-treatment 55 2.2.8.2 Electroporation 55 2.2.8.3 Centrifugation 56 2.2.8.4 Sonication 56 2.2.8.5 Osmotic shock 56 2.2.8.6 n-butanol treatment 56
2.2.9 Embryo development 56 2.2.10 Tracking experimental results 57
2.3 Results 59 2.3.1 Correlation between pea bud length and microspore developmental stage 59 2.3.2 Effect of bud harvesting procedure and cold pre-treatment
duration on microspore viability 59 2.3.3 Effect of culture medium plant growth regulators on androgenesis
induction 61 2.3.4 Effect of culture conditions on callusing frequency 61 2.3.5 Effect of individual and combined stress treatments on elicitation of androgenesis 62
2.3.6 Embryo maturation and germination 65
2.4 Discussion 67 2.5 References 71
Chapter 3: Flow cytometry enables identification of sporophytic eliciting stress treatments in gametic cells 75
3.1 Introduction . 75 3.2 Materials and methods 79
3.2.1 Plant material . 79 3.2.2 Bud sterilization and microspore isolation . 79 3.2.3 Stress treatments studied to elicit and/or enhance androgenesis 80 3.2.3.1 80 3.2.3.2 81
xi
3.2.3.3 81 3.2.4 Flow cytometry analysis . 81
3.2.5 Fluorescein diacetate viability test 82 3.2.6 Culture conditions 82
3.3 Results 83 3.3.1 Flow cytometry analysis of extracted anthers 83 3.3.2 Flow cytometry analysis of isolated microspores 86 3.3.3 Microspore fluorescein diacetate viability test 87 3.4 Discussion 88 3.5 References 92 Chapter 4: Antigibberellin-induced reduction of internode length favors in vitro flowering and seed-set in different pea genotypes 95
4.1 Introduction 95 4.2 Materials and methods 98
4.2.1 Plant material 98 4.2.2 Flurprimidol experiments 98 4.2.2.1 In vivo experiments 98 4.2.2.2 In vitro experiments . 99 4.2.3 Light quality treatments 100
4.3 Results . 102 4.3.1 Effect of Flurprimidol on internode length and flowering time 102 4.3.2 Interaction Flurprimidol x environment . 102 4.3.3 Interaction Flurprimidol x genotype 103 4.3.4 Effect of Flurprimidol on different culture explant sources 103 4.3.5 Light quality effect . 104 4.3.6 Culture system . 104 4.3.7 Comparison of growth environments . 106
4.4 Discussion .107 4.5 References . .112
Chapter 5: General discussion and future directions . 115
5.1 Doubled haploidy 115 5.1.1 Future directions .118
5.2 In vitro based Single-Seed-Descent . 121 5.2.1 Future directions . 124
5.3 Final conclusions .126 5.4 References . 128
Appendixes . 131
Appendix 1 131
Appendix 2 . 132
Appendix 3 . 133
Appendix 4 134
Appendix 5 135
xii
List of figures
Chapter 1
Figure 1.1 Evolutionary and taxonomic relationships within major clades of crop
legume subfamily Papilionoideae. MYA: million years ago (adapted from
Choi et al., 2004).
Figure 1.2 Pea plants with conventional leaf type (a) and semi-leafless type (b).
Figure 1.3 Dry pea production by country in 2010 (percentage of total global
production). Source: FAOSTAT, 2012.
Figure 1.4 Worldwide and Australian dry pea average yields over the past 25 years.
Source: FAOSTAT, 2012.
Figure 1.5 Induction of microspore embryogenesis in B.napus. Culture of late
unicellular microspores or early bicellular pollen at 32°C leads to
embryogenic development. Culture at 18°C allows pollen maturation to
proceed in vitro (modified from Cordewener et al., 1996).
Chapter 2
Figure 2.1 Glasshouse grown pea donor plants (cvs. Frisson and Kaspa).
Figure 2.2 Electro cell manipulator used for the application of the electric shock
treatment.
Figure 2.3 Determination of microspore developmental stage. Pea flower buds
containing anther with microspores predominantly at the uni-nucleate
stage (A and B). DAPI-stained microspores at the optimum uni-nucleated
stage for androgenesis induction (C) and at a later, non inductive, stage
(D), after first asymmetric mitosis.
Figure 2.4 Microspore viability estimated by fluorescein diacetate (FDA). Analysis of
the effect of harvest number on microspore viability at D0 in cv. Frisson
(results are expressed as mean ± SD).
Figure 2.5 Microspore viability estimated by fluorescein diacetate (FDA). Analysis of
the effect of bud cold pre-treatment duration on microspore viability
(results are expressed as mean ± SD; P = 0.05).
Figure 2.6 Transversal cut of cultured anther showing callus production from microspores within the anther (cv. Frisson).
xiii
Figure 2.7 Effect of culture light conditions (light vs. dark) and electroporation field strength (0, 500 and 1000 V/cm) on callusing percentage from extracted anthers of pea cvs. CDC April and Frisson (Results are expressed as mean ± SD; P = 0.05).
Figure 2.8 DAPI-stained pea microspores showing symmetrical division of the nucleus (A) and multinucleate synctium (B). Inverted microscope image showing pea microspore derived callus production (C).
Figure 2.9 Initial androgenetic response observed in cultured anthers at D7 of culture (cv. Frisson). Response obtained after a cold pre-treatment of buds combined with centrifugation and electroporation of intact anthers. A) Unresponsive and responsive (hyperplasia) anthers; B) Embryoid development from intact anther.
Figure 2.10 Effect of electroporation voltage on percentage of callusing from anthers of pea cvs. Frisson and CDC April at optimum developmental stage (results are expressed as mean ± SD; P = 0.05).
Figure 2.11 Effect of electroporation parameters (voltage and capacitance) on callusing percentage from extracted anthers of pea cvs. Frisson and CDC April after 28 days of culture.
Figure 2.12 Effect of different combinations of stress treatments on percentage of callusing after 28 days of culture from cultured anthers of cv. Kaspa (O: osmolarity; S: sonication; C: centrifugation; E: electroporation). In all treatments buds were previously submitted to a cold treatment for 7 d at 4°C. Results are expressed as mean ± SD; P=0.05.
Figure 2.13 Anther culture derived somatic embryos of pea cv. Frisson. Arrow colour indicates different embryo developmental stage (white: pro-embryo; red: globular; blue: heart; black: torpedo).
Chapter 3
Figure 3.1 Determination of microspore developmental stage. Cytological development
of male gametophyte in pea. DAPI-stained microspores showing uni-
nucleated (A), bi-nucleated (B), and multi-nucleated (C and D). Flower buds
and anthers at the optimal developmental stage for androgenesis (E-G).
Figure 3.2 Effect of stress treatments on relative DNA content per nucleus of pea
microspores within anthers after 14 d of culture. C-DNA profiles showing
1C, 2C and 4C ploidy levels. (A) Field pea leaves; (B) Cold + Osmolarity
treatments; (C) Cold + Osmolarity + Sonication treatments; (D) Cold +
Osmolarity + Centrifugation treatments; (E) Cold + Osmolarity +
Electroporation treatments; (F) Cold + Osmolarity + Electroporation +
Centrifugation + Sonication treatments. Note in figure 3.2A the scale of the
xiv
vertical axis is different to the other profiles as the data was obtained from
leaf tissue.
Figure 3.3 Flow cytometry profiles obtained immediately after submission to cold,
electroporation, centrifugation and sonication stress treatments (A) or
after 7 d (B) and 14 d (C) of culture.
Figure 3.4 Flow cytometry profiles of cold pre-treated extracted anthers submitted to
a sonication treatment alone (A) or combined with electroporation (B) or
electroporation and sonication (C).
Figure 3.5 Pyramiding eliciting factors (red) and enhancing factors (blue) enhances
androgenetic response from pea anthers.
Figure 3.6 Effect of treatments on relative nuclear DNA content of pea isolated
microspores after 14 d of culture. (A) Pea leaves; (B) Cold + Osmolarity +
Electroporation treatments; (C) Cold + Osmolarity + Sonication treatments;
(D) Cold + Osmolarity + Centrifugation + Sonication treatments.
Figure 3.7 FDA viability test from isolated microspores of pea. HV: Electroporation (1500 V/cm); HVS: Electroporation (1500 V/cm) + Sonication; LV: Electroporation (750 V/cm); LVS: Electroporation (750 V/cm) + Sonication; S: Sonication.
Chapter 4
Figure 4.1 Effect of Flurprimidol on internode length in vitro for different explant
sources of cv. Kaspa at day 40 (results are expressed as mean ± SE; P =
0.05).
Figure 4.2 Percentage of plants that set seeds under white fluorescent light (results
are expressed as mean ± SE; T: tubes; C: containers).
Figure 4.3 Number of flowers per plant produced in vitro from different explant
sources for A - cv. Frisson and B - cv. Excell (Results are expressed as mean
± SE; n = 30; P = 0.05; MR: meristem removed; STE: shoot tip explants; IP:
intact plants). Flurprimidol concentration: Intact plants and plants with the
meristem (30 cm3 dm-3), shoot tip explants: (10 cm3 dm-3).
Figure 4.4 Comparison between growth environments for estimates of average
generation cycle length of different cultivars. Numbers within bars
indicate average flowering time in days (data are means from three
repetitions with at least 10 individuals). Flowering time in the field source:
Department of Agriculture and Food of Western Australia, Australia.
xv
List of tables
Chapter 1
Table 1.1 Most commonly applied stress treatments for androgenesis induction and
their proposed mechanisms of action.
Table 1.2 Summary of key publications on haploid research in pea (AC: anther culture;
IMC: isolated microspore culture).
Chapter 2
Table 2.1 Pea cultivars used for the anther culture protocol development.
Chapter 4
Table 4.1 Pea genotypes used in this study and their main characteristics.
1
Chapter 1
Introduction and literature review
1.1 Introduction
Pea (Pisum sativum L.) is one of the world’s oldest domesticated crops. It is the
third most widely grown legume, as its seeds serve as a protein-rich food for humans
and livestock alike (Smýkal et al., 2011). It is also important in dry-land cropping
systems both as a cash crop and for the provision of soil nitrogen from the fixation of
atmospheric nitrogen (Redden et al., 2005). Peas are a useful component in cropping
rotations, providing overall economic value in the cropping system (Muehlbauer,
1996).
Breeding against diseases, insect pests and abiotic factors has played a
significant role in improving yields in pea (Khan and Croser, 2004); however, these
breeding objectives have been constrained by the absence of biotechnology tools
applicable to the crop (Croser et al., 2006; Smýkal et al., 2012). Pea cultivars are nearly
homozygous and homogeneous when they are released. The traditional method of
selfing is very slow and inefficient, as it takes up to ten generations of inbreeding
following a cross to achieve a high level of homozygosity (Kasha and Maluszynski,
2003). Current single-seed-descent (SSD) methodologies enable a maximum of three
generations per year to be developed in pea (Ochatt and Sangwan, 2010). Decreasing
the length of the generation cycle will assist in overcoming this breeding bottleneck
and result in the accelerated genetic improvement of pea.
Doubled haploidy has been utilised in important crops such as, tobacco
(Nicotiana tabacum L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.), and
canola (Brassica napus L.) for the rapid achievement of homozygosity (Maluszynski et
2
al., 2003). In responsive species, doubled haploid (DH) technology has enabled the
delivery of improved cultivars significantly faster due to the development of
homozygosity in a single laboratory-based generation. Doubled haploidy is thus the
fastest known route to homozygosity (Chase, 1952; Maluszynski et al., 2003). Legumes
in general have remained recalcitrant to this technology (Croser et al., 2006; Lülsdorf
et al., 2011). In recent years, significant progress has been made toward a DH protocol
from isolated microspores of pea (Croser et al., 2006; Ochatt et al., 2009), however
our understanding of the mechanisms by which isolated legume microspores shift
their developmental pathways to divide and differentiate is limited (Croser et al.,
2005; Sidhu and Davies, 2005; Croser et al., 2006; Ochatt et al., 2009; Lülsdorf et al.,
2011). Doubled haploidy is not currently used in any breeding program and the
development of a pea DH protocol would improve the efficiency of the time-
consuming, laborious and often inefficient conventional breeding methods (Germanà,
2011). Further progress in the development of robust DH protocols, including the
expansion to unresponsive species such as legumes can be expected with a more
thorough understanding of the processes involved in haploid embryogenesis.
In the absence of an efficient DH system, an in vitro based SSD system has been
proposed as a method to accelerate generation turnover across a range of species
(Franklin et al., 2000; Ochatt et al., 2002; Asawaphan et al., 2005; Zhang, 2007; Ochatt
and Sangwan, 2008; Ochatt and Sangwan, 2010). This technique involves the
shortening of generation time by culturing immature seeds developed after forced in
vitro flowering. There have been three in vitro flowering protocols proposed for pea
(Fujioka et al., 1999; Franklin et al., 2000; Ochatt et al., 2002); however, each of these
protocols was developed for a limited number of early-flowering cultivars. To enable
3
the widespread use of the in vitro flowering technology within pea improvement
programs, it is necessary to gain a better understanding of the effect of physiological
mechanisms operating in vitro on the efficient and reproducible induction of flowering
and seed-set across a range of pea genotypes.
It is clear the development of an efficient DH system in pea will provide a
valuable breeding technique. Given the well-documented difficulties associated with
the development of DHs in the Fabaceae, it appears prudent to concurrently
investigate the development of a simple, reliable and widely applicable in vitro based
SSD system. An in vitro SSD system will offer an alternative method for the rapid
achievement of homozygosity by shortening each generation cycle. In light of the
potential benefits of these techniques to pea breeding, the aim of the research within
this thesis was twofold. Firstly, to develop an anther culture based protocol for the
production of haploid plants and ultimately doubled haploid populations and
secondly, to develop an in vitro based SSD system across a range of genetically diverse
pea genotypes. The overall goal was to provide pea breeders with biotechnology tools
to rapidly achieve a high level of homozygosity and, in the process, gain a better
understanding of the physiological mechanisms involved in these processes.
4
1.2 Literature review
1.2.1 Pea background
1.2.1.1 Classification
Cultivated pea (Pisum sativum L.) is an annual plant in the genus Pisum, within
tribe Fabeae (syn. Vicieae) in the Fabaceae family (syn. Leguminoseae) (Fig. 1.1). Pea is
grouped with faba bean (Vicia faba L.), lentil (Lens culinaris Medik.), grasspea
(Lathyrus sativus L.) and chickpea (Cicer arietinum L.) as a cool-season food legume
(Muehlbauer, 1996). Peas are diploid (2n=2x=14) and highly self-pollinating (Gritton,
1980). Peas can be conventional-leaved, or semi-leafless, in which the leaflets on the
conventional type are replaced by tendrils (Khan and Croser, 2004) (Fig. 1.2).
Figure 1.1 Evolutionary and taxonomic relationships within major clades of crop legume subfamily Papilionoideae. MYA: million years ago (adapted from Choi et al., 2004).
Figure 1.2 Pea plants with conventional leaf type (a) and semi-leafless type (b).
5
1.2.1.2 Origin of cultivated pea
The earliest date for the discovery of domesticated pea in archaelogical sites is
7,000 to 6,000 BC from Turkey to Iran, and 1,000 – 2,000 years later in Greece and
Europe (Zohary and Hopf, 1973; Ambrose, 1995). The Near East, Central Asia including
Afghanistan, the Mediterranean, and Ethiopia abound with primitive forms and are
sources of genetic diversity (Khan and Croser, 2004). Although the wild progenitor of
pea is uncertain, Zohary and Hopf (1973) proposed P. sativum subsp. ‘humile’ found in
steppelike habitats, under open conditions not very different from those prevailing in
the cultivated field. Changes with domestication included the development of larger
seed, loss of pod dehiscence and a shorter compact habit (Davies, 1976).
1.2.1.3 Distribution and production
Peas are an important crop in many arable regions of the world. Cultivation of
pea includes diverse types and systems widely dispersed around the world in
temperate to elevated subtropical environments (Redden et al., 2005). It requires
moderate temperatures in the range of 12-18°C with a relatively humid climate for
optimum growth (Khan and Croser, 2004). Peas are a valuable break crop providing
disease and weed control in cereal-based cropping systems. In addition, as with all
legumes, a symbiotic relationship with Rhizobium bacteria in the soil enables pea to fix
atmospheric nitrogen (ca. 181-262 Kg N/ha) (McCallum et al., 2000) and make it
available to the succeeding crop. Thus, peas are a useful component in cropping
rotations, providing overall economic value in the cropping system (Muehlbauer,
1996).
Peas can be classified into two main groups: those harvested at the green or
immature stage of development (vegetable type peas) and those harvested at the
6
physiologically mature stage (field or dry peas) (Redden et al., 2005). Green peas are
one of the most popular vegetable items throughout the world, while dry peas are
mostly used in the feed industry, particularly in the diet of pigs and poultry (Khan and
Croser, 2004). Dry peas are also used as a whole in confectionery and snacks, milled to
produce split peas for making soups, flour, and canned products (Khan and Croser,
2004). In Eastern Europe and Canada, peas are also grown for fodder or for green
manure (Makasheva, 1983). Pea seeds are a good source of dietary protein and
energy, containing 18-30% protein, 30-50% starch and 4-7% fibre. Pea protein is
deficient in sulphur-containing amino acids, but contains relatively high levels of lysine
making it a good dietary complement to cereals (McPhee, 2003).
In 2010, the world production of dry peas was 10.13 million tonnes (FAOSTAT,
2012). Major producers were Canada (28.2%), Russia (11.8%), France (10.8%) and
China (8.8%). Australia is the eighth largest dry pea producer globally with 2.7% of the
total production (FAOSTAT, 2012) (Fig. 1.3). Pea is the third most important pulse crop
in Australia after lupin (Lupinus angustifolius L.) and chickpea, where in 2010, the pea
cultivated area was 277,000 ha with a production of 280,000 Tonne and an average
yield of 1.01 T/ha (FAOSTAT, 2012). The majority of the Australian product is exported,
mainly for human consumption in Asia and the Middle East and to stockfeed markets
in Asia and Europe (Pulse-Australia, 2012).
7
Figure 1.3 Dry pea production by country in 2010 (percentage of total global production). Source: FAOSTAT, 2012.
1.2.2 Genetic improvement in pea
1.2.2.1 Pea yield and breeding constraints
A major remaining challenge for pea breeders is the increase of yield potential
and stability. Dry peas have high yields potentially reaching 8 tonnes per hectare
(Cousin, 1997). However, the world average dry pea yield has remained relatively
unchanged over the past 25 years, varying seasonally between 1.5 and 2.0 tonnes per
hectare, on average (Fig. 1.4) (FAOSTAT, 2012). Many biotic and abiotic factors limit
pea production. Diseases are considered the most important causes of reduced
biomass production and seed yield. Abiotic stresses caused by adverse environmental
conditions such as heat, drought and frost are also responsible for heavy pea
production losses (Ali et al., 1994).
28.2
11.8
10.8 8.8
6.9
6.3
4.4
2.7
19.8
Canada
Russia
France
China
India
USA
Ukraine
Australia
Othercountries
8
Figure 1.4 Worldwide and Australian dry pea average yields over the past 25 years. Source: FAOSTAT, 2012.
The development of improved germplasm in pea has been slow due to the low
level of resistance/ tolerance found in the available germplasm collections, low
reliability of screening methods and polygenic nature of inheritance (Wroth, 1998).
Over the years, biotechnology has emerged as a promising tool to overcome stresses
in plants, but to date, progress has been limited in pea and legumes in general
(McPhee, 2003; Dita et al., 2006). Peas have been described as recalcitrant in tissue
culture and to biotechnological approaches (Ochatt et al., 2000), including doubled
haploidy (Croser et al., 2006; Dita et al., 2006). Tissue culture protocols developed to
date are either genotype-specific, hence not widely applicable, or they are slow and
inefficient (Ochatt et al., 2000; Sidhu and Davies., 2005; Croser et al., 2006). In
addition, in vitro root development can be difficult, often requiring several subculture
steps (Tzitzikas et al., 2004) or grafting procedures (Bean et al., 1997). The
development of biotechnology approaches in pea such as marker assisted breeding,
tissue culture techniques, mutagenesis, and genetic transformation would assist
breeders to overcome major biotic/ abiotic constraints (Christou, 1993; Dita et al.,
2006; Smýkal et al., 2012). The more efficient tissue culture techniques recently
0.0
0.5
1.0
1.5
2.0
2.5
1986 1991 1996 2001 2006 2011
Yie
ld (
Tn/
ha)
Year
World
Australia
9
established for many legume species should encourage legume researchers to resume
the use of techniques such as doubled haploidy, wide hybridisation and mutagenesis
in breeding programs (Dita et al., 2006).
1.2.2.2 Genetic improvement techniques
Various techniques are used in conventional pea breeding programs, and their
application depends on the type and amount of genetic variation in crosses, objectives
of breeding, and available genetic sources (Khan and Croser, 2004). Breeding
strategies in pea generally involve hybridisation among cultivars or between cultivars,
landraces or primitive forms, followed by combinations of pedigree selection, bulk
population, backcross, or single-seed-descent (SSD) methods of selection (McPhee,
2003; Redden et al., 2005).
In traditional breeding, the development of plant varieties with resistance to
abiotic and biotic stress is a very costly and time consuming process (Germanà, 2006).
In addition, a bias of dominance on the phenotype is created due to the selection of
individuals in the early stages of the process, where the non-competitive ones are
rapidly discarded. This early selection is based on individuals grown in non-crop
conditions without replication, as intensive selection cannot be applied until lines
approach homozygosity and sufficient seed is available for field trials (Maluszynski et
al., 2003; Thomas et al., 2003; Germanà, 2006). To overcome this problem, breeders
may delay intense selection until individual genetic lineages approach homozygosity
(Caligari et al., 1987).
Two in vitro technologies have been utilised in economically important crops for
the rapid achievement of homozygosity, and thus acceleration of the breeding
process:
10
a- Doubled haploidy (Jain et al., 1996-1997; Maluszynski et al., 2003; Croser et al.,
2006; Wędzony et al., 2009; Dunwell, 2010; Germanà, 2011; Lülsdorf et al.,
2011); and
b- In vitro based modified SSD system (Narasimhulu and Reddy, 1984; Tisserat and
Galletta, 1988; Zhong et al., 1992; Franklin et al., 2000; Ochatt et al., 2002;
Ochatt and Sangwan, 2008; Ochatt and Sangwan, 2010).
1.2.2.3 Double haploidy
1.2.2.3.1 Introduction
The term ‘haploid plant’ refers to a plant derived from either the male
(androgenesis) or female (gynogenesis) gametes. As there is no fertilisation between
pollen and egg, the resulting haploid plants have only a single copy of the genetic
information of the plant. When the chromosome complement is chemically or
spontaneously doubled, these haploid plants become fertile doubled haploids (DHs)
(Jain et al., 1996-1997; Maluszynski et al., 2003; Wędzony et al., 2009). The production
of haploids and DHs through gametic embryogenesis allows the development of
completely homozygous lines from heterozygous parents in a single laboratory-based
step, shortening the time required to produced homozygous plants compared to
conventional breeding methods that employ several to many generations of self-
pollination. Doubled haploidy is thus the fastest route to homozygosity (Chase, 1952;
Maluszynski et al., 2003).
In responsive species, DH technology has now become the most widely-applied
and beneficial biotechnological tool for crop improvement (Dunwell, 2010). Its use in
cereal and oilseed breeding programs has led to impressive gains in productivity (Jain
et al., 1996-1997; Maluszynski et al., 2003). More than 280 varieties have been
11
produced with the use of various DH methods in crops including barley (Hordeum
vulgare L.), asparagus (Asparagus officinalis L.), wheat (Triticum aestivum L.), canola
(Brassica napus L.), rice (Oryza sativa L.) and tobacco (Nicotiana spp.) (Germanà,
2011).
Despite the development of protocols for more than 200 species, only four
species (canola, tobacco, barley and wheat) are responsive enough to be used as
model systems for the study of androgenesis elicitation (Touraev et al., 1997;
Maraschin et al., 2005). Other scientifically or economically interesting species like
Arabidopsis thaliana (L.) Heynh. and members of the Fabaceae family remain
recalcitrant to doubled haploidy and little is known about the reasons for that (Croser
et al., 2006; Dita et al., 2006; Seguí-Simarro and Nuez, 2008; Lülsdorf et al., 2011).
The poor fundamental understanding of the molecular and biochemical basis for
plant gametophyte to sporophyte transition and morphogenesis makes the current
empirical efforts to adapt DH production techniques to recalcitrant species highly
time-consuming and difficult (Lülsdorf et al., 2011). The establishment in recent years
of efficient in vitro androgenesis systems in species like canola and tobacco has
opened the possibility of investigating the mechanisms underlying the switch from
normal gametophytic pollen development towards the alternative sporophytic
pathway and to isolate and characterise the genes and gene products involved in the
regulation of this process (Touraev et al., 1996). Further improvement in existing DH
protocols, and expansion to more recalcitrant species such as legumes, can be
expected with the growing understanding of the processes involved in haploid
embryogenesis.
12
1.2.2.3.2 Production of doubled haploids (DHs) via androgenesis
Several methods for haploid induction have been succesfully used in many
species. Modified pollination methods in vivo, such as wide hybridisation (interspecific
and intergeneric hybridisation) and chromosome elimination (H. bulbosum technique),
have been extensively used in cereal breeding programs (Dunwell, 2010). In addition,
various methods have been developed for the in vitro development of haploid plants
through the culture of immature gametophyes (gynogenesis and androgenesis)
(Germanà, 2011). Androgenesis is the most deeply studied and the most widely
effective technology (Wędzony et al., 2009). It is based on culture of male organs
(anthers) or gametes (microspores). Under certain optimised conditions, microspores
can shift development from gametophytic cells to sporophytic cells and then on to
form haploid derived embryos. Inducing divisions and cell differentiation to produce
such embryos is modulated by factors including genotype, growth conditions of donor
plants, developmental stage of microspores, pre-treatment of flower buds and culture
medium(Jain et al., 1996-1997; Dunwell, 2010; Germanà, 2011) (Fig. 1.5).
The culture of extracted anthers is the preferred method for DH production in
many crops due to its simplicity, which permits large scale experiments in a wide
range of genotypes (Kush and Virmani, 1996; Sopory and Munshi, 1996). The culture
of isolated microspores requires better equipment and more skills compared to the
culture of anthers. The isolation of pollen grains, asepsis, viability of the isolated
pollen, culture vessels, and desiccation are all factors that need to be considered
(Heberle-Bors, 1989). The advantage of the culture of isolated microspores is that it
provides a better method for investigating cellular, physiological, biochemical and
molecular processes of cellular totipotency using isolated single cells that can be
readily staged for analysis (Nitsch, 1977; Reynolds, 1997).
13
Figure 1.5 Induction of microspore embryogenesis in B.napus. Culture of late unicellular microspores or early bicellular pollen at 32°C leads to embryogenic development. Culture at 18°C allows pollen maturation to proceed in vitro (modified from Cordewener et al., 1996).
Different species, as well as different cultivars within a species, have diverse
requirements for androgenesis induction and no single standard protocol has been
developed. Despite this, a number of general steps have been identified (Croser et al.,
2006; Ferrie and Caswell, 2011; Germanà, 2011). Firstly, responsive genotypes and
appropriate growing conditions for the production of healthy donor plants must be
identified. These genotypes can then be used to optimise the conditions for the
protocol, prior to widening its application to other genotypes. The pollen
developmental stage is one of the most crucial factors of the culture procedure as
small differences in developmental age can significantly affect the androgenesis
induction response (Dunwell, 2010). The exact pollen developmental stage for best
androgenesis induction varies with species. This period has been defined between the
early uni-nucleate and early bi-nucleate stage of pollen development (Sangwan and
Sangwan-Norreel, 1996).
Once microspores at the right developmental stage have been identified, a
trigger needs to be developed to induce the switch from a gametic to a sporophytic
pathway. Triggers are usually stress factors such as low or high temperature pre-
14
treatment of immature flower buds, carbon starvation pre-treatment, osmotic shock
and centrifugation(Jain et al., 1996-1997; Touraev et al., 1997; Croser et al., 2006;
Shariatpanahi et al., 2006; Wędzony et al., 2009). Other important factors that require
optimisation include: media composition for microspore culture, embryo induction
and maturation, plant regeneration, medium osmolarity, culture temperature, light
intensity and culture container (Jain et al., 1996-1997; Wędzony et al., 2009;
Germanà, 2011).
1.2.2.3.3 Role of abiotic stress treatments on androgenesis elicitation
Abiotic stress treatments play a key role in the induction of androgenesis as was
first established for tobacco (Duncan and Heberle-Bors, 1976; Heberle-Bors and
Reinert, 1981). Stress agents are widely used to stimulate the switch from gametic to
sporophytic developmental for induction of microspore embryogenesis (Jain et al.,
1996-1997; Touraev et al., 1997; Shariatpanahi et al., 2006; Wędzony et al., 2009).
There is a large variety of proposed stress factors, with a review by Shariatpanahi et al.
(2006) listing 16 different stresses. Stress can be applied via the donor plant, to
immature flower buds prior to isolation and culture of anthers or microspores, or to
anthers or microspores post-culture using nutritional, physical or chemical means.
Physical stress treatments can be applied via temperature, atmospheric pressure,
centrifugation and photoperiod, whereas chemical stress treatments can include
carbon starvation of flower buds, anther or pollen, and ethanol treatments. Table 1.1
shows the most commonly applied stress treatments and their proposed mechanisms
of action.
15
Table 1.1 Most commonly applied stress treatments for androgenesis induction and their proposed mechanisms of action.
Stress treatment
Proposed mechanism of action
Cold pre-treatment
Slows down degradation processes in the anther tissue thus protecting microspores from toxic compounds released in the decaying anthers (Duncan and Heberle-Bors, 1976).
Assures survival of a greater proportion of embryogenic pollen grains compared to heat treatment (Sunderland and Roberts, 1979).
Increases frequency of endo-reduplication leading to an increased number of spontaneous DH plants (Amssa et al., 1980).
Microspores in cold-treated anthers disconnect from the tapetum resulting in starvation (Sunderland and Xu, 1982).
Favours the synchronisation of the developmental process of microspores (Hu and Kasha, 1999).
Induces many morphological and physiological rearrangements in plant cells as well as hormonal changes (Żur et al., 2008).
Heat shock
Shown to cause changes in microtubule and cytoskeleton in cultured Brassica microspores (Hause et al., 1993; Cordewener et al., 1994; Simmonds and Keller, 1999).
Induces synthesis of heat-shock-proteins (HSP) in microspores (Pechan et al., 1991; Cordewener et al., 1995; Seguí-Simarro et al., 2003).
Carbon starvation
Cytoplasmatic and nuclear changes observed in starved microspores (Kyo and Harada, 1990; Garrido et al., 1995).
Decrease of RNA synthesis and protein kinase activities observed in tobacco microspores (Zarsky et al., 1990; Garrido et al., 1993).
Colchicine
Colchicine, a microtubule-depolymerizing agent, appears to be related to the disruption of the asymmetric spindle formation in gametophyte development and subsequent failure of cell wall formation which could permit fusion of the two nuclei and thus chromosome doubling (Kasha, 2005).
Osmotic stress
Accumulation of ABA observed in osmotic stressed tobacco (Imamura and Harada, 1980) and barley (Van Berger et al., 1999) anthers which may be involved in the reprogramming of microspore development (Wang et al., 2000; Żur et al., 2008).
Improves callusing from microspores, by favouring a slight detachment of membranes from the microspore wall facilitating initial division, and induces differentiation (Ochatt et al., 2009).
Electroporation
Shown to stimulate DNA synthesis in cultured plant protoplasts (Rech et al., 1988).
Creates temporary pores that allow nutrients from the culture medium to get into the cells (Zimmermann et al., 1976; Kinosita and Tsong, 1977), thus improving regeneration competence from microspores (Delaitre et al., 2001; Ochatt et al., 2009) and protoplasts (Rech et al., 1987; Ochatt et al., 1988) of various species.
Centrifugation
Affects auxin to cytokinin ratios, thus facilitating the induction of embryos from microspores of tobacco and fourfold increasing the haploid plant regeneration rate (Tanaka, 1973).
16
Upon stress treatment, isolated microspores swell and their cytoplasm
undergoes structural reorganization. The nucleus moves to a more central position
and cytoplasmic strands are formed that pass through the vacuole and connect the
perinuclear cytoplasm with the subcortical cytoplasm (Touraev et al., 1997). Induction
brings on an altered synthesis and accumulation of RNA and proteins in potentially
embryogenic microspores, leading to the sporophytic divisions (Reynolds, 1997). The
induction of sporophytic development is only possible at early developmental stages,
when the gametic cell appears to be totipotent (Touraev et al., 2001).
The induction of androgenesis through the application of stress treatments has
been linked with changes in endogenous hormone content (Wang et al., 2000; Seguí-
Simarro and Nuez, 2008; Żur et al., 2008; Lülsdorf et al., 2012). Accumulation of
abscisic acid (ABA) during androgenesis induction has been observed in a number of
species after the application of stress treatments (Imamura and Harada, 1980; Van
Berger et al., 1999; Żur et al., 2008). Lülsdorf et al. (2012) observed that androgenesis-
inducing stress treatments in pea, chickpea and lentil anthers caused a greater
proportional increase in active auxins, particularly indol-3-acetic acid-asparagine (IAA-
asp), compared to the other hormones measured. They also observed that the
concentration of ABA and its catabolites spiked in control and cold-treated anthers
and decreased after osmotic shock treatment in the three species studied. Also, no
bioactive gibberellins (G1, G3, G4 and G7) were detected in this study for any of the
studied species, indicating that this hormone group is likely not linked to androgenetic
elicitation in the legumes.
To date, the mechanism for the developmental switch of microspores from
gametophytic to sporophytic development has not been established for any species
17
(Hosp et al., 2007). However, over the last few years, significant progress has been
made toward the discovery of genes involved in haploid embryogenesis and their
mechanisms of action, mostly from three model species: canola, tobacco and barley
(Boutilier et al., 2005; Hosp et al., 2007). Also, the sequencing of the Arabidopsis
genome has permitted the isolation of genes related to embryo induction in
microspores and their subsequent tagging and isolation in crop species (Kasha, 2005).
Functional genomics approaches have revealed several hundreds of up-
regulated and down-regulated genes associated with androgenic induction (Hosp et
al., 2007). The induction of changes in development is characterised by the activation
of specific transcription factors, which in turn cause an altered pattern of gene
expression. In several cases these genes appear to be stress-related (Elmaghrabi et al.,
2013) or associated with zygotic embryogenesis (Verdier and Thompson, 2008;
Elmaghrabi et al., 2013). The stress-related genes may be concerned with a general
reprogramming of the cell or providing some type of protection from that stress
(Raghavan, 1986). This involves a profound remodelling of the transcriptome and up-
regulation of genes involved in primary metabolism and biosynthesis of lipids,
carbohydrates and proteins (Gallardo et al., 2007; Seguí-Simarro and Nuez, 2008;
Verdier and Thompson, 2008; Elmaghrabi et al., 2013). Other gene groups related to
mitochondria, cytoskeleton, epigenetic chromosomal remodelling, signal transduction
and stress response are also up-regulated when compared with non-embryogenic
commitment (Seguí-Simarro and Nuez, 2008). In both tobacco and canola, a close
relationship has been observed between the induction of embryogenesis and the
expression of heat shock proteins (HSP) (Cordewener et al., 1998; Pechan and Smýkal,
18
2001). There are many small HSPs produced by stress, with HSP70 being the
predominant one in treated microspores (Kasha, 2005).
Despite the significant progress made in the last few years toward the discovery
of the genes involved in haploid embryogenesis and their mechanisms of action, the
genetic basis of the process of doubled haploidy is still largely unknown. The new
approaches in discovering gene function are expected to have an impact in the future
(Wędzony et al., 2009). Further progress in the identification and characterisation of
genes specifically expressed during early stages of microspore embryogenesis will lead
to a better fundamental understanding of the mechanisms involved in androgenic
induction and the subsequent formation of haploid plants. This will have a positive
impact in the development of improved protocols in many plant species, particularly
in legumes. For the moment, further improvements in microspore/ anther culture
protocols can be expected mostly from empirical testing of new variants of known
protocols and from the optimisation of factors identified as critical in androgenic
development.
1.2.2.3.4 Application of haploids and DHs in plant breeding, genetics and functional genomics
The instant production of true-breeding lines in diploid or allopolyploid species
saves a number of generations in the breeding program. With this technology it is
possible to cross parental lines and conduct field trials on DH derived progeny within
two years (Thomas et al., 2003) and in self-pollinating crops such as pea, cultivar
developmental time can be reduced by up to five years. The homozygous nature of
the DH population ensures a more efficient and reliable selection compared to
conventional early-generation segregating lines as there are no dominance-related
effects (Thomas et al., 2003). Also, quantitative trait evaluation can begin earlier in the
19
program, saving time and space (Kasha and Maluszynski, 2003). Using a combination
of backcrossing, marker assisted selection and doubled haploidy, one can rapidly
introduce new genes into a variety. Due to these advantages, DH technology is
currently an integral part of the breeding programs of agronomically important crops
such as wheat, barley and canola (Wędzony et al., 2009; Germanà, 2011).
In addition to the exploitation of DH lines in practical breeding, they have
widespread application in such research areas as gene mapping and genomics,
mutation studies, and as targets for transformation. The advent of molecular markers
revolutionised the construction of genetic maps of plant genomes and doubled
haploidy has played a major role in facilitating the generation of maps in a range of
species, notably barley, rice, canola and wheat (Forster and Thomas, 2005). Doubled
haploid populations are ideal for genetic mapping as a population of recombinant
inbred lines (RILs) can be ready for DNA extraction and mapping less than two years
after the initial cross. These RILs can be re-grown and distributed in seed form with
certainty as to their homozygosity making it easier to screen with a large number of
markers (Forster and Thomas, 2003; Lionneton et al., 2004). Map construction from a
DH population derived from the F1 of a cross is relatively simple because the expected
segregation ratio is the same as that of a backcross (Snape, 1976).
Microspores provide an excellent single-celled target for genetic manipulation
through mutation or transformation, enabling enhancement of genetic variability
(Szarejko, 2003). The single-celled nature of the starting material means that chimerae
or heterozygosity are avoided and the mutation/ transgene is expressed throughout
the entire plant (Germanà, 2011). Since the advent of haploid systems, their use for
mutant induction and selection has been listed among its most important applications
20
(Kasha, 1974). This approach has been undertaken for the selection of lines with high
photosynthetic activity in haploid tobacco plants (Medrano and Primo-Millo, 1985),
for the isolation of disease resistance in melon (Cucumis melo L.) (Kusuya et al., 2003),
and for isolation of dwarf potato (Solanum tuberosum L.) mutants with gibberellin
biosynthesis defficiency from anther culture-derived potato lines (Valkonen et al.,
1999). More recently studies have been conducted to select lines tolerant to oxidative
stress in DH maize (Zea mays L.) derived from microspores exposed to paraquat
(Darko et al., 2009) and to isolate lines with increased aluminium tolerance in
aluminium-treated wheat anther cultures (Bakos et al., 2008).
Transformation and DH technology have been used to create homozygous lines
for the trait of interest, thereby speeding up the breeding process (Ferrie and Möllers,
2011). Isolated microspores not only provide a good target for bombardment but also
are readible amenable to transgene in vitro selection (Shim and Kasha, 2003). In 1994,
Jähne et al. were the first to obtain homozygous plants for the transgenes using
biolistic bombardment of freshly-isolated barley microspores. Transgenic embryos or
calli can be selected from cultures by using resistance selection, either to herbicides or
to an antibiotic resistance marker or with a visual marker gene such as the green
fluorescent protein (GFP) (Shim and Kasha, 2003). Transformation and DH technology
have permitted the creation of homozygous Brassica lines with enhanced tolerance to
clubroot (Plasmodiophora brassicae) (Reiss et al., 2009) or pollen beetle (Åhman et al.,
2006).
1.2.2.3.5 Pea doubled haploid research status
The production of DHs via androgenesis should be feasible in most plant species
but it has taken a long time to develop protocols robust enough to be used in
21
breeding programs and these have been developed for a limited number of crops.
Each crop has different requirements and thus, the need for extensive research to
develop an efficient DH system (Kasha and Maluszynski, 2003). Leguminous species,
including pea, have been described as recalcitrant to DH techniques (Croser et al.,
2006) and progress towards this goal has been very slow (Table 1.2). In 1972, Gupta
et al. were the first to report haploid callus induction from anthers of pea breeding
line B22. Microspores were at the uni-nucleate stage at the moment of culture. No
plant regeneration was achieved nor was the ploidy level of the callus cells confirmed.
A few years later, Gupta (1975) reported the production of a few roots, shoots and
torpedo-shaped embryos, again with no confirmation of ploidy level. Gosal and Bajaj
(1988) successfully induced callus by applying a cold pre-treatment to extracted
anthers of the pea cv. Bonneville and the breeding lines T163 and P88. A few heart-
shaped embryos were developed but no plant regeneration was obtained. About 90%
of the cells were diploid indicating that callus might have developed from maternal
anther tissue rather than from microspores.
More recently, Sidhu and Davies (2005) cultured anthers of cvs. Mukta, Gorokh
40, Pelican and P. fulvum genotype ATC113 using media supplemented with casein
hydrolysate and various plant growth regulators. Thirty to 40% of anthers produced
callus and two putative haploid/ DH shoots were recovered, one each from cvs.
Gorokh and Pelican. In pea cultivars CDC April and Highlight a cold pre-treatment of
buds coupled with electroporation of isolated microspores led to symmetrical
microspore nuclear divisions and early-stage embryo production (Croser and Lülsdorf,
2004; Croser et al., 2005; Croser et al., 2007; Grewal et al., 2007). A combination of
multiple stress factors including cold-pre-treatment of buds, osmotic and electric
22
shock, successfully improved androgenetic divisions from extracted anthers (Ochatt et
al., 2007) and isolated microspores (Ochatt et al., 2009) of cultivars Frisson, Terese,
Victor, Cheyenne and CDC April. Calli and haploid derived embryos were obtained and
a few weak plantlets recovered. The haploid condition of these materials was
confirmed by flow cytometry. In 2010, Bobkov reported the development of callus
tissues and globular embryoids in pea anther culture on a medium containing 2,4-D
after a cold (4°C) treatment of buds but no haploid plants were recovered with this
protocol.
Despite the recent encouraging results, no DH protocol has been developed for
pea or any other cool-season legume crop that can be routinely exploited in a
breeding program. The fundamental understanding of the molecular and biochemical
basis for microspore developmental transition from gametophytic to sporophytic
pathway remains elusive (Croser et al., 2006; Ochatt et al., 2009; Germanà, 2011;
Lülsdorf et al., 2011). More research is therefore required to improve our
understanding of the molecular and biochemical mechanisms involved in
androgenesis elicitation in order to facilitate the development of a robust protocol
that can be applied in extensive pea breeding programs and genetic studies.
23
Table 1.2 Summary of key publications on haploid research in pea (AC: anther culure; IMC: isolated microspore culture).
Author/s Target Cultivar/line Stress treatment Result
1Gupta et al. 1972
AC
B22
--
Callus formation
1Gupta 1975
AC
B22
--
Few shoots/ torpedo-shaped
embryos; No plants recovered
1Gosal and Bajaj
1988
AC
Boneville, T163
and P88
Anthers: Cold for 72 h
Callus/ heart shaped-embryos
2Croser and
Lülsdorf 2004
IMC
CDC April; Highlight
Buds: Cold or heat
Symmetrical microspore nuclear divisions to multinucleate stage
3Sidhu and Davies
2005
AC
Gorock 40;
Pelican
Anthers: Cold for 72 h
Callus production;
Two putative haploid shoots recovered
3Croser et al.
2005; 2Croser et
al. 2007
IMC
CDC April; Highlight
Buds: Cold for 48 h
Symmetrical microspore nuclear
divisions; Early-stage embryos
2Grewal et al.
2007
IMC
CDC April
Buds: Cold for 4 d
Microspores: electroporation
Multinucleate microspores; Pro-
embryos
2Ochatt et al.
2007
AC
Frisson; Terese,
Victor; Cheyenne
Buds: Cold > 48 h
Anthers: osmolarity and electroporation
Calli, somatic embryos and buds;
A few enfeebled plantlets recovered
1Ochatt et al.
2009
IMC
Frisson; Terese;
Cameor; Frisson x Cameor
Buds: cold for 2-30 d
Microspores: osmolarity and electroporation
Few flow cytometry confirmed
doubled haploid plants
1Bobkov 2010
AC
Faraon, Vizir, Mul’tik,
Madonna, Stabil and
BL101
Buds: Cold
Anthers: Heat
Green morphogenetic calli;
Globular embryoids
1Journal publications
2Non refereed conference proceedings
3Refereed conference proceedings
24
1.2.2.3.6 Application of flow cytometry in plant breeding
Flow cytometry is a high-throughput analytical tool that simultaneously detects
and quantifies multiple optical properties (fluorescence, light scatter) of single
particles, usually cells or nuclei labelled with fluorescent probes, as they move in a
narrow liquid stream through a powerful beam of light (Kron et al., 2007). Flow
cytometry facilitates a wide range of parameters to be simultaneously recorded from
individual particles, in contrast to other techniques that generate average values of
the whole population of measured particles (Doležel et al., 2007a). Also, relationships
between different properties can be determined after examining multiple parameters
for each cell thus providing a better understanding of the factors responsible for the
characteristics of cells and cell cultures (Yanpaisan et al., 1999). As a result, flow
cytometry is now used routinely in a number of fields ranging from basic cell biology
to genetics, immunology, molecular biology, and environmental science (Kron et al.,
2007).
Flow cytometry has been used for the analysis of the composition of various
chemical components of different tissues (Cvikrová et al., 2003; Loureiro et al., 2006),
or cell wall fractions (Ochatt, 2006), chromosome sorting for physical mapping of
genomes (Ng and Carter, 2006; Doležel et al., 2007b; Ng et al., 2007), characterisation
of true-to-typeness (Geoffriau et al., 1997; Roux et al., 2003; Elmaghrabi and Ochatt,
2006; Kone et al., 2007) and for the distinction between closely related genotypes
(Ochatt et al., 2013). This tool has also been used as a predictor of regeneration
competence from single-cell derived cultures (Ochatt et al., 2000; Ochatt et al., 2010)
or of the occurrence of hyperhydricity in regenerants (Ochatt et al., 2002), as well as
25
for the assessment of developmental status of immature seeds and embryos (Atif et
al., 2013).
In terms of determination of ploidy level in plants, chromosome counting using
root tip cells arrested in metaphase remains the unambiguous method to define the
chromosomal complement. However, this technique is very time-consuming; it
requires highly skilled operators and the supply of tissues containing discrete numbers
of dividing cells, which may not be easily available (Doležel et al., 2007a; Ochatt, 2008;
Ochatt et al., 2011). Alternative methods that do not require mitotically-active cells
include leaf stomatal density and size (van Duren et al., 1996) or the size of pollen
grain (Zonneveld and van Iren, 2001) and cell size (Hao et al., 2002). However, these
techniques have generally proved to be insufficiently reliable (Ochatt, 2008). Since the
nuclear DNA content correlates with ploidy, a high-throughput solution was provided
by the flow cytometry estimation of DNA content (Bohanec, 2003; Doležel et al.,
2007a; Ochatt, 2008). Flow cytometry has been extensively used to efficiently
determine the relative nuclear DNA content and ploidy level across a large number of
species including the legumes pea, Medicago truncatula Gaernt. and Lathyrus spp.
(Ochatt et al., 2009) and chickpea (Grewal et al., 2009). When optimised protocols are
used, a high number of samples, 100 or more per day, can be accurately analysed
(Bohanec, 2003; Cousin et al., 2009).
Despite the wide applications of flow cytometry to plant cell analysis, its use for
the analysis of embryogenic potential in plant cell populations has been relatively
limited (Ochatt, 2008). Flow cytometry has been used to study the embryogenic
potential from protoplasts of pea (Ochatt et al., 2000) and cultured microspores of
canola (Schulze and Pauls, 1999; Schulze and Pauls, 2002) and brown mustard
26
(Brassica juncea L. Czern.) (Lionneton et al., 2001). Flow cytometry promises
quantitative and qualitative advances in the understanding of plant growth,
development and function at subcellular, cellular and organismal levels (Doležel et al.,
2007a), and particularly of the factors affecting haploid embryogenesis.
1.2.2.4 In vitro based Single-Seed-Descent (SSD)
1.2.2.4.1 Introduction
Given the obvious difficulty inherent in the development of DH technology in
pea, I decided to concurrently explore an alternative in vitro approach for the rapid
achievement of homozygosity. The in vitro based modified SSD system has been
proposed as an alternative method to accelerate generation turnover across a number
of species. Conventional SSD consists of randomly selecting one seed per plant from
an F2 population following a cross, and advancing it through the early segregating
generations, thus permitting the rapid fixation of genes in breeding lines (Goulden,
1939). As only one seed is needed to produce the next generation, plants can be
grown under conditions that accelerate flowering and seed-set, but do not encourage
high yield. The technique of SSD makes it possible to advance a population to a
homozygous generation in a much shorter time than the conventional plant breeding
methods, which generally use one generation per year (Brim, 1966). Current
conventional SSD methodologies enable a maximum of three generations per year to
be developed in pea breeding programs (Ochatt and Sangwan, 2010), provided
breeders have access to controlled temperature environments. The in vitro SSD
technique involves the shortening of generation time by culturing immature seeds
developed after forced in vitro flowering under conditions for accelerated flowering
27
and seed-set. This leads to rapid generation turnover by reducing the length of the
generation cycle, enabling multiple generations per year.
1.2.2.4.1 In vitro flowering in pea
In vitro flowering has been reported in many plant species, including
Amaranthus spp. (Tisserat and Galletta, 1988), potato (Al Wareh et al., 1989),
Japanese pear (Pyrus serotina Rehd) (Tsujikawa et al., 1990), Kalanchoe blossfeldiana
Poelln. (Dickens and Van Staden, 1990), cranberry (Vaccinium macrocarpon Ait.
‘Stevens’) (Serres and McCown, 1994), bamboo (Bambusa arundinacea (Retz.) Willd.)
(Joshi and Nadgauda, 1997), ginseng (Panax ginseng C.A. Meyer) (Lin et al., 2005),
Kniphofia leucocephala Baijnath. (Taylor et al., 2005), Perilla frutescens (L.) Britton
(Zhang, 2007), A. thaliana (Ochatt and Sangwan, 2008; Ochatt and Sangwan, 2010),
and tomato (Lycopersicon esculentum Mill.) (Bhattarai et al., 2009). However, a limited
number of publications are available in legumes species e.g. soybean (Glycine max (L.)
Merr.) (Dickens and van Staden, 1985), peanut (Arachis hypogaea L.) (Narasimhulu
and Reddy, 1984; Chengalrayan et al., 1995; Asawaphan et al., 2005), Vigna mungo L.
(Ignacimuthu et al., 1997) and Vigna aconitifolia (Jacq) Marechal (Saxena et al., 2008).
In pea, there have been three in vitro flowering protocols proposed. In 2000,
Franklin et al. successfully obtained in vitro flowering and seed set with cv. ‘PID’. In
this protocol an in vitro rooting phase, a reduced concentration of ammonium nitrate
and the addition of auxins to the culture medium were key factors for the induction of
flowering in vitro. Ochatt et al. (2002) designed a strategy to reduce the length of the
generation cycles in vitro. Neither adding plant growth regulators (1 mg/L of 1-
naphtaleneacetic acid, NAA), nor reducing salt strength in the medium by half or the
rooting of in vitro shoots were essential to induce in vitro flowering and seed-set.
28
Using this protocol, 6.87 generations per year were obtained with cv. ‘Frisson’ and
5.24 generations per year with cv. ‘Terese’. Fujioka et al. (1999) also studied the
effects of culture conditions on in vitro flowering and seed-set for the Japanese pea
cultivars ‘Misasa’ and ‘Kishu-usui’. They observed that by growing plants in 30 x 200
mm glass tubes covered with a cotton stopper and culturing at 25°C with a light
intensity of 135 µmol m-2 s-1 and a photoperiod of 24 h, in vitro flowering could be
achieved in 34.6 days for cultivar ‘Misasa’ (100% pod-set) and in 70.8 days for cultivar
‘Kishu-usui’ (57.7% pod-set). Whilst providing an excellent platform for future studies,
these protocols were developed only for early-flowering pea cultivars. The
identification of a simple, robust and widely applicable in vitro system to accelerate
generation turnover by shortening the length of each cycle will be of great value in
pea breeding programs.
1.2.2.4.3 Factors affecting flowering in pea
The pea plant is botanically indeterminate and continues to grow provided
sufficient moisture is available (McPhee, 2003). The first nodes, some of which give
rise to branches, are vegetative, while the subsequent nodes are reproductive.
Generally two flowers, from which the pods develop, are present at each reproductive
node (Cousin, 1997). The flowering habit of peas is a genetic characteristic of each
variety. All known genotypes produce a number of vegetative nodes before flowering,
ranging from as few as four in the earliest varieties to over 100 in the latest varieties
under non-inductive conditions (Weller et al., 1997). The onset of flowering and the
number of flowering nodes is triggered by environmental conditions, such as
temperature, light quality, photoperiod, carbon dioxide level, humidity and nutrients
(Gottschalk, 1988; Cerdan and Chory, 2003; Iannucci et al., 2008; Nelson et al., 2010).
29
Natural environment, and even glasshouse conditions can be highly variable making it
difficult to examine the influence of environmental factors on flowering (Cummings et
al., 2007). Controlled environment growth chambers are commonly used in order to
minimise environmental variation and to provide plants with the best conditions to
accelerate flowering and seed-set.
Light and temperature are the main environmental factors that influence
flowering in pea (Murfet and Reid, 1974; Moe and Heins, 1990; Nelson et al., 2010).
Light provides environmental information for the plant and consequently affects a
wide range of photomorphogenic responses, including germination, de-etiolation,
elongation, leaf expansion and flowering (Weller, 2004; Spalding and Folta, 2005).
Plants detect light quality by at least three families of photoreceptors: phytochromes,
cryptochromes, and one or more unidentified ultraviolet light receptor(s) (Runkle and
Heins, 2001). Phytochrome absorbance peaks are in the red light (R, 600-700 nm), and
far-red (FR, 700-800 nm) and to a lesser extent in blue (B, 400-500 nm). Blue light is
also absorbed by cryptochromes. As plants preferentially absorb R light for
photosynthesis, the light under canopies or in crowded conditions has low red to far-
red (R:FR) ratio, which can result in the shade avoidance response: elongation of
stems, reduced leaf size and earlier flowering (Smith, 1994). Conversely, light rich in R
light (high R:FR ratio) signals non-competitive conditions, and can result in relatively
reduced plant height and later flowering (Runkle and Heins, 2001). A R:FR ratio of 1:1
seems to be the most effective for flower induction in long-day pea plants (Cummings
et al., 2007).
The influence of temperature on flowering is mediated through at least two
mechanisms in addition to the obvious effect on growth rate (Reid and Murfet, 1975).
30
Firstly, it is proposed that the inhibitor pathway has a higher temperature coefficient
than the floral stimulus pathway leading to promotion of flowering by low
temperatures. Secondly, low temperature (vernalisation) treatment seems to have a
specific effect at the shoot apex rendering it more responsive to the flowering signal
(Reid et al., 1996).
The growth photoperiod also has an important effect on the initiation of
flowering in pea. Most pea cultivars are facultative long-day plants. However, some
are day-neutral and others are essentially obligate long-day plants that may not flower
at all in photoperiods shorter than 12 h if unvernalised (Murfet, 1985). In addition to
the effect on flower initiation, photoperiod also affects a number of other
characteristics in pea (Weller et al., 1997). Relative to long-day conditions, short-day
conditions promote branching of the shoot and elongation of peduncles, and inhibit
stem elongations, apical arrest and the growth of flowers and pods (Murfet, 1985;
Murfet and Reid, 1993). Environmental gas composition (ethylene levels and carbon
dioxide concentration) and humidity levels, through the use of different type of
culture vessels and closure systems, have an important effect on flowering under in
vitro conditions (Fujioka et al., 1999; Asawaphan et al., 2005). In 1999, Fujioka et al.
observed a higher proportion of in vitro flowering in pea when a 30 x 200 mm test
tube and a cotton stopper were used. In peanut, Asawaphan et al. (2005) observed a
higher number of flowers produced in vitro, as well as an increase in carbon dioxide
concentrations when culturing seedling in vessels with reduced ventilation. These
results suggest that carbon dioxide levels may have a promoting effect on flowering.
In contrast, in Brassica campestris L., in vitro accumulation of ethylene was observed
when tightly sealed culture containers were used (Lentini et al., 1988). The
31
accumulation of ethylene in sealed containers was associated with inhibition of plant
development, with the production of abortive flowers or no floral buds.
Along with environmental factors and physiological state of the plant, the use of
different types of growth regulators has been shown to influence the induction of
flowering in vitro in several species. The requirement of auxins for flower induction
has been reported in many plants species, either alone (Ignacimuthu et al., 1997;
Zhang, 2007) or in combination with cytokinins (Narasimhulu and Reddy, 1984).
However, in some species auxins are either ineffective (Rastogi and Sawhney, 1987) or
inhibitory (Deaton et al., 1984; Lin et al., 2005) for floral development. Cytokinins have
also shown a promoting effect on in vitro flowering (Lin et al., 2005; Taylor et al.,
2005). Gibberellins can promote seed germination, internode elongation and
flowering (particularly in species that require long days and/ or cold) (Gaspar et al.,
1996). Gibberellins are essential for floral bud growth in general, and stamen
development in particular (Sawhney and Rastogi, 1990). Gibberellic acid (GA3)
combined with auxins has been used to induce in vitro flowering in various species
(Pharis et al., 1987; Evans et al., 1990; Sankhla et al., 1994), including pea (Franklin et
al., 2000). However, Murfet and Reid (1987) affirmed there is no evidence to suggest
gibberellins have any facilitating role in the flowering process as pea mutants with a
severe block in the gibberellin synthesis pathway still flower.
1.2.2.4.3 Genetics of flowering in pea
Pea has been a model for the genetics of flowering for several decades and
numerous flowering loci have been identified, but until recently little was known
about the molecular nature of these loci. To date, more than 20 flowering-related loci
have been identified in pea from natural genetic variation and induced mutations
32
(Weller et al., 2009). The ability to respond to photoperiod in pea is conferred by the
action of three complimentary dominant genes Sn, Dne and Ppd (Barber, 1959;
Murfet, 1971a; King and Murfet, 1985; Reid et al., 1996). The Sn Dne Ppd system is
responsible for the formation of a graft-transmissible inhibitor of flowering under non-
inductive, short day conditions, resulting in the delay of flower initiation (Murfet,
1971b; Murfet and Reid, 1973; King and Murfet, 1985; Reid et al., 1996; Taylor and
Murfet, 1996). Two major genes, E and Hr, influence ontogenetic expression of the
system. The dominant allele E reduces inhibitor synthesis in the cotyledons (Murfet,
1971b; Murfet, 1971c; Murfet and Reid, 1973; Reid et al., 1996; Taylor and Murfet,
1996), while Hr prolongs inhibitor synthesis in the shoot (Murfet, 1973; Reid, 1979;
Reid et al., 1996). Flowering behaviour in pea is also determined by the genotype at
the Lf locus, which acts at the shoot apex to determine sensitivity to the flowering
signal (Murfet, 1971b). In addition to genes controlling inhibitor synthesis, a gene (Gi)
has been identified in pea, which acts on the synthesis pathway of the floral stimulus
(Beveridge and Murfet, 1996).
1.2.2.5 Doubled haploidy vs. Single-Seed-Descent
Plant breeders have always been interested in shortening the period for
inbreeding. Both DH and SSD breeding methods attempt to reduce the time required
for inbreeding by rapidly advancing generations without breeder or natural selections.
Conventional breeding methods consist of several backcrossing or self-pollination
steps, thus they are labour-intensive and time-consuming procedures. The SSD
strategy was devised to speed-up the development of homozygous lines, but it suffers
from the same handicaps as the conventional breeding technique of pedigree
inbreeding in terms of time delays and competitive interactions among plants
33
(Germanà, 2006). In contrast, gametic embryogenesis makes the production of
homozygous lines feasible and shortens the time required to produce such lines,
allowing the single-step development of completely homozygous lines from
heterozygous parents (Germanà, 2011).
Production of DHs from an F1 hybrid limits the opportunity for recombination
between loci to a single meiosis. In contrast, random inbred lines produced from the
F2 generation by SSD will have passed through several rounds of potential
recombination (Caligari et al., 1986). Hence, the linkage bias in F∞ samples produced
by SSD will be less than that of similar samples produced by DH from the F1 (Caligari et
al., 1987). In barley anther culture, a clear segregation distortion in the absence of
selection was observed (Zivy et al., 1992). On the other hand, Thomas et al. (2003)
affirmed that a comparison of theoretical properties of DH and SSD progenies from a
range of species, principally cereals, showed that in the absence of linkage there is no
difference between these two strategies (Thomas et al., 2003), so the choice of the
strategy to adopt will depend on whether linkage blocks are to be preserved (F1DH) or
broken-up (SSD). The adoption of doubled haploidy does not lead to any bias of
genotypes in populations, and random DHs were even found to be compatible to
selected lines produced by conventional methods (Winzeler et al., 1987; Jain et al.,
1996-1997; Germanà, 2006).
Some researchers argue that the theoretical benefits are not enough to deploy
DH technology in all breeding programs. Indeed, cost efficiency, fixation of rare and
useful alleles, conservation of genetic variability in selection lines, uniformity and
stability of new released varieties must be considered (Thomas et al., 2003; Germanà,
2006). The main difficulty in improving androgenesis lies in the large range of factors
34
that control and influence the process (Wędzony et al., 2009). Conversely, the SSD
technique is very efficient on account of selection effort, is less expensive and labour
intensive, and can be utilised across a wider range of genotypes. The SSD strategy
offers the greatest benefits in situations where simultaneous selection is required for
several characteristics with different heritability.
1.2.2.6 Benefits of pea research to the Australian industry sector
The pea production area has significantly decreased in the last few decades
worldwide (FAOSTAT, 2012) predominantly as a result of the unstable yields caused by
many biotic and abiotic limitations. Further adoption of pea by farmers is dependent
on the availability of superior cultivars with improved abiotic and biotic stress
resistance, which will lead to more reliable yield and quality. One of the main
limitations to pea genetic improvement is the length of the cultivar development
process and the absence of biotechnology tools applicable to the crop. This prevents a
timely response to emerging threats and end-user’ needs. The development of in vitro
technologies in pea, like doubled haploidy and in vitro SSD, has the potential to
contribute exciting new dimensions to the conventional exploitation of genetic
resources and breeding strategies for improved varieties (Redden et al., 2005). The
availability of new biotechnology tools will help overcome some of the major
constraints to increasing productivity in pea. By accelerating the development of
better adapted cultivars these techniques will greatly assist pea breeders in the
development of new germplasm and confer the ability to quickly respond to novel
abiotic and biotic threats. Economically, this could lead to substantial increases in pea
production area which would offset fertiliser and pesticide input costs and assist with
maintaining viable margins in cropping-based agricultural operations. This would have
35
important social ramifications, enabling smaller farm operations to remain viable
leading to critical population mass in rural areas to support important community
infrastructure.
1.3 Research objectives and thesis structure
The aim of this research is to accelerate the breeding process in pea by
developing in vitro methods to more rapidly achieve a high level of homozygosity and
to gain a better understanding of the physiological mechanisms involved in these
processes. Chapter 2 presents the work undertaken on the development of a DH
protocol from extracted anthers in selected pea genotypes of varying genetic
backgrounds. Chapter 2 includes the study of factors known to be involved in the
elicitation of androgenesis. This Chapter leads into Chapter 3, which describes the
flow cytometry study of the effect of abiotic stress factors on androgenesis elicitation
from extracted anthers and isolated microspores of pea. In Chapter 4, an alternative
method of obtaining rapid homozygosity is studied. This chapter focuses on the study
of the effect of physiological mechanisms operating in vitro on the efficient and
reproducible induction of flowering and seed-set across a range of pea genotypes for
use in SSD breeding strategies. Finally, Chapter 5 provides a summary of the research
undertaken and outlines proposed future directions.
36
1.4 References
Åhman IM, Kazachkova NI, Kamnert IM, Hagberg PA, Dayteg CI, Eklund GM, Meijer LJO, Ekbom B (2006) Characterisation of transgenic oilseed rape expressing pea lectin in anthers for improved resistance to pollen beetle. Euphytica 151: 321-330
Al Wareh H, Trolinder NL, Goodin JR (1989) In vitro flowering of potato. Hort. Sci. 24: 827-829 Ali SM, Sharma B, Ambrose MJ (1994) Current status and future strategy in breeding pea to
improve resistance to biotic and abiotic stresses. Euphytica 73: 115-126 Ambrose MJ (1995) From Near East center of origin, the prized pea migrates throughout the
world. Diversity 11: 118-119 Amssa M, De Buyser J, Henry Y (1980) Origin of diploid plants obtained by in vitro culture of
anthers of young wheat (Triticum aestivum L.). Cr. Acad. Sci. D. Nat. 290: 1095-1097 Asawaphan P, Mangkita W, Kachonpadungkitti Y, Matsuyama S, Satake T, Hisajima S (2005)
Efficient flower induction from peanut (Arachis hypogaea L.) seedling in vitro. SABRAO J. Breed. Genet. 37: 131-140
Atif RM, Boulisset F, Conreux C, Thompson R, Ochatt SJ (2013) In vitro auxin treatment promotes cell division and delays endoreduplication in developing seeds of the model legume species Medicago truncatula. Physiol. Plant.: Published online DOI 10.1111/j.1399-3054.2012.01719.x
Bakos F, Darko E, Ascough G, Gaspar L, Ambrus H, Barnabas B (2008) A cytological study on aluminium-treated wheat anther cultures resulting in plants with increased Al tolerance. Plant Breed. 127: 235-240
Barber HN (1959) Physiological genetics of Pisum. II. The genetics of photoperiodism and vernalisation. Heredity 13: 33-60
Bean SJ, Gooding PS, Mullineaux PM, Davies DR (1997) A simple system for pea transformation. Plant Cell Rep. 16: 513-519
Beveridge CA, Murfet IC (1996) The gigas mutant in pea is deficient in the floral stimulus. Physiol. Plant. 96: 637-645
Bhattarai SP, de la Pena RC, Midmore DJ, Palchamy K (2009) In vitro culture of immature seed for rapid generation advancement in tomato. Euphytica 167: 23-30
Bobkov SV (2010) Isolated pea anther culture. Russian Agric. Sci. 36: 413-416 Bohanec B (2003) Ploidy determination using flow cytometry. In M Maluszynski, K Kasha, BP
Forster, I Szarejko, eds, Doubled Haploid Production in Crop Plants. Kluwer Acad. Publ., Dordrecht, The Netherlands, pp 397-403
Boutilier K, Fiers M, Liu CM, Geest ALM (2005) Biochemical and Molecular Aspects of Haploid Embryogenesis. In CE Don Palmer, W Keller, K Kasha, eds, Haploids in Crop Improvement II, Vol 56. Springer Berlin Heidelberg, pp 73-95
Brim CA (1966) A modified pedigree method of selection in soybeans. Crop Sci. 6: 220 Caligari PDS, Powell W, Jinks JL (1986) The use of double haploids for detecting linkage and
pleiotropy between quantitatively varying characters in spring barley. J. Agric. Sci. 106: 75-80
Caligari PDS, Powell W, Jinks JL (1987) A comparison of inbred lines derived by doubled haploidy and single seed descent in spring barley (Hordeum vulgare). Ann. Appl. Biol. 111: 667-675
Cerdan PD, Chory J (2003) Regulation of flowering time by light quality. Nature 423: 881-885 Chase SS (1952) Production of homozygous diploids of maize from monoploids. Agron. J. 44:
263-267 Chengalrayan K, Mhaske VB, Hazra S (1995) In vitro regulation of morphogenesis in peanut
(Arachis hypogaea L.). Plant Sci. 110: 259-268 Choi HK, Mun JH, Kim DJ, Zhu H, Baek JM, Mudge J, Roe B, Ellis N, Doyle J, Kiss GB, Young
ND, Cook DR (2004) Estimating genome conservation between crop and model legume species. Proc. Nat. Acad. Sci.USA 101: 15289-15294
Christou P (1993) The biotechnology of crop legumes. Euphytica 74: 165-185
37
Cordewener JHG, Busink R, Traas JA, Custers JBM, Dons HJM, Van Lookeren Campagne MM (1994) Induction of microspore embryogenesis in Brassica napus L. is accompanied by specific changes in protein synthesis. Planta 195: 50-56
Cordewener JHG, Custers JBM, Dons HJM, Van Lookeren Campagne MM (1996) Molecular and biochemical events during the induction of microspore embryogenesis. In SM Jain, SK Sopory, RE Veilleux, eds, In Vitro Haploid Production in Higher Plants, Vol 1. Kluwer Acad. Publ., Dordrecht, The Netherlands, pp 111-124
Cordewener JHG, Custers JBM, Van Lookeren Campagne MM (1998) Microspore culture. A model for investigating the role of stress in the induction of embryogenesis. In Y Chupeau, M Caboche, Y Henry, eds, Androgenesis and haploid plants. Springer, Berlin Heidelberg Ney York, pp 54-68
Cordewener JHG, Hause G, Görgen E, Busink R, Hause B, Dons HJM, Van Lammeren AAM, Van Lookeren Campagne MM, Pechan P (1995) Changes in synthesis and localization of members of the 70-kDa class of heat-shock proteins accompany the induction of embryogenesis in Brassica napus L. microspores. Planta 196: 747-755
Cousin A, Heel K, Cowling WA, Nelson MN (2009) An efficient high-throughput flow cytometric method for estimating DNA ploidy level in plants. Cytometry 75A: 1015-1019
Cousin R (1997) Peas (Pisum sativum L.). Field Crops Res. 53: 111-130 Croser J, Lülsdorf M, Davies P, Wilson J, Sidhu P, Grewal R, Allen K, Dament T, Warkentin T,
Vandenberg A, Siddique K (2005) Haploid Embryogenesis from Chickpea and Field Pea- Progress Towards a Routine Protocol. In IJ Bennett, E Bunn, H Clarke, JA McComb, eds, Proceedings of the Australian Branch of the IAPTC&B - ‘Contributing to a Sustainable Future’ Perth, Australia, pp 71-82
Croser JS, Clarke HJ, Lülsdorf MM, Mallikarjuna N, Edwards K, Usher K, Bowers HM, Kuo JS, Khan TN, Siddique KHM (2007) Haploids and hybrids. In 6th European Conference on Grain Legumes "Integrating legume biology for sustainable agriculture", 12-16 November 2007, Lisbon congress centre, Portugal: AEP Association Europeenne des proteagineux
Croser JS, Lülsdorf MM (2004) Progress towards haploid division in chickpea (Cicer arietinum L.), field pea (Pisum sativum L.) and lentil (Lens culinaris Medik.) using isolated microspore culture. In European Grain Legume Conference, AEP, Dijon, France, p 189
Croser JS, Lülsdorf MM, Davies PA, Clarke HJ, Bayliss KL, Mallikarjuna N, Siddique KHM (2006) Toward doubled haploid production in the Fabaceae: Progress, constraints, and opportunities. Crit. Rev. Plant Sci. 25: 139-157
Cummings IG, Reid JB, Koutoulis A (2007) Red to far-red ratio correction in plant growth chambers - growth responses and influence of thermal load on garden pea. Physiol. Plant. 131: 171-179
Cvikrová M, Binarová P, Cenklová V, Eder J, Doležel J, Machácková I (2003) Effect of 2-aminoindan-2-phosphonic acid on cell cycle progression in synchronous meristematic cells of Vicia faba roots. Plant Sci. 164: 823-832
Darko E, Ambrus H, Fodor J, Zoltan Kiraly Z, Barnabas B (2009) Enhanced tolerance to oxidative stress with elevated antioxidant capacity in doubled haploid maize derived from microspores exposed to paraquat. Crop Sci. 49: 628-636
Davies DR (1976) Peas Pisum sativum (Leguminosae-papilionoideae). In DR Davies, NW Simmonds, eds, Evolution of Crop Plants. Longman, London, pp 294-296
Deaton MH, Buxton JW, Hamilton-Kemp TR (1984) The effects of growth regulators on development of Nicotiana affinis flowers in vitro. Hort. Sci. 19: 509-511
Delaitre C, Ochatt S, Deleury E (2001) Electroporation modulates the embryogenic responses of asparagus (Asparagus officinalis L.) microspores. Protoplasma 216: 39-46
Dickens CWS, van Staden J (1985) In vitro flowering and fruiting of soybean explants. J. Plant Physiol. 120: 83-86
38
Dickens CWS, Van Staden J (1990) The in vitro flowering of Kalanchöe blossfeldiana Poellniz. II. The effects of growth regulators and gallic acid. Plant Cell Physiol. 31: 757-762
Dita MA, Rispail N, Prats E, Rubiales D, Singh KB (2006) Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes. Euphytica 147: 1-24
Doležel J, Greilhuber J, Suda J (2007a) Flow cytometry with plants: an overview. In J Doležel, J Greilhuber, J Suda, eds, Flow Cytometry with Plant Cells: Analysis of Genes, Chromosomes and Genomes. Wiley Online Library, Weinheim, pp 41-66
Doležel J, Kubaláková M, Paux E, Bartoš J, Feuillet C (2007b) Chromosome-based genomics in the cereals. Chromos. Res. 15: 51-66
Duncan EJ, Heberle E (1976) Effect of temperature shock on nuclear phenomena in microspores of Nicotiana tabacum and consequently on plantlet production. Protoplasma 90: 173-177
Dunwell JM (2010) Haploids in flowering plants: origin and exploitation. Plant Biotechnol. J. 8: 377-424
Elmaghrabi A, Ochatt S (2006) Isoenzymes and flow cytometry for the assessment of true-to-typeness of calluses and cell suspensions of barrel medic prior to regeneration. Plant Cell Tiss. Organ Cult. 85: 31-43
Elmaghrabi AM, Ochatt S, Rogers HJ, Francis D (2013) Enhanced tolerance to salinity following cellular acclimation to increasing NaCl levels in Medicago truncatula. Plant Cell Tiss. Organ Cult. Published online DOI 10.1007/s11240-013-0306-2
Evans LT, King RW, Chu A, Mander LN, Pharis RP (1990) Gibberellin structure and florigenic activity in Lolium temulentum, a long-day plant. Planta 182: 97-106
FAOSTAT (2012) Food and Agriculture Organization of the United Nations. In http://faostat3.fao.org, accessed in December 2012
Ferrie AMR, Caswell KL (2011) Isolated microspore culture techniques and recent progress for haploid and doubled haploid plant production. Plant Cell Tiss. Organ Cult. 104: 301-309
Ferrie AMR, Möllers C (2011) Haploids and doubled haploids in Brassica spp. for genetic and genomic research. Plant Cell Tiss. Organ Cult. 104: 375-386
Forster BP, Thomas WTB (2003) Doubled haploids in genetic mapping and genomics. In M Maluszynski, K Kasha, BP Forster, I Szarejko, eds, Doubled Haploid Production in Crop Plants. Kluwer Acad. Publ., Dordrecht, The Netherlands, pp 367-390
Forster BP, Thomas WTB (2005) Doubled haploid in genetics and plant breeding. Plant Breed. Rev. 25: 57-88
Franklin G, Pius PK, Ignacimuthu S (2000) Factors affecting in vitro flowering and fruiting of green pea (Pisum sativum L.). Euphytica 115: 65-74
Fujioka T, Fujita M, Miyamoto Y (1999) In vitro flowering and pod setting of non-symbiotically germinated pea. J. Japan. Soc. Hort. Sci. 68: 117-123
Gallardo K, Firnhaber C, Zuber H, Héricher D, Belghazi M, Henry C, Küster H, Thompson R (2007) A combined proteome and transcriptome analysis of developing Medicago truncatula seeds, evidence for metabolic specialization of maternal and filial tissues. Mol.Cell. Prot. 6: 2165-2179
Garrido D, Eller N, Heberle-Bors E, Vicente O (1993) De novo transcription of specific mRNAs during the induction of tobacco pollen embryogenesis. Sex. Plant Reprod. 6: 40-45
Garrido D, Vicente O, Heberle-Bors E, Rodriguez-Garcia I (1995) Cellular changes during acquisition of embryogenic potential in isolated pollen grains of Nicotiana tabacum. Protoplasma 186: 220-230
Gaspar T, Kevers C, Penel C, Greppin H, Reid DM, Thorpe TA (1996) Plant hormones and plant growth regulators in plant tissue culture. In Vitro Cell Dev. Biol. : Plant 32: 272-289
Geoffriau E, Kahane R, Bellamy C, Rancillac M (1997) Ploidy stability and in vitro chromosome doubling in gynogenic clones of onion (Allium cepa L.). Plant Science 122: 201-208
Germanà MA (2006) Doubled haploid production in fruit crops. Plant Cell Tiss. Organ Cult. 86: 131-146
39
Germanà MA (2011) Anther culture for haploid and doubled haploid production. Plant Cell Tiss. Organ Cult. 104: 283-300
Gosal SS, Bajaj YPS (1988) Pollen embryogenesis and chromosomal variation in anther culture of three food legumes - Cicer arietinum, Pisum sativum and Vigna mungo. SABRAO J. Breed. Genet. 20: 51-58
Gottschalk W (1988) Phytotron experiments in Pisum. Influence of the photoperiod on the flowering behavior of different genotypes Theor. Appl. Genet. 75: 344-349
Goulden CH (1939) Problems in plant selection. In RC Burnett, ed, Proc. 7th Int. Genet. Cong. Cambridge University Press, England, pp 132-133
Grewal R, Lülsdorf M, Croser J, Ochatt SJ, Vandenberg A, Warkentin T (2007) Pro-embryos of field pea produced from electroporated microspores. In 6th European Conference on Grain Legumes "Integrating legume biology for sustainable agriculture", 12-16 November 2007, Lisbon congress centre, Portugal: AEP Association Europeenne des proteagineux, p 138
Grewal RK, Lülsdorf M, Croser J, Ochatt S, Vandenberg A, Warkentin TD (2009) Doubled-haploid production in chickpea (Cicer arietinum L.): role of stress treatments. Plant Cell Rep. 28: 1289-1299
Gritton ET (1980) Field Pea. In WR Fehr, HH Hadley, eds, Hybridization of Crop Plants. American Society of Agronomy, Madison, pp 347-356
Gupta S (1975) Morphogenetic response of haploid callus tissue of Pisum sativum (Var. B22). Indian Agric. 19: 11-21
Gupta S, Ghosal KK, Gadgil VN (1972) Haploid tissue culture of Triticum aestivum var. Sonalika and Pisum sativum var. B22. Indian Agric. 16: 277-278
Hao J, You C, Deng X (2002) Cell size as a morphological marker to calculate the mitotic index and ploidy level of citrus callus. Plant Cell Rep. 20: 1123-1127
Hause B, Hause G, Pechan P, Van Lammeren AAM (1993) Cytoskeletal changes and induction of embryogenesis in microspore and pollen cultures of Brassica napus L. Cell Biol. Int. 17: 153-168
Heberle-Bors E, Reinert J (1981) Environmental control and evidence for predetermination of pollen embryogenesis in Nicotiana tabacum pollen. Protoplasma 109: 249-255
Heberle-Bors E (1989) Isolated pollen culture in tobacco: plant reproductive development in a nutshell. Sex Plant Reprod. 2:1-10
Hosp J, de Maraschin SF, Touraev A, Boutilier K (2007) Functional genomics of microspore embryogenesis. Euphytica 158: 275-285
Hu TC, Kasha KJ (1999) A cytological study of pretreatments used to improve isolated microspore cultures of wheat (Triticum aestivum L.) cv. Chris. Genome 42: 432-441
Iannucci A, Terribile MR, Martiniello P (2008) Effects of temperature and photoperiod on flowering time of forage legumes in a Mediterranean environment. Field Crops Res. 1006: 156-162
Ignacimuthu S, Franklin G, Melchias G (1997) Multiple shoot formation and in vitro fruiting from cotyledonary nodes of Vigna mungo (L.) Hepper. Curr. Sci. 73: 733-735
Imamura J, Harada H (1980) Effects of abscisic acid and water stress on the embryo and plantlet production in anther culture of Nicotiana tabacum cv. Samsun. Z. Pflanzen-physiol. 100: 285-289
Jähne A, Becker D, Brettschneider R, Lorz H (1994) Regeneration of transgenic, microspore-derived, fertile barley. Theor. Appl. Genet. 89: 525-533
Jain SM, Sopory SK, Veilleux RE (1996-1997) In Vitro Haploid Production in Higher Plants, Vol 1-5. Kluwer Acad. Publ., Dordrecht, The Netherlands
Joshi M, Nadgauda RS (1997) Cytokinins and in vitro induction of flowering in bamboo: Bambusa arundinacea (RetZ.) Willd. Curr. Sci. 73: 523-526
Kasha KJ (1974). Haploids in higher plants. In 1st International Symposium on Haploids in Higher Plants: Advances and Potential, Univ. of Guelph, Guelph, Ontario, p 421
40
Kasha KJ (2005) Chromosome doubling and recovery of doubled haploid plants. In CE Palmer, WA Keller, KJ Kasha, eds, Biotechnology in Agriculture and Forestry, Vol 56. Springer-Verlag, Berlin, Heidelberg, pp 123-152
Kasha KJ, Maluszynski M (2003) Production of doubled haploids in crop plants. An introduction. In M Maluszynski, KJ Kasha, BP Forster, I Szarejko, eds, Doubled Haploid Production in Crop Plants. A Manual. Kluwer Acad. Publ., Dordrecht, The Netherlands, pp 1-4
Khan TN, Croser JS (2004) Pea: Overview. In C Wrigley, H Corke, C Walker, eds, Encyclopedia of Grain Science. Academic Publishers, pp 287-295
King WM, Murfet IC (1985) Flowering in Pisum: a sixth locus, Dne. Ann. Bot. 56: 835-846 Kinosita K, Tsong TY (1977) Voltage-induced pore formation and hemolysis of human
erythrocytes. Biochim. Biophys. Acta 471: 227-242 Kone M, Patat-Ochatt E, Conreux C, Sangwan R, Ochatt S (2007) In vitro morphogenesis from
cotyledon and epicotyl explants and flow cytometry distinction between landraces of Bambara groundnut [Vigna subterranea (L.) Verdc], an under-utilised grain legume. Plant Cell Tiss. Organ Cult. 88: 61-75
Kron P, Suda J, Husband BC (2007) Applications of flow cytometry to evolutionary and population biology. Annu. Rev. Ecol. Evol. Syst. 38: 847-876
Kush GS, Virmani SS (1996) Haploids in plant breeding. In SM Jain, SK Sopory, RE Veilleux, eds, In Vitro Haploid Production in Higher Plants, Vol 1. Kluwer Acad. Publ., Dordrecht, The Netherlands, pp 11-33
Kusuya M, Hosoya K, Yashiro K, Tomita K, Ezura H (2003) Powdery mildew (Spaerotheca fuliginea) resistance in melon is selectable at the haploid level. J. Exp. Bot. 54: 1069-1074
Kyo M, Harada H (1990) Specific phosphoproteins in the initial period of tobacco pollen embryogenesis. Planta 182: 58-63
Lentini Z, Mussell H, Mutschler MA, Earle ED (1988) Ethylene generation and reversal of ethylene effects during development in vitro of rapid-cycling Brassica campestris L. Plant Sci. 54: 75-81
Lin CS, Chen CT, Hsiao HW, Chang WC (2005) Effects of growth regulators on direct flowering of isolated ginseng buds in vitro. Plant Cell Tiss. Organ Cult. 83: 241-244
Lionneton E, Aubert G, Ochatt S, Merah O (2004) Genetic analysis of agronomic and quality traits in mustard (Brassica juncea). Theor. Appl. Genet. 109: 792-799
Lionneton E, Beuret W, Delaitre C, Ochatt S, Rancillac M (2001) Improved microsopore culture and doubled-haploid plant regeneration in the brown condiment mustard (Brassica juncea) Plant Cell Rep. 20: 126-130
Loureiro J, Rodriguez E, Doležel J, Santos C (2006) Flow cytometric and microscopic analysis of the effect of tannic acid on plant nuclei and estimation of DNA content. Ann. Bot. 98: 515-527
Lülsdorf M, Yuan HY, Slater S, Vandenberg A, Han X, Zaharia LI (2012) Androgenesis-inducing treatments change phytohormone levels in anthers of three legume species (Fabaceae). Plant Cell Rep. 31: 1255-1267
Lülsdorf MM, Croser JS, Ochatt S (2011) Androgenesis and doubled-haploid production in food legumes. In A Pratap, J Kumar, eds, Biology and breeding of food legumes. CABI Publishing, pp 159-177
Makasheva RK (1983) The Pea. Amerind Publishing Co. Pvt. Ltd., New Delhi, India, 267 Maluszynski M, Kasha KJ, Szarejko I (2003) Published doubled haploid protocols in plant
species. In M Maluszynski, KJ Kasha, BP Forster, I Szarejko, eds, Doubled haploid production in crop plants. A manual. Kluwer Acad. Publ., Dordrecht, The Netherlands, pp 309-335
Maraschin SF, de Priester W, Spaink HP, Wang M (2005) Androgenic switch: an example of plant embryogenesis from the male gametophyte perspective. J. Exp. Bot. 56: 1711-1726
41
McCallum MH, Peoples MB, Connor DJ (2000) Contributions of nitrogen by field pea (Pisum sativum L.) in a continuous cropping sequence compared with a lucerne (Medicago sativa L.)-based pasture ley in the Victorian Wimmera. Aust. J. Agric. Res. 51: 13-22
McPhee K (2003) Dry pea production and breeding - A mini-review. Food, Agric. & Environ. 1: 64-69
Medrano H, Primo-Millo E (1985) Selection of Nicotiana tabacum haploids of high photosynthetic efficiency. Plant Physiol. 79: 505-508
Moe R, Heins R (1990) Control of plant morphogenesis and flowering by light quality and temperature Acta Hortic. 272: 81-89
Muehlbauer FJ (1996) Advances in the production of cool season food legumes. Amer. J. Altern. Agr. 11: 71-76
Murfet IC (1971c) Flowering in Pisum. Three distinct phenotypic classes determined by the interaction of a dominant early and a dominant late gene. Heredity 26: 243-257
Murfet IC (1971a) Flowering in Pisum: a three gene system. Heredity 27: 93-110 Murfet IC (1971b) Flowering in Pisum: Reciprocal grafts between known genotypes. Aust. J.
Biol. Sci. 24: 1089-1102 Murfet IC (1973) Flowering in Pisum. Hr, a gene for high response to photoperiod. Heredity
31: 157-164 Murfet IC (1985) Pisum sativum. In AH Halevy, ed, Handbook of flowering, Vol 4. CRC Press, pp
97-126 Murfet IC, Reid JB (1973) Flowering in Pisum: evidence that gene Sn controls a graft-
transmissible inhibitor. Aust. J. Biol. Sci. 26: 675-677 Murfet IC, Reid JB (1974) Flowering in Pisum: The influence of photoperiod and vernalising
temperatures on the expression of genes Lf and Sn. Z. Pflanzenphysiologie 71: 323-331
Murfet IC, Reid JB (1987) Flowering in Pisum: gibberelins and the flowering genes. J. Plant Physiol. 127: 23-29
Murfet IC, Reid JB (1993) Developmental mutants, in Peas. In R Casey, DR Davies, eds, Genetics, Molecular Biology and Biotechnology. CAB International, pp 165-216
Narasimhulu SB, Reddy GM (1984) In vitro flowering and pod formation from cotyledons of groundnut (Arachis hypogaea L.). Theor. Appl. Genet. 69: 87-89
Nelson MN, Berger JD, Erskine W (2010) Flowering time control in annual legumes: Prospects in a changing global climate. CAB Rev: Perspect. Agric. Vet. Sci. Nutr. Nat. Resour. 5: 49-62
Ng BL, Carter NP (2006) Factors affecting flow karyotype resolution. Cytometry part A 69: 1028-1036
Ng BL, Yang F, Carter NP (2007) Flow analysis and sorting of microchromosomes (< 3 Mb). Cytometry part A 71: 410-413
Nitsch C (1977) Culture of isolated microspores. In J Reinert, YPS Bajaj, eds, Applied and fundamental aspects of plant cell, tissue and organ culture. Springer, Berlin, pp 268-278
Ochatt S, Pech C, Conreux C, Jacas L (2007) Toward the efficient production of androgenetic doubled haploid plants from pea (Pisum sativum L.) anthers. In 6th European Conference on Grain Legumes "Integrating legume biology for sustainable agriculture", 12-16 November 2007, Lisbon congress centre, Portugal: AEP Association Europeenne des proteagineux, p 136
Ochatt S, Pech C, Grewal R, Conreux C, Lülsdorf M, Jacas L (2009) Abiotic stress enhances androgenesis from isolated microspores of some legume species (Fabaceae). J. Plant Physiol. 166: 1314-1328
Ochatt SJ (2006) Flow cytometry (ploidy determination, cell cycle analysis, DNA content per nucleus). In Medicago truncatula Handbook, version 2006, 13 p. Available at http://www.noble.org/medicagohandbook/index.htnl. ISBN 0-9754303-1-9
Ochatt SJ (2008) Flow cytometry in plant breeding. Cytometry part A 73: 581-598
42
Ochatt SJ, Atif RM, Patat-Ochatt EM, Jacas L, Conreux C (2010) Competence versus recalcitrance for in vitro regeneration. Notulae Botanicae Horti Agrobotanici Cluj-Napoca 38: 102-108
Ochatt SJ, Chand PK, Rech EL, Davey MR, Power JB (1988) Electroporation-mediated improvement of plant regeneration from colt cherry (Prunus avium × pseudocerasus) protoplasts. Plant Sci. 54: 165-169
Ochatt SJ, Conreux C, Jacas L (2013) Flow cytometry distinction between species and between landraces within Lathyrus species and assessment of true-to-typeness of in vitro regenerants. Plant Syst. Evol. 299: 75-85
Ochatt SJ, Mousset-Declas C, Rancillac M (2000) Fertile pea plants regenerate from protoplasts when calluses have not undergone endoreduplication. Plant Sci. 156: 177-183
Ochatt SJ, Muneaux E, Machado C, Jacas L, Pontecaille C (2002b) The hyperhydricity of in vitro regenerants of grass pea (Lathyrus sativus L.) is linked with an abnormal DNA content. J. Plant Physiol. 159: 1021-1028
Ochatt SJ, Patat-Ochatt EM, Moessner A (2011) Ploidy level determination within the context of in vitro breeding. Plant Cell Tiss. Organ Cult. 104: 329-341
Ochatt SJ, Pontecaille C, Rancillac M (2000) The growth regulators used for bud regeneration and shoot rooting affect the competence for flowering and seed set in regenerated plants of protein peas. In Vitro Cell. Dev. Biol.: Plant 36: 188-193
Ochatt SJ, Sangwan RS (2008) In vitro shortening of generation time in Arabidopsis thaliana. Plant Cell Tissue Organ Cult. 93: 133-137
Ochatt SJ, Sangwan RS (2010) In vitro flowering and seed set: Acceleration of generation cycles. In MR Davey, P Anthony, eds, Plant Cell Culture: Essential Methods. John Wiley & Sons, Ltd., Chichester, UK, pp 97-110
Ochatt SJ, Sangwan RS, Marget P, Ndong YA, Rancillac M, Perney P (2002a) New approaches towards the shortening of generation cycles for faster breeding of protein legumes. Plant Breed. 121: 436-440
Pechan PM, Bartels D, Brown DCW, Schell J (1991) Messenger-RNA and protein changes associated with induction of Brassica microspore embryogenesis. Planta 184: 161-165
Pechan PM, Smýkal P (2001) Androgenesis: affecting the fate of the male gametophyte. Physiol. Plant. 111: 1-8
Pharis RP, Evans LT, King RW, Mander LN (1987) Gibberellins, endogenous and applied, in relation to flower induction in the long-day plant Lolium temulentum. Plant Physiol. 84: 1132-1138
Pulse-Australia (2012) Pulse Australia - In http:pulseaus.com.au, accessed in November 2012 Raghavan V (1986) Embryogenesis in Angiosperms: A Developmental and Experimental Study.
Developmental and Cell Biology series; 17. Cambridge University Press, Cambridge, United Kingdom
Rastogi R, Sawhney VK (1987) The role of plant growth regulators, sucrose and pH in the development of floral buds of tomato (Lycopersicon esculentum Mill.) culture in vitro. J. Plant Physiol. 128: 285-295
Rech EL, Ochatt SJ, Chand PK, Davey MR, Mulligan BJ, Power JB (1988) Electroporation increases DNA-synthesis in cultured plant-protoplasts. Biotech. 6: 1091-1093
Rech EL, Ochatt SJ, Chand PK, Power JB, Davey MR (1987) Electro-enhancement of division of plant protoplast-derived cells. Protoplasma 141: 169-176
Redden B, Leonforte T, Ford R, Croser J, Slattery J (2005) Pea (Pisum sativum L.). In RJ Singh, PP Jauhar, eds, Genetic Resources, Chromosome Engineering, and Crop Improvement, Vol 1. Boca Raton, FL : Taylor & Francis, pp 49–83.
Reid JB (1979) Flowering in Pisum: the effect of age on the gene Sn and the site of action of gene Hr. Ann. Bot. 44: 163-173
Reid JB, Murfet IC (1975) Flowering in Pisum: the sites and possible mechanisms of the vernalisation response. J. Exp. Bot. 26: 860-867
43
Reid JB, Murfet IC, Singer SR, Weller JL, Taylor SA (1996) Physiological-genetics of flowering in Pisum. Semin. Cell Dev. Biol. 7: 455-463
Reiss E, Schubert J, Scholze P, Kramer R, Sonntag K (2009) The barley thaumatin-like protein Hv-TLP8 enhances resistance of oilseed rape plants to Plasmodiophora brassicae. Plant Breed. 128
Reynolds TL (1997) Pollen embryogenesis. Plant Mol. Biol. 33: 1-10 Roux N, Toloza A, Radecki Z, Zapata-Arias FJ, Doležel J (2003) Rapid detection of aneuploidy
in Musa using flow cytometry. Plant Cell Reports 21: 483-490 Runkle ES, Heins RD (2001) Specific functions of red, far red, and blue light in flowering and
stem extension of long-day plants. J. Amer. Soc. Hort. Sci. 126: 275-282 Sangwan RS, Sangwan-Norreel BS (1996) Cytological and biochemical aspects of in vitro
androgenesis in higher plants. In SM Jain, SK Sopory, RE Veilleux, eds, In Vitro Haploid Production in Higher Plants, Vol 1. Kluwer Acad. Publ., Dordrecht, The Netherlands, pp 95-109
Sankhla D, Davis TD, Sankhla N, Upadhyaya A (1994) In vitro production of flowering shoots in 'German Red' carnation: effect of uniconazole and gibberellic acid. Plant Cell Rep. 13: 514-518
Sawhney VK, Rastogi R (1990) In vitro flower development of the normal and a male sterile, stamenless-2 mutant of tomato (Lycopersicon esculentum Mill.). Acta Hort. 280: 562-568
Saxena SN, Kaushik N, Sharma R (2008) Effect of abscisic acid and proline on in vitro flowering in Vigna aconitifolia. Biol. Plant. 52: 181-183
Schulze D, Pauls KP (2002) Flow cytometric analysis of cellulose tracks development of embryogenic Brassica cells in microspore cultures. New Pythol. 154: 249-254
Schulze D, Pauls P (1999) Flow cytometric characterization of embryogenic and gametophytic development in Brassica napus microspore cultures. Plant Cell Physio. 39: 226-234
Seguí-Simarro JM, Nuez F (2008) How microspores transform into haploid embryos: changes associated with embryogenesis induction and microspore-derived embryogenesis. Physiol. Plant. 134: 1-12
Seguí-Simarro JM, Testillano PS, Risueno MC (2003) Hsp70 and Hsp90 change their expression and subcellular localization after microspore embryogenesis induction in Brassica napus L. J. Struct. Biol. 142: 379-391
Serres R, McCown B (1994) Rapid flowering of microcultured cranberry plants. HortScience 29: 159-161
Shariatpanahi ME, Bal U, Heberle-Bors E, Touraev A (2006) Stresses applied for the reprogramming of plant microspores towards in vitro embryogenesis. Physiol Plant. 127: 519-534
Shim YS, Kasha KJ (2003) Barley microspore transformation protocol by biolistic gun. In M Maluszynski, K Kasha, BP Forster, I Szarejko, eds, Doubled Haploid Production in Crop Plants. Kluwer Acad. Publ., Dordrecht, The Netherlands, pp 363-366
Sidhu P, Davies P (2005) Pea anther culture: callus initiation and production of haploid plants. In IJ Bennett, E Bunn, H Clarke, JA McComb, eds, Proceedings of the Australian Branch of the IAPTC&B - Contributing to a sustainable future, Perth, Australia, pp 180-186
Simmonds DH, Keller WA (1999) Significance of preprophase bands of microtubules in the induction of microspore embryogenesis of Brassica napus. Planta 208: 383-391
Smith H (1994) Phytochrome-mediated responses: implications for controlled environment research facilities. In TW Tibbits, ed, International Lighting in Controlled Environments Workshop, NASA-CP-95-3309
Smýkal P, Aubert G, Burstin J, Coyne CJ, Ellis NTH, Flavell AJ, Ford R, Hýbl M, Macas J, Neumann P, McPhee KE, Redden RJ, Rubiales D, Weller JL, Warkentin TD (2012) Pea (Pisum sativum L.) in the genomic era. Agronomy 2: 74-115
44
Smýkal P, Kenicer G, Flavell AJ, Corander J, Kosterin O, Redden RJ, Ford R, Coyne CJ, Maxted N, Ambrose MJ (2011) Phylogeny, phylogeography and genetic diversity of the Pisum genus. Plant Genet. Resour. 9: 4-18
Snape JW (1976) A theoretical comparison of diploidised haploid and single seed descent populations. Heredity 36: 275-277
Sopory SK, Munshi M (1996) Anther culture. In SM Jain, SK Sopory, RE Veilleux, eds, In Vitro Haploid Production in Higher Plants, Vol 1. Kluwer Acad. Publ., Dordrecht, The Netherlands, pp 145-176
Spalding EP, Folta KM (2005) Illumination topics in plant photobiology. Plant Cell Environ. 28: 39-53
Sunderland N, Roberts M (1979) Cold pre-treatment of excised flower buds in float culture of tobacco anthers. Ann. Bot. 43: 405-414
Sunderland N, Xu ZH (1982) Shed pollen culture in Hordeum vulgare. J. Exp. Bot. 136: 1086-1095
Szarejko I (2003) Doubled haploid mutant production. In M Maluszynski, K Kasha, BP Forster, I Szarejko, eds, Doubled Haploid Production in Crop Plants. Kluwer Acad. Publ., Dordrecht, The Netherlands, pp 351-361
Tanaka M (1973) The effect of centrifugal treatment on the emergence of plantlets from cultured anther of tobacco. Japan. J. Breed. 23: 171-174
Taylor NJ, Light ME, Van Staden J (2005) In vitro flowering of Kniphofia leucocephala: influence of cytokinins Plant Cell Tiss. Organ Cult. 83: 327-333
Taylor SA, Murfet IC (1996) Flowering in Pisum: Identification of a new ppd allele and its physiological action as revealed by grafting. Physiol. Plant. 97: 719-723
Thomas WTB, Forster BP, Gertsson B (2003) Doubled haploids in breeding. In M Maluszynski, K Kasha, BP Forster, I Szarejko, eds, Doubled Haploid Production in Crop Plants. Kluwer Acad. Publ., Dordrecht, The Netherlands, pp 337-449
Tisserat B, Galletta PD (1988) In vitro flowering in Amaranthus. Hort. Sci. 23: 210-212 Touraev A, Ilham A, Vicente O, Heberle-Bors E (1996) Stress-induced microspore
embryogenesis in tobacco: an optimized system for molecular studies. Plant Cell Rep. 15: 561-565
Touraev A, Pfosser M, Heberle-Bors E (2001) The microspore: A haploid multipurpose cell. In Advances in Botanical Research, Vol 35. Acad. Press, pp 53-109
Touraev A, Vicente O, Heberle-Bors E (1997) Initiation of microspore embryogenesis by stress. Trends Plant Sci. 2: 297-302
Tsujikawa T, Ichii T, Nakanishi T, Ozaki T, Kawai Y (1990) In vitro flowering of Japanese pear and the effect of GA4+ 7. Sci. Hort. 41: 233-245
Tzitzikas EN, Bergervoet M, Raemakers K, Vincken JP, van Lammeren A, Visser RGF (2004) Regeneration of pea (Pisum sativum L.) by a cyclic organogenic system. Plant Cell Rep. 23: 453-460
Valkonen JPT, Moritz T, Watanabe KN, Rokka VM (1999) Dwarf (di)haploid pito mutants obtained from a tetraploid potato cultivar (Solanum tuberosum subsp. tuberosum) via anther culture are defective in gibberellin biosynthesis. Plant Sci. 149: 51-57
Van Berger S, Kottenhagen MJ, Van Der Meulen RM, Wang M (1999) The role of abscisic acid in induction of androgenesis: a comparative study between Hordeum vulgare L. cvs. Igri and Digger J. Plant Growth Regul. 18: 135-143
van Duren M, Morpurgo R, Doležel J, Afza R (1996) Induction and verification of autotetraploids and diploid banana (Musa acuminata) by in vitro techniques. Euphytica 88: 25-35
Verdier J, Thompson RD (2008) Transcriptional regulation of storage protein synthesis during dicotyledon seed filling. Plant Cell Physiol. 49: 1263-1271
Wang M, Van Berger S, Van Duijn B (2000) Insights into a key developmental switch and its importance for efficient plant breeding. Plant Physiol. 124: 523-530
45
Wędzony M, Forster BP, Żur I, Golemiec E, Szechyńska-Hebda M, Dubas E, Gotębiowska G (2009) Progress in Doubled Haploid Technology in Higher Plants. In A Touraev, BP Forster, SM Jain, eds, Advances in Haploid Production in Higher Plants. Springer Netherlands, pp 1-33
Weller JL (2004) Phytochrome and chrytochrome functions in crop plants. In R Goodman, ed, Encyclopedia of Plant and Crop Science. Marcel Dekker Inc., New York, pp 920-923
Weller JL, Hecht V, Chee Liew L, Sussmilch C, Wenden B, Knowles CL, Vander Schoor JK (2009) Update on the genetic control of flowering in garden pea. J. Exp. Bot. 60: 2493-2499
Weller JL, Reid JB, Taylor SA, Murfet IC (1997) The genetic control of flowering in pea. Trends Plant Sci. 2: 412-428
Winzeler H, Schmid J, Fried PM (1987) Field performance of androgenetic doubled haploid spring wheat lines in comparison with lines selected by the pedigree system. Plant Breed. 99: 41-48
Wroth JM (1998) Possible role for wild genotypes of Pisum spp. to enhance ascochyta blight resistance in pea. Aust. J. Exp. Agric. 38: 469-479
Yanpaisan W, King NJC, Doran PM (1999) Flow cytometry of plant cells with applications in large-scale bioprocessing. Biotech. Adv. 17: 3-27
Zarsky V, Rihova L, Tupy J (1990) Biochemical and cytological changes in young tobacco pollen during in vitro starvation in relation to pollen embryogenesis. In HJJ Nijkamp, LHW Van der Plas, J Van Aartrijk, eds, Progress in plant cellular and molecular biology, Kluwer, Dordrecht, Boston, London, pp 228-233
Zhang T (2007) In vitro flowering of Perilla frutescens. In Vitro Cell. Dev. Biol.: Plant 43: 91-94 Zhong H, Srinivasan C, Sticklen MB (1992) In vitro morphogenesis of corn (Zea mays L.).
Planta 187: 490-497 Zimmermann U, Pilwat G, Beckers F, Riemann F (1976) Effects of external dielectric fields on
cell membranes. Bioelectrochem. Bioenerg 3: 53-83 Zohary D, Hopf M (1973) Domestication of Pulses in the Old World. Science 182: 887-894 Zonneveld BJM, van Iren F (2001) Genome size and pollen viability as taxonomic criteria:
Application to the genus Hosta. Plant Biol. 3: 176-185 Zivy M, Devaux P, Blaisonneau J, Jean R, Thiellement H (1992) Segregation distortion and
linkage studies in microspore-derived doubled haploid lines of Hordeum vulgare L. Theor. Appl. Genet. 83: 919-924
Żur I, Dubas E, Golemiec E, Szechyńska-Hebda M, Janowiak F, Wędzony M (2008) Stress-induced changes important for effective androgenic induction in isolated microspore culture of triticale (× Triticosecale Wittm.). Plant Cell Tiss. Organ Cult. 94: 319-328
47
Chapter 2 1
The application of multiple stress treatments and the 2
optimisation of culture conditions lead to routine induction of 3
androgenesis from intact anthers of pea (Pisum sativum L.) 4
2.1 Introduction 5
Doubled haploid (DH) technology is a well-established technique for accelerating 6
the genetic progress of key cereal, oilseed and horticultural crop species (Jain et al., 7
1996-1997; Wędzony et al., 2009; Dunwell, 2010; Germanà, 2011). The production of 8
DHs through gametic embryogenesis allows the single-step development of 9
completely homozygous lines from heterozygous parents, shortening the time 10
required to produce homozygous plants in comparison to the conventional breeding 11
methods that require generations of selfing (Germanà, 2011). Doubled haploidy is 12
thus the fastest known route to homozygosity (Chase, 1952; Maluszynski et al., 2003). 13
However, to date, no DH protocol is routinely used in any crop, pasture or tree legume 14
breeding program, and a fundamental understanding of the mechanisms involved in 15
the developmental transition of microspores from the gametophytic to the 16
sporophytic pathway remains elusive (Croser et al., 2006; Ochatt et al., 2009; 17
Germanà, 2011; Lülsdorf et al., 2011). It is therefore the aim of this research to study 18
the effects of key factors known to be involved in androgenesis elicitation in order to 19
develop a robust anther culture protocol across a range of pea (Pisum sativum L.) 20
genotypes. 21
Haploid embryogenesis requires the reprogramming of microspores, diverting 22
them from their original pathway, toward embryogenesis (Jain et al., 1996-1997). It is 23
clear from the literature that this switch in development is achieved by the application 24
48
of stress treatments and the manipulation of in vitro culture conditions (Touraev et al., 1
1997; Shariatpanahi et al., 2006; Wędzony et al., 2009; Dunwell, 2010; Ferrie and 2
Caswell, 2011; Germanà, 2011). Genotype, microspore developmental stage, and 3
culture conditions are the most important factors affecting microspore developmental 4
fate (Jain et al., 1996-1997). Genotype plays a major role in androgenesis, with 5
important differences in response observed between species but also within cultivars 6
of the same species (Jain et al., 1996-1997; Maluszynski et al., 2003; Seguí-Simarro 7
and Nuez, 2008a; Ochatt et al., 2009; Germanà, 2011). The pollen developmental 8
stage is a complex factor that has a large bearing on the success of androgenesis 9
(Germanà, 2011). Optimal responses are normally obtained from the early uni-10
nucleate to the early bi-nucleate stage (Sangwan and Sangwan-Norreel, 1996). 11
Microphenological traits such as bud length and anther size can be used as indicators 12
of the microspore developmental stage. For pea, uni-nucleate microspores appear to 13
be the most appropriate stage for initiation of haploid cultures (Croser et al., 2006; 14
Ochatt et al., 2009; Lülsdorf et al., 2011). The uni-nucleate stage corresponds to a 15
flower bud length of 6 to 7 mm and an anther size of 1 mm. 16
There is no clear consensus in the literature on the specific culture conditions 17
required for doubled haploidy of grain legumes (Croser et al., 2006; Lülsdorf et al., 18
2011). However, key factors that require optimisation in the development of every 19
protocol have been identified. Some of these factors include light conditions (i.e. light 20
intensity, light quality and photoperiod), culture temperature, microspore culture 21
density, medium composition for embryo induction, maturation and regeneration (i.e. 22
macro and micro salt composition, plant growth regulators, osmolarity, pH, 23
carbohydrate source) (Jain et al., 1996-1997; Croser et al., 2006; Ochatt et al., 2009; 24
49
Wędzony et al., 2009; Bobkov, 2010; Dunwell, 2010; Germanà, 2011; Lülsdorf et al., 1
2011). 2
The first report of haploid callus induction in pea was by Gupta et al. in 1972, 3
with the later recovery of a few plants (Gupta, 1975), however, the haploid condition 4
of these plants was not confirmed and the results were not reproduced. More 5
recently, a cold pre-treatment of pea buds successfully induced symmetrical divisions, 6
callus formation and early-stage embryo development from extracted anthers (Gosal 7
and Bajaj, 1988; Sidhu and Davies, 2005; Bobkov, 2010) and isolated microspores 8
(Croser and Lülsdorf, 2004; Croser et al., 2005). In 2009, Ochatt et al. were the first to 9
recover a small number of flow cytometry-confirmed haploid plants from isolated 10
microspores of pea cultivars Frisson, CDC April and Victor. However, these plants were 11
weak and did not survive greenhouse transfer. The combined application of multiple 12
stress treatments viz. cold, electroporation and osmotic shock, was critical to the 13
successful elicitation of haploid divisions and subsequent haploid embryogenesis. 14
Also, the initial microspore culture density had a significant effect on microspore 15
division rate. A density of 2 x 105 microspores/ ml provided the most consistent 16
responses. On the other hand, other culture factors such as temperature or light 17
conditions, as well as medium basal salts and hormonal composition had little 18
influence on androgenetic responses in pea. It is clear the DH protocols published to 19
date are not robust enough to be used in a pea breeding program and have been 20
applicable to a very limited number of cultivars. 21
A wide variety of stress treatments have been successfully applied across 22
species for the reprogramming of microspores towards haploid embryogenesis 23
(Shariatpanahi et al., 2006). A review of the literature has shown cold pre-treatment 24
50
of buds, electroporation of anthers or isolated microspores, stepwise osmotic 1
modification of the induction medium and centrifugation of microspores appear to be 2
the most effective for legumes. The positive effect on androgenesis of a bud/ anther 3
cold pre-treatment has been reported for the legumes pigeonpea (Cajanus cajan L.) 4
(Kaur and Bhalla, 1998), chickpea (Cicer arietinum L.) (Grewal et al., 2009) and pea 5
(Gosal and Bajaj, 1988; Croser et al., 2005; Ochatt et al., 2009). Electroporation of 6
isolated microspores improved regeneration competence in Asparagus officinalis L., 7
canola (Brassica napus L.), tobacco (Nicotiana tabacum L.), chickpea, pea, Lathyrus 8
spp. and Medicago truncatula Gaernt. (Mishra et al., 1987; Jardinaud et al., 1993; 9
Delaitre et al., 2001; Grewal et al., 2009; Ochatt et al., 2009). Modification of osmotic 10
pressure was essential for the continued development of induced microspores of 11
chickpea, pea, Lathyrus spp. and M. truncatula (Croser et al., 2006; Grewal et al., 12
2009; Ochatt et al., 2009). In pea, an osmotic stress treatment has been shown to 13
improve callusing of microspores by favouring a slight detachment of membranes 14
from the microspore wall and thus facilitating initial divisions (Ochatt et al., 2009). A 15
centrifugal shock has been used for the successful induction of androgenesis in 16
chickpea (Grewal et al., 2009). Centrifugation of chickpea anthers at 168 g for 10 min 17
resulted in early embryo formation and a greater proportion of embryos per anther 18
compared to untreated anthers. More recently, the addition of n-butanol in the 19
induction medium has been reported to stimulate androgenesis in wheat (Triticum 20
aestivum L.) (Soriano et al., 2008; Broughton, 2011). It has been proposed that the 21
increase in number of haploid embryos and plants may be due to the disruption of 22
cortical microtubules by n-butanol (Soriano et al., 2008). Unpublished results from our 23
laboratory at UWA indicated that n-butanol also effectively enhances the switch from 24
gametic to sporophytic development in chickpea (Croser pers. comm. 2010). I have 25
51
therefore selected these factors as the basis for my initial experiments to improve 1
robustness of the DH process for pea. 2
The aim of the research presented in this chapter was to use the existing body 3
of literature to develop a protocol for the regeneration of haploid plants from cultured 4
anthers of pea. I reason that the application of key stress factors and the optimisation 5
of the culture medium and culture conditions will lead to a robust in vitro protocol for 6
the routine induction of sporophytic development from extracted anthers in a range 7
of pea cultivars. 8
9
10
11
12
13
14
15
16
17
18
19
52
2.2 Materials and methods 1
2.2.1 Plant material 2
A range of genetically diverse pea (Pisum sativum L.) genotypes were selected 3
for this research, viz. the Australian cultivars Kaspa, Mukta, Dunwa, Helena, Excell and 4
Bundi; the French cv. Frisson; and the Canadian cv. CDC April (Table 2.1). 5
Table 2.1 Pea cultivars used for the anther culture protocol development 6
SL: semi-leafless type; C: conventional leaf type; T: tall type; SD: semi-dwarf type. 7 *The Dun type is variously dimpled with greenish-brown testa and yellow cotyledons (Khan and Croser, 2004). 8 **In the recessive mutants designated as afila (af), leaflets are modified into tendrils. Plants with afila 9 characteristics are popularly known as semi-leafless (Khan and Croser, 2004). 10 ***Le: internode length locus. The principal effect of le is to reduce the length of the upper internodes and cause 11 a zig-zag appearance of the stem (semi-dwarf type) (Reid et al., 1983). 12
2.2.2 Donor plant growth conditions 13
Seeds were sown in a glasshouse in 180 mm pots filled with potting mix (UWA 14
Plant Bio Mix – Richgro Garden Products Australia Pty. Ltd.) (Fig. 2.1). The 15
temperature was controlled at 20°C day/ 18°C night under natural light conditions (lat: 16
31°58’49” S; long: 115°49’7” E). Plants were watered daily and fertilised fortnightly 17
with a water soluble N-P-K (19-8.3-15.8) fertiliser with micronutrients (Poly-feed, 18
Greenhouse Grade, Haifa Chemicals Ltd.) at a rate of 3 g/ pot. 19
20 Figure 2.1 Glasshouse grown pea donor plants (cvs. Frisson and Kaspa). 21
Kaspa
Mukta
Dunwa
Helena
Excell
Bundi
Frisson
CDC April
Seed type*
Dun, round
White, square
Dun, dimpled
Dun, dimpled
Blue, round
White, round
White, round
Dun, dimpled
Leaf type**
SL (af)
SL (af)
C (Af)
C (Af)
SL (af)
SL (af)
C (Af)
SL (af)
Flowering/ Maturity
Late
Late
Mid/Late
Mid
Early/Mid
Early
Early
Late
Flower colour
Pink
White
Purple
Purple
White
White
White
Purple
Plant height***
SD (le)
SD (le)
T (Le)
T (Le)
SD (le)
SD (le)
SD (le)
SD (le)
53
2.2.3 Harvest of buds and determination of microspore developmental stage 1
To confirm the correlation of the bud size to the uni-nucleate stage of 2
microsporogenesis, flower buds at various sizes (4-4.9, 5-5.9, 6-6.9 mm of length) 3
were harvested daily for each of the genotypes studied and the developmental stage 4
of microspores immediately determined by DAPI (4’-6’-Diamidino-2-5
phenylindole.2HCl) stain. Buds of various sizes were opened and the anthers 6
contained within were placed in 1.5 ml centrifuge tubes containing 500 µl of Carnoy’s 7
fixative (3: 1 90% Ethanol: Glacial Acetic Acid) and stored at 4°C for 24 h for fixation. 8
After 24 h, the Carnoy’s fixative was carefully removed using a glass pipette. One 9
anther per treatment was then placed on a slide and 20 µl of a freshly prepared DAPI 10
(Sigma D-8417) working solution (25 µl of 1 mg/ ml DAPI stock solution + 1 ml of 0.3 M 11
mannitol) was added. Anthers were quickly dissected in this solution to release the 12
microspores, anther debris was removed and a coverslip applied. Samples were then 13
incubated in the dark for at least 30 min at room temperature and the development of 14
at least 100 microspores per treatment was examined with a Zeiss fluorescent 15
microscope (Carl Zeiss, Germany) fitted with DAPI wavelength appropriate filter set. 16
Nuclei were stained bright blue. Images were obtained using an Axiocam camera and 17
annotated using AxioVision Imaging System software (Carl Zeiss, Germany). 18
2.2.4 Effect of bud harvest number on microspore viability 19
As an indeterminate species, the pea plants could be harvested many times. An 20
experiment was established to determine the effect of multiple harvests on 21
microspore viability. Buds of cv. Frisson containing microspores at the uni-nucleate 22
stage were harvested sequentially six times and the microspore viability immediately 23
determined by fluorescein diacetate (FDA) staining (Heslop-Harrison and Heslop-24
54
Harrison, 1970). A minimum of 200 microspores per treatment were counted using a 1
Zeiss fluorescence microscope (Carl Zeiss, Germany). Viability was determined as 2
percentage of microspores fluorescing yellow-green under UV light. Images were 3
obtained using an Axiocam camera and annotated using AxioVision Imaging System 4
software (Carl Zeiss, Germany). 5
2.2.5 Bud sterilisation 6
Flower buds (approximately 100 buds) were placed in a 100 ml beaker and 7
surface sterilised by treatment for 1 min in 70% v/v ethanol, followed by 10 min 1% 8
v/v commercial sodium hypochlorite (White King bleach, Pental Products Pty Ltd, 9
Australia). Buds were rinsed three times with sterile water for 1 min. 10
2.2.6 Culture media 11
Different media composition and plant growth regulators were used in order to 12
induce haploid divisions. The media used in these experiments were HSO (Ochatt et 13
al., 2009), NLN (Lichter, 1982) and JFK (see Appendix 1). These media were all tested 14
with no hormones or with the addition of 2,4-Dichlorophenoxyacetic acid (2,4-D) 15
(1mg/L), picloram (0.25 mg/L) and 6-Benzylaminopurine (6-BAP) (0.1mg/L). All media 16
components were autoclaved and only plant growth regulators were filter sterilised. 17
2.2.7 Anther extraction procedure 18
Anthers were extracted using forceps and needles under sterile conditions using a 19
dissecting microscope and making sure any remnants of filament were removed. Ten 20
anthers per treatment were cultured into a 10 x 35 mm Petri dish containing 2 ml of 21
liquid medium. Dishes were sealed with Parafilm® and incubated for four weeks at 22
24°C under dark or light conditions (90 µmol m-2 s-1 and a 16/8 h light/ dark 23
photoperiod). 24
55
2.2.8 Stress treatments 1
A series of experiments were undertaken to study the effect of the individual 2
and combined application of stress treatment/s on the elicitation of androgenesis 3
from extracted anthers of a range of pea genotypes (Table 2.1). 4
2.2.8.1 Cold pre-treatment 5
Flower buds containing microspores at the uni-nucleate stage were placed in 6
sealed Petri dishes (approximately 30 buds per treatment) and stored under dry 7
conditions in the fridge at 4°C in the dark for 0, 7, 14 and 21 days before culture. 8
Microspore viability was determined after each cold pre-treatment, by fluorescein 9
diacetate (FDA) staining (Heslop-Harrison and Heslop-Harrison, 1970) as described in 10
Section 2.2.4. Cold pre-treated buds were then sterilised as described in Section 2.2.5. 11
Anthers were extracted and directly cultured (see Section 2.2.7) or cultured after 12
submission to the various combinations of stress treatments. 13
2.2.8.2 Electroporation 14
Thirty anthers were dispensed into a cuvette with electrodes 2 mm apart 15
containing 1300 µL of Rech et al. (1987) electroporation buffer. Three successive 16
exponential pulses of 0, 750, 1000 or 1500 V/cm with a capacitance of 0, 75, 100, 150 17
µF and a constant resistance of 50 were delivered at 10 s intervals using an Electro 18
Cell Manipulator ECM630 (BTX Gentronics Biomedical Ltd., San Diego, USA) (Fig. 2.2). 19
20 21 Figure 2.2 Electro cell manipulator used for the application of the electric shock 22
treatment. 23
24
56
2.2.8.3 Centrifugation 1
Thirty intact anthers per treatment were placed in 10 ml glass tubes containing 2
8 ml of electroporation buffer (Rech et al., 1987) and centrifuged at 170 g for 10 min, 3
at 4°C using a Jouan CR4-22 Benchtop Centrifuge (Jouan Inc, Winchester, VA, USA). 4
2.2.8.4 Sonication 5
Thirty intact anthers per treatment were sonicated in 10 ml glass tubes 6
containing 8 ml of electroporation buffer (Rech et al., 1987) at 38 KHz for 30 s, at 7
room temperature using a Branson 1210 Sonicator (Bransonic Ultrasonic Co, Danbury, 8
CT, USA). 9
2.2.8.5 Osmotic shock 10
Anthers were plated into 35 x 10 mm Petri dishes (10 anthers/ dish) with 2 ml of 11
NLN, HSO or JFK medium containing 17% (w/v) of sucrose. After 7 days of culture, 12
anthers were transferred to the same medium containing 10% (w/v) of sucrose 13
(Ochatt et al., 2009). 14
2.2.8.6 n-butanol treatment 15
Thirty anthers of cv. Frisson per n-butanol treatment were extracted and 16
cultured in Petri dishes (10 anthers per dish) containing 2 ml of hormone-free HSO 17
medium. Various concentrations (0, 0.1, 0.3 and 0.5%) of n-butanol (99.8% 1-butanol, 18
anhydrous, Sigma-AldrichTM Co.) were added to the medium for 5 h at D0, D2 and D5 19
of culture. During the treatments, dishes were covered with aluminium foil and left 20
unsealed in the laminar flow cabinet (Broughton,2011). After the treatment, the 21
culture medium with the n-butanol was removed by pipette and 2 ml of the same 22
fresh medium were added to each dish. 23
24
25
57
2.2.9 Embryo development 1
After four weeks of culture in liquid medium, at least 30 early embryoids per 2
treatment were transferred to two alternative media sequences in order to support 3
embryo development and germination and cultured as described in Section 2.2.7: 1- 4
Media sequence devised by Lehminger-Mertens and Jacobsen (1989) (LMJ media); or 5
2- Media sequence designed by Loiseau et al. (1995). 6
LMJ media sequence: The basal medium consists of MS salts (Murashige and Skoog, 7
1962) + B5 vitamins (Gamborg et al., 1968) plus 2% sucrose, 5% mannitol, 0.1% (w/v) 8
casein hydrolysate, 0.6% of agar and pH: 5.6. For embryo induction 2,4D (4 µM) is 9
added to the basal medium. Embryos are then transferred to basal medium with no 10
added hormones. Embryo maturation is promoted by culturing in a medium with GA3 11
(1.5 µM) and NAA (0.12 µM). Finally, embryo germination is obtained by adding GA3 12
(2.9 µM) to the basal medium. Embryo transfer from one medium to another was 13
done based on the visual assessment of the developing embryos, as per Ochatt et al., 14
2002. 15
Loiseau et al. media sequence: The composition of the basal medium is: MS salts 16
(Murashige and Skoog, 1962) + B5 vitamins (Gamborg et al., 1968) plus 0.1% (w/v) 17
casein hydrolysate, 0.7% agar, 9% sucrose and pH: 5.6. Calli are cultured for 4 weeks in 18
90 mm Petri plates containing 50 ml of the basal medium with 4.5 µM of 2,4-D. After 19
that period, calli/ embryos are transferred to the same medium with no hormones. 20
2.2.10 Tracking experimental results 21
Microspore embryo development was checked every 48 h from culture using an 22
Olympus CK2 inverted microscope (Olympus Optical Co. Ltd., Japan). In case of 23
observation of embryo development/ callusing, two anthers per treatment were 24
58
sampled and split open on a slide, and a few drops of 2% acetocarmine stain were 1
added. Samples were then observed under 40x magnification (light microscope) in 2
order to detect if microspores within anthers were developing. Also, DAPI-staining 3
was used to track microspore development within anthers during culture: two anthers 4
per treatment were collected at D0, D7 and D14 and DAPI-stained and analysed as 5
previously described in Section 2.2.3. 6
The experimental design was completely randomised and all treatments were 7
repeated at least three times with a minimum of 10 replicates per treatment per 8
genotype. Statistical analysis was performed by analysis of variance (Microsoft Office 9
Excel 2007 software). 10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
59
2.3 Results 1
2.3.1 Correlation between pea bud length and microspore developmental stage 2
A correlation between bud size and microspore developmental stage was 3
observed in most of the pea cultivars studied. For cultivars Frisson, Kaspa, Dunwa, 4
Helena, Excell, Bundi and Mukta, flower buds 6-7 mm long contained microspores 5
predominantly at the optimum, uni-nucleated stage (Fig. 2.3). However, in cv. CDC 6
April, 4-5 mm long flower buds contained mainly microspores at the uni-nucleate 7
stage. This was the optimal stage for induction of androgenesis divisions. 8
9
Figure 2.3 Determination of microspore developmental stage. Pea flower buds 10
containing anthers with microspores predominantly at the uni-nucleate stage (A and 11
B). DAPI-stained microspores at the optimum uni-nucleated stage for androgenesis 12
induction (C) and at a later, non inductive, stage (D), after first asymmetric mitosis. 13
14
2.3.2 Effect of bud harvesting procedure and cold pre-treatment duration on 15
microspore viability 16
The number of times each donor plant was harvested (up to the 6th harvest) had 17
no significant effect on microspore viability at the day of harvesting for cv. Frisson (Fig. 18
2.4). 19
60
1
Figure 2.4 Microspore viability estimated by fluorescein diacetate (FDA). Analysis of 2
the effect of harvest number on microspore viability at D0 in cv. Frisson (results are 3
expressed as mean ± SD). 4
The cold pre-treatment of buds for up to 7 days did not significantly affect the 5
microspore viability rate (Fig. 2.5). However, storing buds in the cold for more than 7 6
days significantly reduced the viability rate compared to the control treatment (0 days 7
in cold). A 14 day cold pre-treatment reduced microspore viability to less than 60% 8
while a 21 d pre-treatment further reduced it to less than 30%. 9
10
Figure 2.5 Microspore viability estimated by fluorescein diacetate (FDA). Analysis of 11
the effect of bud cold pre-treatment duration on microspore viability in cv. Frisson 12
(results are expressed as mean ± SD; P = 0.05). 13
14
0
10
20
30
40
50
60
70
80
90
100
1st 2nd 3rd 4th 5th 6th
Mic
rosp
ore
via
bili
ty (
%)
Harvest number
0
10
20
30
40
50
60
70
80
90
100
D0 D7 D14 D21
Mic
rosp
ore
via
bili
ty (
%)
Cold pre-treatment duration
ab
b
c
a
61
2.3.3 Effect of culture medium plant growth regulators on androgenesis induction 1
Continuous culture of anthers in JFK medium, containing 2,4-D (1 mg/l), 2
Picloram (0.25 mg/l) and BAP (0.1 mg/l) for 4 weeks led to the induction of 3
androgenesis with consistent callus production. A transversal cut showed callus 4
production from the microspores contained within the anthers rather than from the 5
anther wall tissue (Fig 2.6). No callusing was observed when culturing anthers in 6
hormone-free HSO and NLN media regardless of the stress treatment applied. 7
8 Figure 2.6 Transversal cut of cultured anther showing callus production from 9
microspores within the anther (cv. Frisson). 10
2.3.4 Effect of culture conditions on callusing frequency 11
The effect of culture light conditions (light vs. dark) and electroporation field 12
strength (0, 500 and 1000 V/cm) on callusing response from extracted anthers of cvs. 13
Frisson and CDC April was studied. In both genotypes, the highest percentage of 14
callusing was obtained by culturing anthers for the first four weeks under dark 15
conditions, disregarding the electroporation treatment applied. No androgenetic 16
response was obtained for cv. CDC April when cultured under light (90 µmol m-2 s-1) 17
(Fig. 2.7). 18
62
1
Figure 2.7 Effect of culture light conditions (light vs. dark) and electroporation field 2
strength (0, 500 and 1000 V/cm) on callusing percentage from extracted anthers of 3
pea cvs. CDC April and Frisson (results are expressed as mean ± SD; P = 0.05). 4
2.3.5 Effect of individual and combined stress treatments on elicitation of 5
androgenesis 6
A number of eliciting factors were studied for their effect on androgenetic 7
competence of pea. The application of multiple stress treatments induced 8
symmetrical divisions of the nuclei leading to the development of multinucleate 9
synctiums (Fig. 2.8A and 2.8B) and callus production (Fig. 2.8C). As a result of these 10
treatments, an enlargement of microspore/ anther size (hyperplasia) was observed 11
after androgenesis induction (Fig. 2.9). 12
0
25
50
75
100
0 V/cm 500 V/cm 1000 V/cm 0 V/cm 500 V/cm 1000V/cm
CDC April Frisson
% c
allu
sin
g
Treatments and genotypes
Light
Dark
b
ab
a
a ab
b b
a
a
63
1
Figure 2.8 DAPI-stained pea microspores showing symmetrical division of the nucleus 2
(A) and multinucleate synctium (B). Inverted microscope image showing pea 3
microspore derived callus production (C). 4 5 6 7
8
Figure 2.9 Initial androgenetic response observed in cultured anthers at D7 of culture 9
(cv. Frisson). Response obtained after a cold pre-treatment of buds combined with 10
centrifugation and electroporation of intact anthers. A) Unresponsive and responsive 11
(hyperplasia) anthers; B) Embryoid development from intact anther. 12
13 Electroporation of anthers containing microspores at the uni-nucleate stage at 14
electrical parameters optimised for each genotype (Fig. 2.7, Fig. 2.10 and Fig. 2.11) 15
promoted the best callusing responses for the genotypes studied. Centrifugation and/ 16
or sonication alone did not suffice to induce androgenesis. For cv. CDC April best 17
A
B
64
responses were obtained with anther electroporation at 1000 V/cm and 100 µF of 1
capacitance, while 500 or 1000 V/cm and 75 or 100 µF were best for cv. Frisson. 2
3
4
Figure 2.10 Effect of electroporation voltage on percentage of callusing from anthers 5
of pea cvs. Frisson and CDC April at optimum developmental stage (results are 6
expressed as mean ± SD; P = 0.05). 7
8
Figure 2.11 Effect of electroporation parameters (voltage and capacitance) on 9
callusing percentage from extracted anthers of pea cvs. Frisson and CDC April after 28 10
days of culture. 11
The combined application of multiple stress treatments, including a cold pre-12
treatment, electroporation, centrifugation and sonication of anthers prior to culture 13
and an osmotic shock during the first week of culture, promoted androgenetic 14
proliferation and subsequent differentiation from microspores within anthers of the 15
0
25
50
75
100
0 V/cm 500 V/cm-100 µF 1000 V/cm-100 µF
Cal
lusi
ng
(%)
Treatments
Frisson
CDC April
a
b b
a
ab b
0
25
50
75
100
0 µF 75 µF 100 µF 150 µF 0 µF 75 µF 100 µF 150 µF
CDC April Frisson
Cal
lusi
ng
(%)
Genotypes and treatments
0 V/cm
500 V/cm
1000 V/cm
65
pea genotypes studied (Fig. 2.12). On the other hand, none of the n-butanol stress 1
treatments applied successfully induced haploid divisions from cultured anthers (data 2
not shown). 3
4
Figure 2.12 Effect of different combinations of stress treatments on percentage of 5
callusing after 28 days of culture from extracted anthers of cv. Kaspa (O: osmolarity; S: 6
sonication; C: centrifugation; E: electroporation). In all treatments buds were 7
previously submitted to a cold treatment for 7 d at 4°C. Results are expressed as mean 8
± SD; P=0.05. 9
2.3.6 Embryo maturation and germination 10
After four weeks of culture in JFK medium, the calli obtained (friable and yellow-11
green in colour) were transferred to the media sequence devised by Lehminger-Mertens 12
and Jacobsen (1989) or to the media sequence devised by Loiseau et al. (1995). No 13
further development was achieved when culturing using the LMJ media sequence. On 14
the other hand, the culture of calli in the Loiseau media sequence led to the production 15
of advanced stage anther-derived embryos (heart-shaped and cotyledonary stages) in 16
cvs. Frisson, Kaspa, Helena and Bundi (Fig 2.13). However, using this protocol, no haploid 17
plantlet germination was achieved for any of the pea cultivars studied. 18
66
1 Figure 2.13 Anther culture derived somatic embryos of pea cv. Frisson. Arrow colour 2
indicates different embryo developmental stage (white: pro-embryo; red: globular; 3
blue: heart; black: torpedo). 4
5
6
7
8
9
10
11
12
13
67
2.4 Discussion 1
In the research presented in this Chapter, a series of key factors in the 2
development of a robust pea anther culture protocol were identified. The combined 3
application of multiple stress treatments and the optimisation of key culture factors 4
led to the development of an efficient protocol for the routine induction of 5
androgenesis from extracted anthers of a range of pea genotypes. Advanced-stage 6
embryos were consistently achieved; however, no haploid plants were recovered with 7
the strategy proposed. Further research is required before a robust and reliable 8
method for doubled haploid plant production across a range of pea genotypes is 9
available. 10
Anther culture conditions (i.e. temperature and light conditions), microspore 11
developmental stage and the combined application of multiple stress treatments were 12
identified as critical factors for the induction of androgenic division in pea anther 13
culture. Induction of sporophytic development was achieved in uni-nucleate 14
microspores within anthers of pea via the application of a cold (4°C) pre-treatment to 15
buds for 2 to 14 days combined with electroporation, centrifugation and sonication 16
treatments before culture and an osmotic shock during the first 7 d of culture. These 17
results are consistent with those published by Ochatt et al. (2009) for isolated 18
microspore culture of pea. In addition, sonication is reported as a novel abiotic stress 19
factor for the elicitation of androgenesis in pea. On the other hand, the n-butanol 20
stress treatment failed to induce androgenic divisions from cultured anthers. 21
Microscope analysis during culture showed symmetrical division of the nuclei was 22
followed by the development of multinucleate synctiums, callus formation and 23
somatic embryo development. With the proposed protocol, torpedo-shaped embryos 24
68
were routinely achieved from extracted anthers of pea but no haploid plantlets were 1
recovered. 2
Little is known about the exact mechanisms underlying the elicitation of 3
androgenesis using stress treatments. However, attempts have been made to explain 4
the possible effects of some of these stress factors. It has been proposed that a cold 5
pre-treatment slows-down the degradation process in the anther tissue thus 6
protecting microspores from toxic compounds released from the decaying anthers 7
(Duncan and Heberle-Bors, 1976) and favours the synchronisation of the microspore 8
developmental stage (Hu and Kasha, 1999). Also, a cold pre-treatment appears to 9
increase the frequency of endo-reduplication during androgenesis induction (Amssa et 10
al., 1980), a phenomenon that has long been suggested as a mechanism to explain the 11
occurrence of higher DNA content in induced microspores (Seguí-Simarro and Nuez, 12
2008b). In fact, Raquin et al. (1982) observed in wheat that a high proportion of uni-13
nucleate microspores at G2-phase (2C DNA content) increased their DNA content to 14
4C after cold induction while maintaining their uni-nucleate status. Electric pulses 15
have been shown to modify the structure of the plasma membrane, presumably by 16
the temporary formation of pore-like openings which facilitate the uptake of media 17
components into the cells (Neumann and Rosenheck, 1972; Zimmermann et al., 1976; 18
Kinosita and Tsong, 1977; Ochatt, 2013). Electroporation has also been shown to 19
stimulate DNA synthesis in cultured plant protoplasts (Rech et al., 1988). An 20
accumulation of ABA has been observed in osmotic stressed anthers of tobacco 21
(Imamura and Harada, 1980) and barley (Hordeum vulgare L.) (Van Berger et al., 22
1999), which may be involved in the reprogramming of microspore development 23
(Wang et al., 2000; Żur et al., 2008). 24
69
Changes in endogenous hormone levels following stress treatments has been 1
observed in a number of species during androgenesis elicitation (Wang et al., 2000; 2
Seguí-Simarro and Nuez, 2008a; Żur et al., 2008). In pea, Lülsdorf et al. (2012) 3
observed important hormonal changes during androgenesis induction from stress-4
treated anthers. They primarily detected an increase in active auxins (mainly IAA-asp) 5
after the application of individual and combined stress treatments. In the research 6
presented in this chapter, the addition of plant growth regulators to the culture 7
medium promoted callus proliferation from microspores of pea within anthers. 8
Transversal cut of cultured anthers showed that callus was originating from dividing 9
microspores within the anthers rather than from the diploid maternal tissue. 10
However, there is a possibility that some of the callus tissue obtained may have 11
originated from the anther wall tissue. From these results it is not possible to infer if 12
exogenous hormones are involved in the elicitation androgenesis from cultured 13
anthers of pea or if they have an effect in promoting callusing after stress treatment 14
induction. For that reason, it will be of great value to identify a strategy to assess the 15
effect of individual and combined stress treatments on the elicitation of androgenesis. 16
The development of a robust pea DH protocol will facilitate the rapid 17
establishment of useful traits and the released of improved cultivars. This will permit a 18
rapid response to changing conditions. However, despite the significant effort to date, 19
there is no universal DH protocol effective in every species and success remains highly 20
genotype-dependent (Ochatt et al., 2009; Wędzony et al., 2009). Legume species in 21
particular, are still regarded as recalcitrant to this technology (Croser et al., 2006; 22
Lülsdorf et al., 2011). The main difficulty in improving existing DH protocols lies in the 23
great variety of factors that influence and control androgenesis (Wędzony et al., 24
70
2009). Much of the research work in DH protocol development is limited to the 1
identification of responsive genotypes, the optimisation of culture conditions and the 2
application of a variety of stress treatments, while the understanding of the 3
fundamental mechanisms involved in haploid induction and the decisive role of stress 4
treatments remains elusive. 5
From the results presented in this chapter I could identify and optimise some of 6
the key factors involved in haploid embryogenesis from cultured anthers of pea. 7
Advanced-stage embryos were consistently achieved but no haploid plants were 8
recovered with the proposed strategy. The application of multiple stress treatments 9
was clearly crucial for the induction of haploid divisions from extracted anthers of pea. 10
The focus of the following chapter will be a flow cytometry study of the effect of these 11
stress treatments on the elicitation of androgenesis from intact anthers and isolated 12
microspores of pea. 13
14
15
16
17
18
19
20
21
71
2.5 References 1
Amssa M, De Buyser J, Henry Y (1980) Origin of diploid plants obtained by in vitro culture of 2 anthers of young wheat (Triticum aestivum L.). Cr. Acad. Sci. D. Nat. 290: 1095-1097 3
Bobkov SV (2010) Isolated pea anther culture. Russian Agric. Sci. 36: 413-416 4 Braybrook SA, Harada JJ (2008) LECs go crazy in embryo development. Trends Plant Sci. 13: 5
624-630 6 Broughton S (2011) The application of n-butanol improves embryo and green plant 7
production in anther culture of Australian wheat (Triticum aestivum L.) genotypes. 8 Crop Past. Sci. 62: 813-822 9
Chase SS (1952) Production of homozygous diploids of maize from monoploids. Agron. J. 44: 10 263-267 11
Croser J, Lülsdorf M, Davies P, Wilson J, Sidhu P, Grewal R, Allen K, Dament T, Warkentin T, 12 Vandenberg A, Siddique K (2005) Haploid Embryogenesis from Chickpea and Field Pea 13 - Progress Towards a Routine Protocol. In IJ Bennett, E Bunn, H Clarke, JA McComb, 14 eds, Proceedings of the Australian Branch of the IAPTC&B - ‘Contributing to a 15 Sustainable Future’ Perth, Australia, pp 71-82 16
Croser JS, Lülsdorf MM (2004) Progress towards haploid division in chickpea (Cicer arietinum 17 L.), field pea (Pisum sativum L.) and lentil (Lens culinaris Medik.) using isolated 18 microspore culture. In European Grain Legume Conference, AEP, Dijon, France, p 189 19
Croser JS, Lülsdorf MM, Davies PA, Clarke HJ, Bayliss KL, Mallikarjuna N, Siddique KHM 20 (2006) Toward doubled haploid production in the Fabaceae: Progress, constraints, 21 and opportunities. Crit. Rev. Plant Sci. 25: 139-157 22
Delaitre C, Ochatt S, Deleury E (2001) Electroporation modulates the embryogenic responses 23 of asparagus (Asparagus officinalis L.) microspores. Protoplasma 216: 39-46 24
Duncan EJ, Heberle-Bors E (1976) Effect of temperature shock on nuclear phenomena in 25 microspores of Nicotiana tabacum and consequently on plantlet production. 26 Protoplasma 90: 173-177 27
Dunwell JM (2010) Haploids in flowering plants: origin and exploitation. Plant Biotechnol. J. 8: 28 377-424 29
Ferrie AMR, Caswell KL (2011) Isolated microspore culture techniques and recent progress for 30 haploid and doubled haploid plant production. Plant Cell Tiss. Organ Cult. 104: 301-31 309 32
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of 33 soybean root cells. Exp. Cell Res. 50: 151-158 34
Germanà MA (2011) Anther culture for haploid and doubled haploid production. Plant Cell 35 Tiss. Organ Cult. 104: 283-300 36
Gosal SS, Bajaj YPS (1988) Pollen embryogenesis and chromosomal variation in anther of 37 three food legumes - Cicer arietinum, Pisum sativum and Vigna mungo. SABRAO J. 38 Breed. Genet. 20: 51-58 39
Grewal RK, Lülsdorf M, Croser J, Ochatt S, Vandenberg A, Warkentin TD (2009) Doubled-40 haploid production in chickpea (Cicer arietinum L.): role of stress treatments. Plant 41 Cell Rep. 28: 1289-1299 42
Gupta S (1975) Morphogenetic response of haploid callus tissue of Pisum sativum (Var. B22). 43 Indian Agric. 19: 11-21 44
Gupta S, Ghosal KK, Gadgil VN (1972) Haploid tissue culture of Triticum aestivum var. Sonalika 45 and Pisum sativum var. B22. Indian Agric. 16: 277-278 46
Heslop-Harrison J, Heslop-Harrison Y (1970) Evaluation of pollen viability by enzymatically 47 induced fluorescence; intracellular hydrolysis of fluorescein diacetate. Biotech. 48 Histochem. 45: 115-120 49
Hu TC, Kasha KJ (1999) A cytological study of pretreatments used to improve isolated 50 microspore cultures of wheat (Triticum aestivum L.) cv. Chris. Genome 42: 432-441 51
72
Imamura J, Harada H (1980) Effects of abscisic acid and water stress on the embryo and 1 plantlet production in anther culture of Nicotiana tabacum cv. Samsun. Z. Pflanzen-2 physiol. 100: 285-289 3
Jain SM, Sopory SK, Veilleux RE (1996-1997) In vitro haploid production in higher plants, Vol 4 1-5. Kluwer Acad. Publ., Dordrecht, The Netherlands 5
Jardinaud MF, Souvre A, Alibert G (1993) Transient GUS gene expression in Brassica napus 6 electroporated microspores. Plant Sci. 93: 177-184 7
Kaur P, Bhalla JK (1998) Regeneration of haploid plants from microspore culture of pigeonpea 8 (Cajanus cajan L.). Indian J. Exp. Biol. 36: 736-738 9
Khan TN, Croser JS (2004) Pea: Overview. In C Wrigley, H Corke, C Walker, eds, Encyclopedia 10 of Grain Science. Academic Publishers, pp 287-295 11
Kinosita K, Tsong TY (1977) Voltage-induced pore formation and hemolysis of human 12 erythrocytes. Biochim. Biophys. Acta 471: 227-242 13
Lehminger-Mertens R, Jacobsen HJ (1989) Plant regeneration from pea protoplasts via 14 somatic embryogenesis. Plant Cell Rep. 8: 379-382 15
Lichter R (1982) Induction of haploid plants from isolated pollen of Brassica napus. Zeitschrift 16 für Pflanzenphysiologie 105: 427-434 17
Loiseau J, Marche C, Deunff Y (1995) Effects of auxins, cytokinins, carbohydrates and amino 18 acids on somatic embryogenesis induction from shoot apices of pea. Plant Cell Tiss. 19 Organ Cult. 41: 267-275 20
Lülsdorf M, Yuan HY, Slater S, Vandenberg A, Han X, Zaharia LI (2012) Androgenesis-inducing 21 treatments change phytohormone levels in anthers of three legume species 22 (Fabaceae). Plant Cell Rep. 31: 1255-1267 23
Lülsdorf MM, Croser JS, Ochatt S (2011) Androgenesis and doubled-haploid production in 24 food legumes. In A Pratap, J Kumar, eds, Biology and Breeding of Food Legumes. CABI 25 Publishing, pp 159-177 26
Maluszynski M, Kasha KJ, Szarejko I (2003) Published doubled haploid protocols in plant 27 species. In M Maluszynski, KJ Kasha, BP Forster, I Szarejko, eds, Doubled Haploid 28 Production in Crop Plants. A Manual. Kluwer Acad. Publ., Dordrecht, The Netherlands, 29 pp 309-335 30
Mishra KR, Joshua DC, Bhatia CR (1987) In vitro electroporation of tobacco pollen. Plant Sci. 31 52: 135-139 32
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco 33 tissue cultures. Physiol. Plant. 15: 473-497 34
Neumann E, Rosenheck K (1972) Permeability changes induced by electric impulses in 35 vesicular membranes. J. Membrane Biol. 10: 279-290 36
Ochatt S (2013) Plant cell electrophysiology: Applications in growth enhancement, somatic 37 hybridisation and gene transfer. Biotechnol. Adv.: Published online - 38 http://dx.doi.org/10.1016/j.biotechadv.2013.1003.1008 39
Ochatt S, Pech C, Grewal R, Conreux C, Lülsdorf M, Jacas L (2009) Abiotic stress enhances 40 androgenesis from isolated microspores of some legume species (Fabaceae). J. Plant 41 Physiol. 166: 1314-1328 42
Raquin C, Amssa M, Henry Y, de Buyser J, Essad S (1982) Origin des plantes polyploides 43 obtenues par culture d'antheres. Analyse cytophotometrique in situ et in vitro des 44 microspores de Petunia et ble tendre. Z. Pflanzenzucht 89: 265-277 45
Rech EL, Ochatt SJ, Chand PK, Davey MR, Mulligan BJ, Power JB (1988) Electroporation 46 increases DNA-synthesis in cultured plant-protoplasts. Biotech. 6: 1091-1093 47
Rech EL, Ochatt SJ, Chand PK, Power JB, Davey MR (1987) Electro-enhancement of division of 48 plant protoplast-derived cells. Protoplasma 141: 169-176 49
Reid JB, Murfet IC, Potts WC (1983) Internode length in Pisum. Additional information on the 50 relationship and action of loci Le, La, Cry, Na and Lm. J. Exp. Bot. 34: 349-364 51
73
Rose R, Nolan K (2006) Invited review: Genetic regulation of somatic embryogenesis with 1 particular reference to Arabidopsis thaliana and Medicago truncatula. In Vitro Cell 2 Dev. Biol. : Plant 42: 473-481 3
Sangwan RS, Sangwan-Norreel BS (1996) Cytological and biochemical aspects of in vitro 4 androgenesis in higher plants. In SM Jain, SK Sopory, RE Veilleux, eds, In Vitro Haploid 5 Production in Higher Plants, Vol 1. Kluwer Acad. Publ., Dordrecht, The Netherlands, pp 6 95-109 7
Seguí-Simarro JM, Nuez F (2008a) How microspores transform into haploid embryos: changes 8 associated with embryogenesis induction and microspore-derived embryogenesis. 9 Physiol. Plant. 134: 1-12 10
Seguí-Simarro JM, Nuez F (2008b) Pathways to doubled haploidy: chromosome doubling 11 during androgenesis. Cytogenet. Genome Res. 120: 358-369 12
Shariatpanahi ME, Bal U, Heberle-Bors E, Touraev A (2006) Stresses applied for the 13 reprogramming of plant microspores towards in vitro embryogenesis. Physiol Plant. 14 127: 519-534 15
Sidhu P, Davies P (2005) Pea anther culture: callus initiation and production of haploid plants. 16 In IJ Bennett, E Bunn, H Clarke, JA McComb, eds, Proceedings of the Australian Branch 17 of the IAPTC&B - Contributing to a sustainable future, Perth, Australia, pp 180-186 18
Soriano M, Cistué L, Castillo AM (2008) Enhanced induction of microspore embryogenesis 19 after n-butanol treatment in wheat (Triticum aestivum L.) anther culture. Plant Cell 20 Rep. 27: 805-811 21
Touraev A, Vicente O, Heberle-Bors E (1997) Initiation of microspore embryogenesis by 22 stress. Trends Plant Sci. 2: 297-302 23
Van Berger S, Kottenhagen MJ, Van Der Meulen RM, Wang M (1999) The role of abscisic acid 24 in induction of androgenesis: a comparative study between Hordeum vulgare L. cvs. 25 Igri and Digger J. Plant Growth Regul. 18: 135-143 26
Wang M, Van Berger S, Van Duijn B (2000) Insights into a key developmental switch and its 27 importance for efficient plant breeding. Plant Physiol. 124: 523-530 28
Wędzony M, Forster BP, Żur I, Golemiec E, Szechyńska-Hebda M, Dubas E, Gotębiowska G 29 (2009) Progress in doubled haploid technology in higher plants. In A Touraev, BP 30 Forster, SM Jain, eds, Advances in Haploid Production in Higher Plants. Springer 31 Netherlands, pp 1-33 32
Zimmermann U, Pilwat G, Beckers F, Riemann F (1976) Effects of external dielectric fields on 33 cell membranes. Bioelectrochem. Bioenerg 3: 53-83 34
Żur I, Dubas E, Golemiec E, Szechyńska-Hebda M, Janowiak F, Wędzony M (2008) Stress-35 induced changes important for effective androgenic induction in isolated microspore 36 culture of triticale (× Triticosecale Wittm.). Plant Cell Tiss. Organ Cult. 94: 319-328 37
38
39
75
Chapter 3
Flow cytometry enables identification of sporophytic eliciting
stress treatments in gametic cells
Disclaimer - The research presented in Chapter 3 has been published in the Journal of
Plant Physiology: Ribalta FM, Croser JS, Ochatt SJ (2012). Flow cytometry enables
identification of sporophytic eliciting stress treatments in gametic cells. J. Plant
Physiol.; Vol 169, Issue 1, pp. 104-110. All the glasshouse and laboratory work related
to this publication was carried out solely by Federico Ribalta. The publication was
written by Federico Ribalta and the co-authors were involved in the experimental
planning phase, discussion of results, structure of the paper and editorial comments
to finalise it.
3.1 Introduction
Flow cytometry is a method to rapidly and reproducibly determine relative
nuclear DNA content and ploidy level of cellular samples. Flow cytometry has been
widely applied for the analysis of animal cells (Curran, 1999) and more recently, plants
cells (Doležel et al., 2007a) following the advent of simple and reproducible methods
for tissue analysis (Galbraith et al., 1983; Doležel et al., 2007b). Applications to plant
cell analysis include, but are not limited to, differentiation between cells within a
mixed cell population, analysis of chemical composition of different tissues,
measurement of secondary metabolite accumulation and ploidy determination
following haplo-diploidisation (Yanpaisan et al., 1999; Doležel et al., 2007a; Ochatt,
2008). Despite this, flow cytometry has been applied on a relatively limited scale for
analysis of embryogenic potential in plant cell populations (Ochatt et al., 2000; Ochatt,
2008; Ochatt et al., 2011).
Flow cytometry analysis has shown a correlation between light-scatter and
fluorescence properties of cultured cells and their embryogenic potential. In 2000,
Ochatt et al. proposed flow cytometry as a tool for early prediction of plant
76
regeneration competence from protoplasts of pea (Pisum sativum L.). Fertile plants
were produced only from calli with a normal relative DNA content profile. Schulze and
Pauls (2002) developed a flow cytometry method to assess embryogenic potential of
cultured microspores of rapeseed (Brassica napus L.). By staining with calcofluor white
the authors were able to follow synthesis of cellulose in microspores, thought to be an
early indicator of embryogenic potential. Later, Ochatt (2008) also showed the
importance of relative nuclear DNA content to assess embryogenic competence from
calli of Medicago truncatula Gaertn. On the other hand, cell wall thickness of
microspores was also shown to be a reliable indicator of embryogenesis competence
in legumes by Ochatt et al. (2008). However, to our knowledge, the use of flow
cytometry to study androgenic potential of microspores of any plant species by
analysing their relative nuclear DNA content has not been reported.
Stress treatments have been widely adopted to induce the switch from
gametophytic to sporophytic development for induction of microspore embryogenesis
(Jain et al., 1996-1997; Touraev et al., 1997; Shariatpanahi et al., 2006). There is a
large variety of proposed stress treatments, with a recent review by Shariatpanahi et
al. (2006) listing 16 different stresses, which did not include the novel stress of
sonication proposed within this report. However, until now, there has been no simple
tool for assessing the actual effect of these stress treatments on subsequent DNA
synthesis.
For legume species, pea, chickpea (Cicer arietinum L.), M. truncatula and grass
pea (Lathyrus spp.), we have demonstrated pyramiding (meaning successively
superimposed) multiple stress treatments viz. cold treatment, electroporation and
osmotic stress, is critical to successful elicitation of haploid division and subsequent
77
haploid embryogenesis* (Grewal et al., 2009; Ochatt et al., 2009). Cold pre-treatment
has been shown to synchronise microspore development and foster microspore
division in a number of species (Jain et al., 1996-1997; Touraev et al., 1997; Delaitre et
al., 2001; Maluszynski et al., 2003), including legumes (Kaur and Bhalla, 1998; Croser
et al., 2006; Skrzypek et al., 2008; Grewal et al., 2009; Ochatt et al., 2009).
Electroporation has been shown to stimulate DNA synthesis (Rech et al., 1988) and
improve regeneration competence from protoplasts of various species (Rech et al.,
1987; Ochatt et al., 1988). Electroporation is an effective stress treatment for
induction of haploid embryogenesis from microspores of tobacco (Nicotiana tabacum
L.) (Mishra et al., 1987), rapeseed (Jardinaud et al., 1993), Ginkgo biloba L. (Laurain et
al., 1993), Asparagus officinalis L. (Delaitre et al., 2001), and legumes (Grewal et al.,
2009; Ochatt et al., 2009). Improved androgenic response after modification of
osmotic pressure has been observed in a number of species (Jain et al., 1996-1997;
Lionneton et al., 2001), including legumes (Croser et al., 2006; Grewal et al., 2009;
Ochatt et al., 2009). Centrifugation is applied as part of many microspore isolation
protocols, but has also been used as a stress treatment for induction of androgenesis
from intact anther culture in chickpea (Grewal et al., 2009), tobacco (Tanaka, 1973),
Datura innoxia Mill. (Sangwan-Norreel, 1977) and rapeseed (Aslam et al., 1990).
However, the need for combining stress treatments for the induction of androgenesis
appears to be confined to species of the Fabaceae.
Observations from our previous research led us to hypothesize stress
treatments may be categorized as either elicitators or enhancers of androgenesis. We
consider as eliciting those stress treatments required to induce the developmental
shift from gametophytic to sporophytic pathway. Enhancing treatments, whilst not
* Refer to Chapter 2 – P.64
78
eliciting, improve androgenetic embryogenesis after elicitacion. To test this, we used
flow cytometry to assess the effect of individual and combined stress treatments on
relative nuclear DNA content of microspores.
79
3.2 Materials and methods
3.2.1 Plant material
Pea (Pisum sativum L.) cv. Frisson and the F1 from a cross cv. Frisson x cv.
Cameor were grown in a glasshouse at 20/18°C, with a 16/8 h light/dark regime of 220
µE/ m2 s from 400 W sodium lamps. Pots were filled with volcanic rock (pozzolanne).
Plants were watered and nourished by capillarity with the standard nutrient solution
used in INRA greenhouses: 14.4 mM NO3, 3.94 mM NH4, 15.8 mM Ca, 17.9 mM K2O,
4 mM MgO, 2.46 mM P2O5, 2 mM SO3 and various micro-elements. Flower buds 7 mm
long containing anthers 1 mm in length and with microspores predominantly at the
uninucleate stage, optimal for induction of microsporogenesis (Fig. 3.1), were
harvested and subjected to a cold treatment at 4°C in the dark for 7 to 21 d.
Figure 3.1 Determination of microspore developmental stage. Cytological development of male gametophyte in pea. DAPI-stained microspores showing uninucleated (A), bi-nucleated (B) and multi-nucleated stages (C and D). Flower buds and anthers at the optimal developmental stage for androgenesis (E, F and G).
3.2.2 Bud sterilization and microspore isolation
Flower buds were placed in a 100 ml beaker and surface sterilized by treatment
for 1 min in 70% v/v ethanol, followed by 10 min in calcium hypochlorite (70 gL-1).
Buds were rinsed three times with sterile water (refer to Chapter 2, Section 2.2.5).
Note: text in italics has been added to the published text
80
Buds were transferred to a sterile mortar and macerated with a pestle in
electroporation buffer (Rech et al., 1987). Three successive steps of suspension and
centrifugation (100 g, 10°C, 5 min each) were used to eliminate debris remaining after
serial sieving through 80, 60 and 40 µm filters (Ochatt et al., 2009).
Density was determined using a Fuchs Rosenthal haemocytometer and adjusted
for culturing to 1 x 105 microspores/ ml or, for electro-stimulation and sonication
treatments, to 1 x 106 in electroporation buffer.
3.2.3 Stress treatments studied to elicit and/or enhance androgenesis
Extracted anthers and isolated microspores were submitted to the following
stress treatments either individually or in combination: cold, electroporation,
sonication, centrifugation and osmotic shock. The relative nuclear DNA content of
extracted anthers and isolated microspores was immediately analyzed using a Partec
PA II flow cytometer or analyzed after 7 d and 14 d of culture. All flow cytometry
assessments were repeated at least three times over a period of two months and a
minimum of 2500 nuclei per run were counted.
3.2.3.1 Electroporation
Isolated microspores and extracted anthers were dispensed into cuvettes of an
Electro Cell Manipulator ECM630 (BTX Gentronics Biomedical Ltd., San Diego, CA,
USA), containing electroporation buffer (Rech et al., 1987). For electroporation of
isolated microspores, a 600 µL aliquot of buffer containing 6 x 105 microspores was
dispensed into a cuvette with electrodes spaced 1 mm apart. For electroporation of
anthers, a 1300 µL aliquot of buffer was dispensed into a cuvette with electrodes
spaced 2 mm apart and 30 anthers were added. Three successive exponential pulses
81
of 1500 V/cm (anthers) or 750 V/cm (anthers and microspores) with a capacitance of
100 µF and a constant resistance of 50 were delivered at 10 s intervals.
3.2.3.2 Centrifugation of extracted anthers
Thirty intact anthers per treatment in 8 mL of electroporation buffer were
centrifuged with brakes off in 10 mL glass tubes at 170 g for 10 min, at 4°C using a
Jouan CR422 centrifuge. For microspores, centrifugation was applied during isolation.
3.2.3.3 Sonication of extracted anthers and isolated microspores
Thirty intact anthers per treatment in 8 mL of electroporation buffer and 1x
105 microspores per treatment in 2 mL of the same buffer were sonicated in 10 mL
glass tubes at 38 kHz for 30 s, at room temperature using a Branson 1210 sonicator
(Bransonic Ultrasonic Corporation, USA).
3.2.4 Flow cytometry analysis
For flow cytometry analysis, a Partec PA-II flow cytometer equipped with a HBO-
100 W mercury lamp and a dichroic mirror (TK420) was used. The relative nuclear DNA
content of 1) extracted anthers and 2) isolated microspores of pea, after submission
to various stress treatments and times of culture, was compared with leaf controls
from seed-derived plants of the two genotypes studied. In addition, the cell-division
cycle was analyzed and mitotic index calculated (Doležel et al., 2007a; Ochatt, 2008).
Nuclei were mechanically isolated by chopping tissues* with a razor blade in 2
mL of single-step isolation + stain buffer (Partec®) containing 4,6 diamidino-2-
phenylindole (DAPI), and the suspension was filtered through a 50 µm nylon mesh
(Galbraith et al., 1983; Elmaghrabi and Ochatt, 2006; Doležel et al., 2007b). The
collected data were analyzed with Partec Flomax software (Partec®, Partec GmbH,
*See Appendix 2.
82
Germany). The key feature of DNA probes is that they are stoichiometric, and hence
the number of molecules of the probe bound is equivalent to the number of
molecules of DNA that are present. The excitation light intercepts the stained particle,
which then emits a fluorescence signal that is transferred via optical system of the
flow cytometer to photomultipliers. This signal is then converted from an analog pulse
waveform to a corresponding digital value and processed by the computer to produce
a one-parameter or two parameter dot-plot histogram.
3.2.5 Fluorescein diacetate viability test
The viability of extracted anthers and isolated microspores following stress
treatments was determined by fluorescein diacetate (FDA) staining (Heslop-Harrison
and Heslop-Harrison, 1970), after counting a minimum of 200 microspores per
treatment using a Leika TMD fluorescence microscope (B1 IF 420-485 filter). Viability
was determined as percentage of microspores fluorescing yellow-green under UV
light. Images were obtained using a 3CCD Sony PowerHAD camera and annotated
using Archimed Pro (Microvision France) software, as detailed elsewhere (Ochatt et
al., 2008; Ochatt and Moessner, 2010).
3.2.6 Culture conditions
Pre-treated anthers and isolated microspores* were plated into 35 x 10 mm
Petri dishes with 2 mL of NLN medium (Lichter, 1982) containing 17% (w/v) of sucrose.
After 7 d of culture samples were transferred to the same medium containing 10%
(w/v) of sucrose (Ochatt et al., 2009)**. The culture conditions were as follows: 22/
19°C, with a 16/ 8 h light/ dark regime of 90 µE/ m² s from warm white fluorescent
tubes (Osram L58W/245 Universal White).
*Refer to Section 3.2.3; ** Refer to Chapter 2, Section 2.2.8.5.
83
3.3 Results 3.3.1 Flow cytometry analysis of extracted anthers
Differences in relative nuclear DNA content of microspores within anthers after
stress treatment/s were clearly evident from analysis of the flow cytometry profiles.
The profiles obtained from a diploid, glasshouse-grown leaf sample (Fig. 3.2A) were
compared with those obtained from anthers submitted to stress treatment/s and then
cultured for 0, 7 and 14 d. A normal (euploid) profile consists of two peaks,
corresponding to nuclei in G1 phase of mitosis (2C DNA) and those in G2/M (4C DNA).
Depending on treatment, profiles showed either two peaks (2C and 4C ploidy level) or
three peaks (1C, 2C and 4C ploidy level). The results were analyzed in terms of
presence/absence of haploid peaks in the profiles, coupled with their intensity
(relative position) and magnitude (expressed by the percentage of the total
population of nuclei analyzed they represent).
Pre-treating anthers at 4°C and transferring from a high to low osmolarity
medium at day 7 of culture did not induce DNA synthesis as only 2C and 4C peaks
could be observed after 14 d of culture (Fig. 3.2B). The application of a cold pre-
treatment, an osmotic treatment plus centrifugation and/ or sonication treatments
also did not induce androgenesis (Fig. 3.2C and 3.2D). However, adding an
electroporation treatment to cold stressed anthers prior to their culture and an
osmotic stress treatment successfully induced DNA synthesis and enhanced
androgenetic proliferation. This was evidenced by a flow cytometry profile with three
peaks (1C, 2C and 4C) showing clear indication of androgenic divisions within
microspores (Fig. 3.2E). The combination of these three treatments was therefore
considered to elicit androgenesis.
84
In an effort to further improve microspore induction, we added a centrifugation
and/ or a sonication treatment to anthers subjected to cold, electroporation and
osmotic stresses. There was a clear synergistic and additive enhancing effect on
androgenesis induction after successively imposing these multiple stress factors (Fig.
3.2F).
Figure 3.2 Effect of stress treatments on relative DNA content per nucleus of pea microspores within anthers after 14 d of culture. C-DNA profiles showing 1C, 2C and 4C ploidy levels. (A) Field pea leaves; (B) Cold + Osmolarity treatments; (C) Cold + Osmolarity + Sonication treatments; (D) Cold + Osmolarity + Centrifugation treatments; (E) Cold + Osmolarity + Electroporation treatments; (F) Cold + Osmolarity + Electroporation + Centrifugation + Sonication treatments. Note in figure 3.2A the scale of the vertical axis is different to the other profiles as the data was obtained from leaf tissue.
85
Differences among treatments in terms of relative nuclear DNA content at 0 d
(Fig. 3.3A) and 7 d (Fig. 3.3B) were not as evident as they were after 14 d of culture
(Fig. 3.3C), indicative of a delayed improvement of responses due to stress
treatments.
Figure 3.3 Flow cytometry profiles obtained immediately after submission to cold, electroporation, centrifugation and sonication stress treatments (A) or after 7 d (B) and 14 d (C) of culture.
Sonication treatment alone (Fig. 3.4A) or combined with other stress treatments
(Fig. 3.4B and 3.4C) increased the production of tetraploid peaks, indicating a possible
doubling effect of sonication treatment.
Figure 3.4 Flow cytometry profiles of cold pre-treated extracted anthers submitted to a sonication treatment alone (A) or combined with electroporation (B) or electroporation and sonication (C).
The analysis of relative nuclear DNA content of microspores was used to predict
whether a combination of stresses were elicitors or enhancers of androgenesis. The
relative nuclear DNA content of microspores was greater after submission to eliciting
factors (cold, electro-stimulation and osmolarity) compared to enhancing factors
(centrifugation and sonication), leading us to propose their roles as shown in Figure
3.5.
86
Figure 3.5 Pyramiding eliciting factors (red) and enhancing factors (blue) enhances androgenetic response from pea anthers.
3.3.2 Flow cytometry analysis of isolated microspores
Flow cytometry profiles obtained from a diploid, glasshouse-grown leaf sample
(Figure 3.6A), were compared with those obtained from isolated microspores
submitted to stress treatment/s and then cultured for 0, 7 and 14 d. All stress
treatments presented profiles indicative of no spontaneous DNA doubling, with one
haploid (1C) peak at day 0, 7 and 14 (Fig. 3.6B, 3.6C and 3.6D). There was no clear
effect of stress treatments on DNA synthesis in isolated microspores.
87
Figure 3.6 Effect of treatments on relative nuclear DNA content of pea isolated microspores after 14 d of culture. (A) Pea leaves; (B) Cold + Osmolarity + Electroporation treatments; (C) Cold + Osmolarity + Sonication treatments; (D) Cold + Osmolarity + Centrifugation + Sonication treatments.
3.3.3 Microspore fluorescein diacetate viability test
Following the stress treatments, a very high (>85%) viability rate was observed
for both isolated microspores or extracted anthers of pea, indicating that there was no
immediate deleterious effect of stress treatments on viability (Fig. 3.7).
Figure 3.7 FDA viability test from isolated microspores of pea. HV: Electroporation (1500V/cm); HVS: Electroporation (1500V/cm) + Sonication; LV: Electroporation (750V/cm); LVS: Electroporation (750V/cm) + Sonication; S: Sonication.
88
3.4 Discussion
Flow cytometry analysis of relative nuclear DNA content of microspores within
anthers has enabled us to divide stress treatments between two categories: a)
elicitors or b) enhancers of androgenesis. Our previous research has clearly shown
pyramiding stress treatments to be the key to sustained androgenic division from pea
(Croser et al., 2006; Ochatt et al., 2009). This is the first report in a plant species to
evaluate the effect of various stress treatments based on relative nuclear DNA content
and to use this information to categorize them as ‘elicitors’ or ‘enhancers’.
The flow cytometry analysis showed that cold and electroporation stress
treatment of androgenic tissue, coupled with an osmotic modification of the medium
during culture had both an individual and an additive eliciting effect on androgenesis
from pea microspores within anthers. Thus, these treatments are required in
combination to induce the developmental shift from gametophytic to sporophytic
pathway. Hence, these stress treatments act as “elicitors” of androgenesis. In
addition, as observed previously (Grewal et al., 2009; Ochatt et al., 2009) there was a
positive effect of pyramiding stress factors, including sonication and centrifugation, on
the induction of embryo formation from intact anthers. Sonication and centrifugation,
whilst not elicitors, improve androgenetic embryogenesis when combined with the
eliciting stresses above and can therefore be described as “enhancing treatments”.
The application of combined stress factors seems to be the way to overcome
recalcitrance of legumes to androgenesis, likely mediated through increases in
hormone levels in stressed anthers (Lülsdorf et al., 2011).
Whilst the mechanism of various more commonly applied stress treatments
such as temperature (Duncan and Heberle, 1976; Amssa et al., 1980; Żur et al., 2008)
89
and osmotic regulation (Dunwell and Thurling, 1985; Ochatt et al., 2009) is relatively
well understood, the effect of these stresses on DNA synthesis has been more difficult
to determine. Previously, a cytological study using fluorochromes such as DAPI has
been the method of choice for showing the effect of treatments on nuclei division
(Ochatt et al., 2011). Whilst we are not advocating the flow cytometry analysis
presented here as a replacement for this type of study, it will have a role to play in
quickly ascertaining the effect of one or more stresses on a population. Flow
cytometry analysis of relative nuclear DNA content of microspores should assist
researchers in making choices about what stress or group of stresses are most
effective in eliciting androgenic development, particularly during the process of
protocol development, not only for legumes but also for other plant species.
The flow cytometry analysis presented here represents a relatively simple, quick
and reliable way to analyze and discriminate the effect of various stress treatments on
elicitation of androgenesis. Sample preparation requires a small amount of plant
tissue (10-20 mg) and takes only a few minutes (approximately 2 min per sample) at a
low cost per sample (less than $2 per sample). Analysis is rapid, and a significant
number of nuclei can be measured in a few minutes (>3000 nuclei per min), which
makes results statistically robust and representative of the whole population. A
constraint for broad application of this type of flow cytometry analysis is complexity
and cost of equipment. However, in recent years, modern, small and more affordable
multiparameter flow cytometers have become readily available in many laboratories
(Ochatt, 2008; Ochatt et al., 2011). Therefore, flow cytometry analysis of relative
nuclear DNA content should be of broad application for assessing effect of stresses on
elicitation of androgenesis of many species.
90
Although several decades have elapsed since the first reports on haploid plants
from anthers/microspores, it is only recently that progress was made in understanding
the molecular mechanisms underlying the switch from a gametophytic to a
sporophytic path (Seguí-Simarro et al., 2006). While the identities of most of these
genes remain unknown, in several cases they appear to be stress-related or are
associated with zygotic embryogenesis. These stress-related genes may be involved
with a general reprogramming of the cell or providing some type of protection from
stress (Reynolds, 1997; Maraschin et al., 2005). Molecular approaches for the
identification of embryogenesis genes have included for instance the use of gene
expression profiling, and revealed the expression of several embryogenesis-related
genes like the BABY BOOM ERF/AP2 transcription factor (Boutilier et al., 2002), LEC1
and LEC2 (Gaj et al., 2005; Braybrook et al., 2006; Fambrini et al., 2006; Alemanno et
al., 2008), which took place as early as 48-72 h after initiating microspore culture.
Already in their early study, Rech et al. (1988) had shown that DNA synthesis was
enhanced by electroporation, and its use as an eliciting factor in our experiments here
is likely to have encompassed an increased expression of some of the genes above.
Assessment of expression profiles of genes and transcription factors known to
be involved in embryo development in the model species arabidopsis (Arabidopsis
thaliana L.) and M. truncatula, is envisaged in order to get a better understanding of
the genetic basis of doubled haploidy in pea. We are also presently analysing the
effects of the various stress treatments on the level of G-C by using Chromomycine
A3, which is GC-specific, and on the concomitant AT/ GC ratio of stressed anthers.
91
The results presented here form a solid basis for further efforts designed to
enhance responses and to extend doubled haploid technology to other pea genotypes
and to other legume species, generally regarded as recalcitrant to this approach.
92
3.5 References
Alemanno L, Devic M, Niemenak N, Sanier C, Guilleminot J, Rio M, Verdeil JL, Montoro P
(2008) Characterization of leafy cotyledon1-like during embryogenesis in Theobroma
cacao L. Planta 227: 853-866
Amssa M, De Buyser J, Henry Y (1980) Origin of diploid plants obtained by in vitro culture of
anthers of young wheat (Triticum aestivum L.). Cr. Acad. Sci. D. Nat. 290: 1095-1097
Aslam FN, MacDonald MV, Loudon PT, Ingram DS (1990) Rapid-cycling Brassica species:
Inbreeding and selection of Brassica napus for anther culture ability and an
assessment of its potential for microspore culture. Ann. Bot. 66: 331-339
Boutilier K, Offringa R, Sharma VK, Kieft H, Ouellet T, Zhang L, Hattori J, Liu CM, van
Lammeren AAM, Miki BLA, Custers JBM, van Lookeren Campagne MM (2002)
Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic
growth. Plant Cell 14: 1737-1749
Braybrook SA, Stone SL, Park S, Bui AQ, Le BH, Fischer RL, Goldberg RB, Harada JJ (2006)
Genes directly regulated by LEAFY COTYLEDON2 provide insight into the control of
embryo maturation and somatic embryogenesis. Proc. Natl. Acad. Sci. USA 103: 3468-
3473
Croser JS, Lülsdorf MM, Davies PA, Clarke HJ, Bayliss KL, Mallikarjuna N, Siddique KHM
(2006) Toward doubled haploid production in the Fabaceae: Progress, constraints,
and opportunities. Crit. Rev. Plant Sci. 25: 139-157
Curran AD (1999) Flow cytometry in the exploration of the physiopathology of occupational
lung disease. Occup. Environ. Med. 56: 742-746
Delaitre C, Ochatt S, Deleury E (2001) Electroporation modulates the embryogenic responses
of asparagus (Asparagus officinalis L.) microspores. Protoplasma 216: 39-46
Doležel J, Greilhuber J, Suda J (2007b) Estimation of nuclear DNA content in plants using flow
cytometry. Nat. Protoc. 2: 2233-2244
Doležel J, Greilhuber J, Suda J (2007a) Flow Cytometry with Plants: An Overview. In J Doležel,
J Greilhuber, J Suda, eds, Flow Cytometry with Plant Cells: Analysis of Genes,
Chromosomes and Genomes. Wiley Online Library, Weinheim, pp 41-66
Duncan EJ, Heberle E (1976) Effect of temperature shock on nuclear phenomena in
microspores of Nicotiana tabacum and consequently on plantlet production.
Protoplasma 90: 173-177
Dunwell JM, Thurling N (1985) Role of sucrose in microspore embryo production in Brassica
napus ssp. oleifera. J. Exp. Bot. 36: 1478-1491
Elmaghrabi A, Ochatt S (2006) Isoenzymes and flow cytometry for the assessment of true-to-
typeness of calluses and cell suspensions of barrel medic prior to regeneration. Plant
Cell Tiss. Organ Cult. 85: 31-43
Fambrini M, Durante C, Cionini G, Durante C, Cionini G, Geri C, Giorgetti L, Michelotti V,
Salvini M, Pugliesi C (2006) Characterization of LEAFY COTYLEDON1-LIKE gene in
Helianthus annuus and its relationship with zygotic and somatic embryogenesis. Dev.
Genes Evol. 216: 253-264
Gaj MD, Zhang S, Harada JJ, Lemaux PG (2005) Leafy cotyledon genes are essential for
induction of somatic embryogenesis of Arabidopsis. Planta 222: 977-988
93
Galbraith DW, Harkins KR, Maddox JR, Ayres NM, Sharma DP, Firrozabady E (1983) Rapid
flow cytometric analysis of the cell cycle in intact plant tissues. Science 220: 1049-
1051
Grewal RK, Lülsdorf M, Croser J, Ochatt S, Vandenberg A, Warkentin TD (2009) Doubled-
haploid production in chickpea (Cicer arietinum L.): role of stress treatments. Plant
Cell Rep. 28: 1289-1299
Heslop-Harrison J, Heslop-Harrison Y (1970) Evaluation of pollen viability by enzymatically
induced fluorescence; intracellular hydrolysis of fluorescein diacetate. Biotech.
Histochem. 45: 115 - 120
Jain SM, Sopory SK, Veilleux RE (1996-1997) In vitro Haploid Production in Higher Plants, Vol
1-5. Kluwer Acad. Publ., Dordrecht, The Netherlands
Jardinaud MF, Souvre A, Alibert G (1993) Transient GUS gene expression in Brassica napus
electroporated microspores. Plant Sci. 93: 177-184
Kaur P, Bhalla JK (1998) Regeneration of haploid plants from microspore culture of pigeon pea
(Cajanus cajan L.). Indian J. Exp. Biol. 36: 736-738
Laurain D, Trémouillaux-Guiller J, Chénieux JC (1993) Embryogenesis from microspores of
Ginkgo biloba L., a medicinal woody species. Plant Cell Rep. 12: 501-505
Lichter R (1982) Induction of haploid plants from isolated pollen of Brassica napus. Z
Pflanzenphysiol 105: 427–434
Lionneton E, Beuret W, Delaitre C, Ochatt S, Rancillac M (2001) Improved microspore culture
and doubled-haploid plant regeneration in the brown condiment mustard (Brassica
juncea). Plant Cell Rep. 20: 126-130
Lülsdorf MM, Croser JS, Ochatt S (2011) Androgenesis and doubled haploid production in
food legumes. CABI, Oxfordshire
Maluszynski M, Kasha KJ, Szarejko I (2003) Published doubled haploid protocols in plant
species. Klewer Acad. Publ.
Maraschin SF, de Priester W, Spaink HP, Wang M (2005) Androgenic switch: an example of
plant embryogenesis from the male gametophyte perspective. J. Exp. Bot. 56: 1711–
1726
Mishra KR, Joshua DC, Bhatia CR (1987) In vitro electroporation of tobacco pollen. Plant Sci.
52: 135-139
Ochatt S, Moessner A (2010) Rounding up plant cells. Int. J. Plant Biol. 1: 40-42
Ochatt S, Muilu R, Ribalta F (2008) Cell morphometry and osmolarity as early indicators of the
onset of embryogenesis from cell suspension cultures of grain legumes and model
systems. Plant Biosyst. 142: 480-486
Ochatt S, Pech C, Grewal R, Conreux C, Lülsdorf M, Jacas L (2009) Abiotic stress enhances
androgenesis from isolated microspores of some legume species (Fabaceae). J. Plant
Physiol. 166: 1314-1328
Ochatt SJ (2008) Flow cytometry in plant breeding. Cytometry Part A 73A: 581-598
Ochatt SJ, Chand PK, Rech EL, Davey MR, Power JB (1988) Electroporation-mediated
improvement of plant regeneration from colt cherry (Prunus avium × pseudocerasus)
protoplasts. Plant Sci. 54: 165-169
Ochatt SJ, Mousset-Declas C, Rancillac M (2000) Fertile pea plants regenerate from
protoplasts when calluses have not undergone endoreduplication. Plant Sci. 156: 177-
183
94
Ochatt SJ, Patat-Ochatt EM, Moessner A (2011) Ploidy level determination within the context
of in vitro breeding. Plant Cell Tiss. Organ Cult. 103: 329-341
Rech EL, Ochatt SJ, Chand PK, Davey MR, Mulligan BJ, Power JB (1988) Electroporation
increases DNA-synthesis in cultured plant-protoplasts. Biotechnol. 6: 1091-1093
Rech EL, Ochatt SJ, Chand PK, Power JB, Davey MR (1987) Electro-enhancement of division of
plant protoplast-derived cells. Protoplasma 141: 169-176
Reynolds TL (1997) Pollen embryogenesis. Plant Mol. Biol. 33: 1-10
Sangwan-Norreel BS (1977) Androgenic stimulating factors in the anther and isolated pollen
grain culture of Datura innoxia Mill. J. Exp. Bot. 28: 843-852
Schulze D, Pauls KP (2002) Flow cytometric analysis of cellulose tracks development of
embryogenic Brassica cells in microspore cultures. New Phytol. 154: 249-254
Seguí-Simarro JM, Barany I, Suarez R, Fadon B, Testillano PS, Risueño MC (2006) Nuclear
bodies domain changes with microspore reprogramming to embryogenesis. Eur. J.
Histochem. 50: 35-44
Shariatpanahi ME, Bal U, Heberle-Bors E, Touraev A (2006) Stresses applied for the re-
programming of plant microspores towards in vitro embryogenesis. Physiol. Plant.
127: 519-534
Skrzypek E, Czyczyło-Mysza I, Marcińska I, Wędzony M (2008) Prospects of androgenetic
induction in Lupinus spp. Plant Cell Tiss. Organ Cult. 94: 131-137
Tanaka M (1973) The effect of centrifugal treatment on the emergence of plantlet from
cultured anther of tobacco. Jap. J. Breed. 23: 171-174
Touraev A, Vicente O, Heberle-Bors E (1997) Initiation of microspore embryogenesis by
stress. Trends Plant Sci. 2: 297-302
Yanpaisan W, King NJC, Doran PM (1999) Flow cytometry of plant cells with applications in
large-scale bioprocessing. Biotech. Adv. 17: 3-27
Żur I, Dubas E, Golemiec E, Szechynska-Hebda M, Janowiak F, Wędzony M (2008) Stress-
induced changes important for effective androgenic induction in isolated microspore
culture of triticale (x Triticosecale Wittm.). Plant Cell Tiss. Organ Cult. 94: 319-328
95
Chapter 4 1
Antigibberellin-induced reduction of internode length favors 2
in vitro flowering and seed-set in different pea genotypes 3
Disclaimer - The research presented in Chapter 4 has been published in Biologia 4
Plantarum: Ribalta FM, Croser JS, Erskine W, Finnegan PM, Lülsdorf MM, Ochatt SJ 5
(2014). Antigibberellin-induced reduction of internode length favors in vitro flowering 6
and seed-set in different pea genotypes. Biol. Plant; Vol 58, Issue 1, pp. 39-43. All the 7
glasshouse and laboratory work related to this publication was carried out solely by 8
Federico Ribalta. The publication was written by Federico Ribalta and the co-authors 9
were involved in the experimental planning phase, discussion of results, structure of 10
the paper and editorial comments to finalise it. 11
12
4.1 Introduction 13
In vitro based modified single-seed-descent (SSD) systems have been proposed 14
as one method to accelerate generation turnover across a number of species (Franklin 15
et al., 2000; Ochatt et al., 2002; Asawaphan et al., 2005; Zhang, 2007; Ochatt and 16
Sangwan, 2008). The in vitro SSD technique involves the shortening of generation time 17
by culturing immature seeds after forced in vitro flowering. Current conventional SSD 18
methodologies enable a maximum of three generations per year to be developed in 19
pea (Pisum sativum L.) (Ochatt and Sangwan, 2010). Pea cultivars are usually released 20
after 8-10 generations of self-pollination to achieve an appropriate level of 21
homozygosity (Kasha and Maluszynski, 2003). Decreasing the length of the generation 22
cycle will overcome this breeding bottleneck and accelerate genetic improvement. 23
Doubled haploidy is the fastest known route to homozygosity (Chase, 1952), however, 24
considerable further research is required before this technology will be available 25
routinely within a pea breeding programme (Croser et al., 2006; Ochatt et al., 2009). 26
There have been three in vitro flowering protocols proposed for pea (Fujioka et al., 27
1999; Franklin et al., 2000; Ochatt et al., 2002); however, these protocols were 28
96
developed for a limited number of early-flowering cultivars. To enable the 1
development of a widely applicable protocol we studied the effect of in vivo versus in 2
vitro application of the antigibberellin Flurprimidol to reduce internode length, of light 3
quality on in vitro flowering and seed-set, and of the optimization of culture 4
methodology through comparative in vitro culture of intact plants, plants with the 5
meristem removed or excised shoot tip explants. Based on our results we report 6
herewith an improved protocol that enables in vitro flowering across a range of pea 7
genotypes, including mid and late flowering types. 8
The antigibberellin Flurprimidol is a chemical used in horticulture to reduce 9
plant height and produce compact plants. It reduces internode elongation through the 10
inhibition of gibberellic acid (GA) biosynthesis (Rademacher, 2000). Flurprimidol has 11
been extensively used to control plant growth under glasshouse conditions in a 12
number of species (Hamid and Williams, 1997; Pobudkiewicz and Treder, 2006; Burton 13
et al., 2007), including pea (Ochatt et al., 2002). However, to our knowledge, 14
Flurprimidol has never been used in any in vitro flowering protocol by adding it to the 15
medium. We believe the addition of Flurprimidol in vitro will result in smaller plants 16
required for in vitro growth. 17
Environmental factors such as light quality (Reid et al., 1996; Cerdan and Chory, 18
2003; Ausín et al., 2005; Nelson et al., 2010), photoperiod length (Reid et al., 1996; 19
Ceunen and Geuns, 2013), growth temperature (Ausín et al., 2005; Nelson et al., 2010) 20
and stress (Wang et al., 2012; Zhou et al., 2012) play a key role in the regulation of the 21
transition to flowering in plants. Among these, in pea the effect of light quality is 22
unclear but exploitable. In general, light containing a mixture of red and far-red light is 23
most effective in causing flowering of long day plants (Reid and Murfet, 1977; Vince-24
97
Prue, 1981; Weller et al., 1997; Ausín et al., 2005; Cummings et al., 2007). In 2001, 1
Runkle and Heins reported the red: far-red ratio had no effect on in vivo flowering 2
time of the long day pea cv. Utrillo. In contrast, Cummings et al. (2007) reported the 3
high red: far-red ratio of light sources like cool white fluorescent and high-intensity 4
discharge lamps delayed pea flowering in vivo and inhibited internode extension in 5
long day genotypes. Thus, we reason the addition of far-red filtered light may help 6
reduce the red: far-red ratio and thus stimulate in vitro flowering and seed 7
development in pea. 8
To develop a protocol that could be widely adopted, it was necessary to 9
optimize culture factors considered important to in vitro flowering and seed initiation 10
and maturation. The factors we identified as critical were the gas exchange and 11
humidity level through the use of different culture vessel types (Lentini et al., 1988; 12
Fujioka et al., 1999; Asawaphan et al., 2005), explant sources (culture of intact plants, 13
plants with the meristem removed or excised shoot tip explants) (Narasimhulu and 14
Reddy, 1984; Franklin et al., 2000; Ochatt et al., 2000; 2002), culture medium 15
composition (Franklin et al., 2000; Ochatt et al., 2000; 2002; Asawaphan et al., 2005), 16
and the addition of plant growth regulators (Narasimhulu and Reddy, 1984; 17
Chengalrayan et al., 1995; Franklin et al., 2000; Ochatt et al., 2002). We believe the 18
optimization of these factors would lead to a robust pea in vitro flowering protocol. 19
The aim of this work is to understand the effect of physiological mechanisms 20
operating in vitro on the efficient and reproducible induction of flowering and seed-21
set across a range of pea genotypes. The final goal is to contribute to the acceleration 22
of generation cycles for faster breeding of novel genotypes. 23
24
98
4.2 Materials and methods 1
4.2.1 Plant material 2
A range of genetically diverse Pisum sativum L. genotypes with varying 3
flowering times were selected for this research (Table 4.1). 4
Table 4.1 Pea genotypes used in this study and their main characteristics. 5
Name
Flowering type
Plant habit
Leaf type
Comment
Frisson Early Semi-dwarf Conventional French cv. (Ochatt et al., 2002 - control)
Excell Early/Mid Semi-dwarf Semi-leafless Australian cv.
Bundi Early/Mid Semi-dwarf Semi-leafless Australian cv.
Victor Mid Semi-dwarf Conventional French cv. (Ochatt et al., 2002)
Dunwa Mid/late Tall Conventional Australian cv.
Kaspa Late Semi-dwarf Semi-leafless Australian cv. (industry standard)
00P016-1 Very late Tall Conventional Ethiopian germplasm accession: landrace with blackspot resistance
6
4.2.2 Flurprimidol experiments 7
4.2.2.1 In vivo experiments 8
Seeds of cv. Kaspa and line 00P016-1 were sown in a glasshouse at The 9
University of Western Australia (Perth, Australia - lat: 31°58’49” S; long: 115°49’7” E) 10
in 1 dm3 pots filled with UWA Plant Bio Mix potting mix (Richgro Garden Products, 11
Perth, Australia). The temperature was controlled at 20/18°C day/night under ambient 12
light conditions. In preliminary experiments it was observed that cv. Kaspa and line 13
00P016-1 did not flower under in vitro conditions; this is the reason why the in vivo 14
approach was taken for these two genotypes. 15
Flurprimidol, 2-methyl-1-pyrimidine-5-yl-1-(4-trifluoromethoxy phenyl) 16
propane-1-ol (Topflor, SePRO Corporation, Carmel, IN, USA), was used to reduce 17
internode elongation. In order to identify the most effective Flurprimidol treatment, a 18
5% w/v solution was applied as a drench at various concentrations (0, 25, 50 and 75 19
99
cm3). Applications were repeated three times at 10-day intervals from the three-leaf 1
stage (Ochatt et al., 2002). 2
4.2.2.2 In vitro experiments 3
Dry seeds were surface-sterilized by treatment for 5 min in 70% ethanol, 4
followed by 10 min in sodium hypochlorite (21 g dm-3). Seeds were rinsed three times 5
with sterile deionised water and imbibed overnight. The coats of imbibed seeds were 6
removed and 10 embryos with both cotyledons intact were cultured in a vessel 7
containing 50 cm3 of B5 salts and vitamins (Gamborg et al., 1968) modified by the 8
addition of 10 mM NH4Cl (Ochatt et al., 2000). 9
After seven days, the shoot apical meristems (1 cm length and comprising two 10
internodes) of 50% of the plants, randomly selected, were removed using a scalpel. 11
Both, the plants with the meristem removed, which developed via axillary branching, 12
and the excised shoot apical meristems were then individually cultured in vitro and 13
compared to cultured intact plants. The three treatments, plants with the meristem 14
removed, excised shoot tip explants and intact plants were cultured into either 150 x 15
25 mm borosilicate glass tubes covered with a polypropylene closure 16
(PhytoTechnology Laboratories, Kansas, USA) and containing 20 cm3 of MS medium 17
(Murashige and Skoog, 1962), or into 150 x 70 mm polycarbonate containers sealed 18
with a screw cap (Sarstedt Australia Pty Ltd, Adelaide, Australia) with a 4 mm diameter 19
hole covered in breathable membrane (Flora Laboratories, Melbourne, Australia) with 20
50 cm3 of MS medium. The pH of the medium was adjusted to 5.6 prior to autoclaving 21
at 121°C for 20 min. 22
All cultures were incubated at 24°C with a light intensity of 145 µmol m-2 s-1 from 23
cool-white-fluorescent tubes (LIFEMAX TL-D 30W/840, Philips Lighting, Bangkok, 24
100
Thailand), unless stated otherwise. Preliminary experiments included culture of 1
explants at photoperiods of 12, 16, 20 and 24 h of light. However, there was no 2
significant difference between treatments. Thus, a photoperiod of 20/4 h light/dark 3
was chosen for all in vitro experiments in this study. 4
To evaluate the effect of Flurprimidol on in vitro growth and flowering, a range 5
of concentrations (0, 5, 10, 15, 20, 30 and 35 cm3 of 5% w/v Flurprimidol per dm3 of 6
medium) were added filter-sterilized to the MS medium immediately after 7
autoclaving. Also, experiments were undertaken to assess the effect of vernalisation on 8
in vitro flowering in pea. However, no significant differences were observed between 9
treatments (Appendix 3). 10
4.2.3 Light quality treatments 11
To study the effect of light quality on in vitro flowering induction, three different 12
light source types were compared: 1- Cool-white fluorescent light only (145 µmol m-2 13
s-1); 2- Cool-white fluorescent combined with red fluorescent light from F30W/GRO 14
Gro-Lux tubes (Sylvania Lighting International, Erlangen, Germany) (120 µmol m-2 s-1); 15
and 3- Cool-white fluorescent plus far-red filtered light (Blood Red 789, LEE filters, 16
Andover, England) (130 µmol m-2 s-1). 17
In all experiments, flowering time and node number of the first flower were 18
recorded. Also, the average internode length (average plant length/ average number 19
of nodes) was measured at day 40. In addition, the efficiency of recovery of immature 20
seeds and embryos was studied. This included the selection of the embryo 21
developmental stage (number of days after flowering) for best in vitro germination 22
and plant development. The immature pods at 16, 18, 20 and 22 d after flowering 23
Note: text in italics has been added to the published text
101
were opened aseptically and the embryos removed and directly transferred on to new 1
glass tubes containing 20 cm3 of hormone-free MS medium. 2
The experimental design was completely randomized and all treatments were 3
repeated at least three times with a minimum 10 replicates per treatment per 4
genotype. Statistical analysis was performed by analysis of variance (Microsoft Office 5
Excel 2007 software). 6
7
102
4.3 Results 1
4.3.1 Effect of Flurprimidol on internode length and flowering time 2
Pea plant height was reduced under all conditions: the addition of the 3
antigibberellin Flurprimidol produced smaller plants by reducing internode length in 4
vivo (Appendix 4). For example, for line 00P016-1 we observed an average reduction 5
in plant height from 77 ± 7.0 cm in the control treatment (0 cm3 of Flurprimidol) to 13 6
± 2.8 cm for the highest Flurprimidol concentration applied (75 cm3). Flurprimidol also 7
reduced internode length in vitro as shown in Fig. 4.1 for cv. Kaspa; although with 8
lower concentrations of antigibberellin. Flurprimidol had no effect on flowering time 9
and seed-set under either in vivo or in vitro conditions and no associated change in 10
node number was observed. 11
12
Figure 4.1 Effect of Flurprimidol on internode length in vitro for different explant 13
sources of cv. Kaspa at day 40 (results are expressed as mean ± SE; n = 30; P = 0.05). 14
4.3.2 Interaction Flurprimidol x environment 15
For both, in vivo and in vitro experiments, an important seasonal effect of 16
Flurprimidol was detected. In the glasshouse under natural light conditions, internode 17
length was reduced more in spring compared to autumn. For any given concentration 18
103
used, Flurprimidol was less effective in reducing plant size during the autumn-winter 1
period (shorter days and lower light intensity) than in the spring-summer period 2
(longer days and higher light intensity) (Appendix 4). Similarly, in controlled 3
environment rooms under low light intensity (110-180 µmol m-2 s-1) Flurprimidol was 4
less effective in reducing plant size in vitro, therefore a higher concentration was 5
required than under a higher light intensity (550-900 µmol m-2 s-1) to produce similar 6
effects. Thus, under low light intensity the addition of Flurprimidol in vitro at a 7
concentration of 30 cm3 dm-3 reduced the average plant size at day 40 of cv. Kaspa by 8
20.9%, while under higher light intensity a concentration of 15 cm3 dm-3 was sufficient 9
to produce similar plant size reduction. 10
4.3.3 Interaction Flurprimidol x genotype 11
The effectiveness of Flurprimidol on reducing plant height varied across 12
genotypes. Flurprimidol had a greater effect on tall genotypes compared to semi-13
dwarf genotypes. For example, for the tall landrace 00P016-1 a 25 cm3 application of 14
the Flurprimidol solution under glasshouse conditions reduced plant height by 73.6% 15
compared to the control treatment (0 cm3 of Flurprimidol). Conversely, the same 16
Flurprimidol treatment reduced plant height by just 37.9% for the semi-dwarf cv. 17
Kaspa. 18
4.3.4 Effect of Flurprimidol on different culture explant sources 19
Flurprimidol significantly reduced in vitro internode length on the three explant 20
sources studied: intact plants; plants with the meristem removed and excised shoot 21
tip explants (Fig. 4.1). Plants with the meristem removed developed through the 22
proliferation of a cotyledonary node axillary meristem. No significant difference was 23
observed on in vitro time to flowering when comparing intact plants and plants with 24
104
the meristem removed. Thus, for plants of cv. Excell grown in tubes, the average 1
flowering time was 32 ± 2.7 d for intact plants and 32 ± 5.9 d for plants with the 2
meristem removed. In contrast, excised shoot tip explants cultured in vitro always 3
took longer to flower, e.g. the average flowering time of excised shoot tip explants of 4
cv. Excell grown in tubes was 44 ± 5.5 d. It was observed that only excised shoot tip 5
explants that produced roots were able to form flowers. 6
4.3.5 Light quality effect 7
No significant effect of light quality in vitro was observed on: a- days to 8
flowering, b- percentage of flowering plants, and c- percentage of podding plants 9
(Appendix 4). White fluorescent light was thus adopted in the subsequent 10
experiments. For all genotypes studied, light quality treatments, and Flurprimidol 11
treatments, the node of first flower production was not affected. For each treatment 12
the mean node of first flower production for intact plants of cv. Excell was 12 ± 0.4. 13
4.3.6 Culture system 14
In general, there was a higher percentage of seed-set in plants grown in tubes 15
compared to plants grown in containers (Fig. 4.2), despite more flowers being 16
produced in containers (Fig. 4.3). The largest difference between treatments was 17
observed for conventional leafed cvs. Frisson and Victor, with a consistently higher 18
percentage of seed setting plants in tubes compared to containers. However, a lower 19
difference between treatments was observed for the semi-leafless cvs. Excell and 20
Bundi. Interestingly, for intact plants of cvs. Excell and Bundi, containers favoured 21
marginally better seed-set than tubes. The type of culture vessel used had no effect 22
on plant architecture irrespectively of the genotype studied. 23
105
1
Figure 4.2 Percentage of plants that set seeds under white fluorescent light (results 2
are expressed as mean ± SE; n = 30; T: tubes; C: containers). 3
4
5
Figure 4.3 Number of flowers per plant produced in vitro from different explant 6
sources for A - cv. Frisson and B - cv. Excell (results are expressed as mean ± SE; n = 30; 7
P = 0.05; MR: meristem removed; STE: shoot tip explants; IP: intact plants). 8
Flurprimidol concentration: Intact plants and plants with the meristem (30 cm3 dm-3), 9
shoot tip explants: (10 cm3 dm-3). 10
In order to shorten the length of the reproductive cycle, immature embryos 11
were removed and cultured in vitro. Best germination rates were obtained by 12
culturing immature seeds between 18-20 d after flowering. The length of the 13
106
generation cycle in vitro was approximately 50 d for most of the genotypes studied 1
(Victor: 48 ± 1.8 d; Bundi: 49 ± 3.6 d; Excell: 50 ± 5.9 d; and Frisson: 54 ± 3.4 d). On the 2
other hand, for the very late flowering landrace 00P016-1, the average generation 3
length in vitro was over 90 d with a very low flowering and seed setting rate (under 4
10%). 5
4.3.7 Comparison of growth environments 6
Significant differences among the studied genotypes were observed in 7
flowering time and in the estimated average generation cycle length when comparing 8
plants grown under different growth environments (Fig. 4.4). With the proposed in 9
vitro flowering protocol, up to seven generations per year can be obtained for the 10
early/mid flowering cvs. Bundi and Excell; and over five generations for the mid/late 11
flowering cv. Dunwa. 12
13 Figure 4.4 Comparison between growth environments for estimates of average 14
generation cycle length for different pea cultivars. Numbers within bars indicate 15
average flowering time in days (data are means from three repetitions with at least 10 16
individuals). Flowering time in the field source: Department of Agriculture and Food of 17
Western Australia, Australia. 18
19
20
21
22
23
24
25
107
4.4 Discussion 1
An optimized in vitro flowering protocol is presented here using Flurprimidol to 2
control in vitro plant size, culturing plants with the meristem removed and excised 3
shoot tip explants into glass tubes under white fluorescent light. In vitro flowering and 4
seed-set was achieved across a genetically diverse range of pea genotypes (Appendix 5
5) with an average generation cycle length of approximately 50 d for the early to mid 6
flowering cultivars. We were able to accept our hypothesis that the antigibberellin 7
Flurprimidol permits in vitro flowering across a range of pea genotypes by reducing 8
internode length. However, the addition of red or far-red light sources did not reduce 9
the flowering time in vitro in the genotypes studied. 10
In order to shorten the generation cycle of pea, it is essential to control plant 11
growth to obtain plants with reduced vegetative development (Ochatt et al., 2002). In 12
horticulture Flurprimidol has been used in vivo to produce compact plants. This 13
chemical acts by blocking cytochrome P450-dependent monooxygenases, catalyzing 14
the oxidation of ent-kaurene into ent-kaurenoic acid, thereby inhibiting GA 15
biosynthesis (Rademacher, 2000). In our experiments, Flurprimidol reduced pea plant 16
size in vivo and in vitro. In terms of logistics, Flurprimidol permits growth in vitro by 17
producing smaller plants, which is particularly important for tall genotypes. 18
Internode length in pea is determined at least by five major loci: Le, La, Cry, Na, 19
Lm (Reid et al., 1983). GA1 is the native GA controlling internode elongation in pea 20
(Ross et al., 1989). Slender phenotypes (la, cry) have long and thin internodes and are 21
not dependent on endogenous GAs. Dwarf plants (La and/or Cry) acquire a similar 22
phenotype to slender types when treated with high concentrations of GA3 (Potts et al., 23
1985). A major gene (Le/le) for internode length is mainly responsible for tall climbing 24
108
(Le/-) versus dwarf bush (le/le) habit (Potts et al., 1982). Application of GA1 can mask 1
the Le/le gene difference. Le plants respond equally to GA20 and GA1, while le plants 2
respond only weakly to GA20, the major biologically active gibberellin found in dwarf 3
peas. These results suggest that the Le gene controls the production of a 3β-4
hydroxylase capable of converting GA20 to GA1 (Ingram et al., 1983). 5
As evoked above, gibberellins are a large group of compounds which share a 6
common gibbane ring structure, and which all have at least some physiological activity 7
(Cleland, 1999). They include both precursors and catabolites and thus, even if GA1 is 8
the main active GA in stem elongation (Ingram et al., 1986), other GAs are as active or 9
more active in processes including, among others, tendril and pod growth in pea 10
(Smith et al., 1992). Thus, while the biosynthesis of GA20 takes place in unfolded leaves 11
and tendrils, its conversion to GA1 occurs in the upper stem (Smith, 1992). The 12
paramount role of GAs on leaf expansion has been proven in cereals (Nelissen et al., 13
2012) and is also known in pea (Hedden, 1999). In this context, the differential 14
responses observed between the different tissues tested and also between the 15
conventional (Frisson and Victor) and semi-leafless (Bundi and Excell) cultivars might 16
be attributed to a differential endogenous content of active GAs within the tissues in 17
conjunction with the antigibberellin effect of the exogenously applied Flurprimidol. 18
For long day plants, a red to far-red ratio close to the natural level of around 1 19
is the most effective for flower induction (Vince-Prue, 1981). Most growth chamber 20
light sources have high red: far-red ratios (greater than 2) which can delay flowering in 21
photoperiodic sensitive species and inhibit internode extension (Whitman et al., 1998; 22
Runkle and Heins, 2001; Cummings et al., 2007). In our experiments, the addition of 23
red or far-red light sources did not reduce the flowering time in vitro in early, mid or 24
109
late flowering pea genotypes. The addition of far-red filtered light probably did not 1
correct the red: far-red ratio sufficiently as to mimic natural light, as was discussed by 2
Runkle and Heins (2001) and Cummings et al., (2007). Also, the light intensity applied 3
may not have been high enough to induce fast in vitro flowering in the genotypes 4
studied (Fujioka et al., 1999). It was then concluded that white fluorescent light was 5
the best of the light sources tested in the in vitro flowering protocol presented here. 6
Growth rates, and many of the physiological characteristics of plants developed 7
in vitro, are influenced by the physical and chemical environment of the culture 8
vessels (Walker et al., 1988; Jackson et al., 1991; Kozai et al., 1992; Majada et al., 9
1997). The type of culture vessel and its closure (use of gas-permeable membranes or 10
lids) influences or alters the in vitro environment by modifying the gas composition. 11
When comparing different culture vessels, we observed a higher percentage of seed-12
setting plants when culturing in tubes covered with polypropylene caps compared to 13
containers sealed with screw caps covered in a breathable membrane. This is likely to 14
be associated with better air exchange (reduced levels of ethylene and CO2 15
concentration) of glass tubes compared to polycarbonate containers, as reported by 16
Jackson et al. (1991) and Lentini et al. (1988). Interestingly, we observed that plants 17
grown in tubes produced a lower number of flowers compared to plants grown in 18
containers, and that this was true irrespectively of their flowering or leaf type, as the 19
early, leafy type cv. Frisson gave the same responses as the early/ mid, semi-leafless 20
type cv. Excell (Fig. 4.3). This may be related to the smaller amount of culture medium 21
used in tubes compared to containers, which may affect the partition of nutrients in 22
the plant, thus affecting the production of flowers, as observed previously with other 23
species (Figueira and Janick, 1994; George et al., 2009). Therefore, tubes were 24
110
considered the best culture vessel in the in vitro flowering protocol proposed in this 1
work, as the reduced number of flowers per plant produced was also associated with 2
a lesser early pod abortion, maybe due to a reduced competition between flowers. 3
Efficient breeding methods are needed to advance hybrid populations and to 4
facilitate selection of lines with desirable combinations of characters (Haddad and 5
Muehlbauer, 1981). The SSD method consists of taking a single seed from each F2 6
plant and advancing each seed to the next generation until a desired level of 7
homozygosity is achieved (F6 to F8), thus saving space and time (Goulden, 1939). 8
However, the extent of plant loss from generation to generation affects the genetic 9
makeup of the SSD populations (Martin et al., 1978). We have calculated that with an 10
attrition rate per generation of 10% of the plant population, after four generations of 11
SSD (from F2 to F6) we would end up with only 34% of the initial population. This 12
indicates the importance for the breeder of the robustness and reliability of any tissue 13
culture system used. In the present study, removing the meristem and culturing it 14
separately permits the production of a second cloned plant, which provides a back-up 15
plant in case of loss. This is particularly important when working with rare and 16
valuable genotypes in a population such as the production of recombinant inbred lines 17
(Soller and Beckmann, 1990). 18
The identification of a system to accelerate generation turnover by shortening 19
each cycle is crucial in breeding programmes. In this publication we present a simple 20
and reliable system to reduce generation time in vitro across a range of pea 21
genotypes. In this protocol Flurprimidol is used to control plant size in vitro, and plants 22
with the meristem removed and shoot tip explants are cultured into glass tubes under 23
cool-white fluorescent light. More than five generations per year can be obtained with 24
111
mid to late flowering genotypes using this protocol and over six generations per year 1
for early to mid flowering genotypes. However, some late/very late flowering 2
genotypes like cv. Kaspa and the landrace accession 00P016-1 remain recalcitrant to 3
this technology. The presented data provides the possibility to answer more basic 4
questions related to in vitro plant growth and development via the exploration of the 5
endogenous changes in phytohormone levels in genetically diverse peas after 6
Flurprimidol application. 7
8
9
10
11
12
13
14
15
16
17
18
19
20
112
4.5 References 1
Asawaphan P, Mangkita W, Kachonpadungkitti Y, Matsuyama S, Satake T, Hisajima S (2005) 2 Efficient flower induction from peanut (Arachis hypogaea L.) seedling in vitro. SABRAO 3 J. Breed. Genet. 37: 131-140 4
Ausín I, Alonso-Blanco C, Martinez-Zapater JM (2005) Environmental regulation of flowering. 5 Int. J. Dev. Biol. 49: 689-705 6
Burton AL, Pennisi SV, van Iersel MW (2007) Morphology and postharvest performance of 7 Geogenanthus undatus C. Koch & Linden ‘Inca’after application of ancymidol or 8 flurprimidol. HortScience 42: 544-549 9
Cerdan PD, Chory J (2003) Regulation of flowering time by light quality. Nature 423: 881-885 10 Ceunen S, Geuns JMC (2013) Glucose, sucrose, and steviol glycoside accumulation in Stevia 11
rebaudiana grown under different photoperiods. Biol. Plant. 57: 390-394 12 Chase SS (1952) Production of homozygous diploids of maize from monoploids. Agron. J. 44: 13
263-267 14 Chengalrayan K, Mhaske VB, Hazra S (1995) In vitro regulation of morphogenesis in peanut 15
(Arachis hypogaea L.). Plant Sci. 110: 259-268 16 Cleland RE (1999) Introduction: Nature, ocurrence and functioning of plant hormones. In PJJ 17
Hooykaas, MA Hall, KR Libbenga, eds, Biochemistry and Molecular Biology of Plant 18 Hormones. Elsevier, Amsterdam, The Netherland, pp 3-22 19
Croser JS, Lülsdorf MM, Davies PA, Clarke HJ, Bayliss KL, Mallikarjuna N, Siddique KHM 20 (2006) Toward doubled haploid production in the Fabaceae: Progress, constraints, 21 and opportunities. Crit. Rev. Plant Sci. 25: 139-157 22
Cummings IG, Reid JB, Koutoulis A (2007) Red to far-red ratio correction in plant growth 23 chambers – growth responses and influence of thermal load on garden pea. Physiol. 24 Plant. 131: 171-179 25
Figueira A, Janick J (1994) Optimizing carbon dioxide and light levels during in vitro culture of 26 Theobroma cacao. J. Amer. Soc. Hort. Sci. 119: 865-871 27
Franklin G, Pius PK, Ignacimuthu S (2000) Factors affecting in vitro flowering and fruiting of 28 green pea (Pisum sativum L.). Euphytica 115: 65-74 29
Fujioka T, Fujita M, Miyamoto Y (1999) In vitro flowering and pod setting of non-symbiotically 30 germinated pea. J. Japan. Soc. Hort. Sci. 68: 117-123 31
Gamborg OL, Miller RA, Ojima K (1968) Nutrient requirements of suspension cultures of 32 soybean root cells. Exp. Cell Res. 50: 151-158 33
George EF, Hall MA, De Klerk GJ (2009) The components of plant tissue culture media II: 34 Organic additions, osmotic and pH effects. In EF George, MA Hall, GJ De Klerk, eds, 35 Plant Propagation by Tissue Culture, 3rd eddition. Springer, Berlin, pp 115-173 36
Goulden CH (1939) Problems in plant selection. In RC Burnett, ed, Proc. 7th Int. Genet. Cong. 37 Cambridge University Press, England, pp 132-133 38
Haddad NI, Muehlbauer FJ (1981) Comparison of random bulk population and single-seed-39 descent methods for lentil breeding. Euphytica 30: 643-651 40
Hamid MM, Williams RR (1997) Effect of different types and concentrations of plant growth 41 retardants on Sturt's desert pea (Swainsona formosa). Scientia Hortic. 71: 79-85 42
Hedden P (1999) Recent advances in gibberellin biosynthesis. J. Exp. Bot. 50: 553-563 43 Ingram TJ, Reid JB, MacMillan J (1986) The quantitative relationship between gibberellin A1 44
and internode elongation in Pisum sativum L. Planta 168: 414-420 45 Ingram TJ, Reid JB, Potts WC, Murfet C (1983) Internode length in Pisum. IV. The effect of the 46
Le gene on gibberellin metabolism. Physiol. Plant. 59: 607-616 47 Jackson MB, Abbot AJ, Belcher AR, Hall KC, Butler R, Cameron J (1991) Ventilation in plant 48
tissue cultures and effects of poor aeration on ethylene and carbon dioxide 49 accumulation, oxygen depletion and explant development. Ann. Bot. 67: 229-237 50
Kasha KJ, Maluszynski M (2003) Production of doubled haploids in crop plants. An 51 introduction. In M Maluszynski, KJ Kasha, BP Forster, I Szarejko, eds, Doubled Haploid 52
113
Production in Crop Plants. A Manual. Kluwer Acad. Publ., Dordrecht, The Netherlands, 1 pp 1-4 2
Kozai T, Fujiwara K, Hayashi M, Aitken-Christie J (1992) The in vitro environment and its 3 control in micropropagation. In K Kurata, T Kozai, eds, Transplant Production Systems. 4 Kluwer Acad. Publ., Dordrecht, The Netherlands, pp 247-282 5
Lentini Z, Mussell H, Mutschler MA, Earle ED (1988) Ethylene generation and reversal of 6 ethylene effects during development in vitro of rapid-cycling Brassica campestris L. 7 Plant Sci. 54: 75-81 8
Majada JP, Fal MA, Sanchez-Tames R (1997) The effect of ventilation rate on proliferation and 9 hyperhydricity of Dianthus caryophyllus L. In Vitro Cell. Dev. Biol. Plant 33: 62-69 10
Martin RJ, Wilcox JR, Laviolette FA (1978) Variability in soybean progenies developed by 11 single seed descent at two plant populations. Crop Sci. 18: 359-362 12
Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco 13 tissue cultures. Physiol. Plant. 15: 473-497 14
Narasimhulu SB, Reddy GM (1984) In vitro flowering and pod formation from cotyledons of 15 groundnut (Arachis hypogaea L.). Theor. Appl. Genet. 69: 87-89 16
Nelissen H, Rymen B, Jikumaru Y, Demuynck K, Van Lijsebettens M, Kamiya Y, Inze D, 17 Beemster GTS (2012) A local maximum in gibberellin levels regulates maize leaf 18 growth by spatial control of cell division. Curr. Biol. 22: 1183-1187 19
Nelson MN, Berger JD, Erskine W (2010) Flowering time control in annual legumes: Prospects 20 in a changing global climate. CAB Rev: Perspect. Agric. Vet. Sci. Nutr. Nat. Resour. 5: 21 49-62 22
Ochatt S, Pech C, Grewal R, Conreux C, Lülsdorf M, Jacas L (2009) Abiotic stress enhances 23 androgenesis from isolated microspores of some legume species (Fabaceae). J. Plant 24 Physiol. 166: 1314-1328 25
Ochatt SJ, Pontecaille C, Rancillac M (2000) The growth regulators used for bud regeneration 26 and shoot rooting affect the competence for flowering and seed set in regenerated 27 plants of protein peas. In Vitro Cell. Dev. Biol.: Plant 36: 188-193 28
Ochatt SJ, Sangwan RS (2008) In vitro shortening of generation time in Arabidopsis thaliana. 29 Plant Cell Tissue Organ Cult. 93: 133-137 30
Ochatt SJ, Sangwan RS (2010) In vitro flowering and seed set: Acceleration of generation 31 cycles. In MR Davey, P Anthony, eds, Plant Cell Culture: Essential Methods. John Wiley 32 & Sons, Ltd., Chichester, UK, pp 97-110 33
Ochatt SJ, Sangwan RS, Marget P, Ndong YA, Rancillac M, Perney P (2002) New approaches 34 towards the shortening of generation cycles for faster breeding of protein legumes. 35 Plant Breed. 121: 436-440 36
Pobudkiewicz A, Treder J (2006) Effects of flurprimidol and daminozide on growth and 37 flowering of oriental lily ‘Mona Lisa’. Scientia Hortic. 110: 328-333 38
Potts WC, Reid JB, Murfet C (1982) Internode length in Pisum. I. The effect of the Le/le gene 39 difference on endogenous gibberellin-like substances. Physiol. Plant. 55: 323-328 40
Potts WC, Reid JB, Murfet IC (1985) Internode length in Pisum. Gibberellins and the slender 41 phenotype. Physiol. Plant. 63: 357-364 42
Rademacher W (2000) Growth retardants: effects on gibberellin biosynthesis and other 43 metabolic pathways. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 501-531 44
Reid JB, Murfet IC (1977) Flowering in Pisum: the effect of light quality on the genotype If e Sn 45 Hr. J. Exp. Bot. 28: 1357-1364 46
Reid JB, Murfet IC, Potts WC (1983) Internode length in Pisum. Additional information on the 47 relationship and action of loci Le, La, Cry, Na and Lm. J. Exp. Bot. 34: 349-364 48
Reid JB, Murfet IC, Singer SR, Weller JL, Taylor SA (1996) Physiological-genetics of flowering 49 in Pisum. Semin. Cell Dev. Biol. 7: 455-463 50
Ross JJ, Reid JB, Gaskin P, MacMillan J (1989) Internode length in Pisum. Estimation of GA1 51 levels in genotypes Le, le and led. Physiol. Plant. 76: 173-176 52
114
Runkle ES, Heins RD (2001) Specific functions of red, far red, and blue light in flowering and 1 stem extension of long-day plants. J. Am. Soc. Hort. Sci. 126: 275-282 2
Smith VA (1992) Gibberellin A1 biosynthesis in Pisum sativum L. II. Biological and biochemical 3 consequences of the le mutation. Plant Physiol. 99: 372-377 4
Smith VA, Knatt CJ, Gaskin P, Reid JB (1992) The distribution of gibberellins in vegetative 5 tissues of Pisum sativum L. I. Biological and biochemical consequences of the le 6 mutation. Plant Physiol. 99: 368-371 7
Soller M, Beckmann JS (1990) Marker-based mapping of quantitative trait loci using replicated 8 progenies. Theor. Appl. Genet. 80: 205-208 9
Vince-Prue D (1981) Daylength and photoperiodism. In H Smith, ed, Plants and the daylight 10 spectrum. Academic Press Inc., London, UK, pp 223-242 11
Walker PN, Heuser CW, Heinemann PH (1988) Micropropagation: studies of gaseous 12 environments. Acta Hortic. 230: 145-151 13
Wang WY, Xu J, Liu XJ, Yu Y, Ge Q (2012) Cadmium induces early flowering in Arabidopsis. 14 Biol. Plant. 56: 117-120 15
Weller JL, Reid JB, Taylor SA, Murfet C (1997) The genetic control of flowering in pea. Trends 16 Plant Sci. 2: 412-418 17
Whitman CM, Heins RD, Cameron AC, Carlson WH (1998) Lamp type and irradiance level for 18 daylight extensions influence flowering of Campanula carpatica 'Blue clips', Coreopsis 19 grandiflora 'Early Sunrise', and Coreopsis verticillata 'Moonbeam' J. Am. Soc. Hortic. 20 Sci. 123: 802-807 21
Zhang T (2007) In vitro flowering of Perilla frutescens. In Vitro Cell. Dev. Biol.: Plant 43: 91-94 22 Zhou B, Li N, Zhang Z, Huang X, Chen H, Hu Z, Pang X, Liu W, Lu Y (2012) Hydrogen peroxide 23
and nitric oxide promote reproductive growth in Litchi chinensis. Biol. Plant. 56: 321-24 329 25
26
27
28
29
30
31
32
33
34
35
36
37
38
115
Chapter 5
General discussion and future directions
In plant breeding, the development of varieties with resistance to abiotic and
biotic stress is a very costly and time consuming process. In order to accelerate the
genetic progress it is essential to provide plant breeders with the broadest variety of
biotechnological tools. Two in vitro technologies have been proposed for the rapid
achievement of homozygosity and thus acceleration of the breeding process in many
economically important crops: a) Doubled haploidy, and b) an in vitro-based modified
single-seed-descent (SSD) system. In pea (Pisum sativum L.), neither of these
technologies is routinely used in a breeding program. The aim of this study was to
accelerate the breeding process in pea by developing in vitro techniques to rapidly
achieve a high level of homozygosity and to better understand the fundamental
mechanisms involved in these processes.
5.1 Doubled haplody
Despite the significant effort undertaken in this area, to date there is no
universal doubled haploid (DH) protocol effective in every species and success remains
highly genotype-dependent. Leguminous species, including pea, have been described
as recalcitrant to this technology (Croser et al., 2006; Dita et al., 2006; Lülsdorf et al.,
2011). Since the first DH studies in pea undertaken by Gupta et al. (1972) many
researchers have worked toward a robust DH protocol with very limited success.
Recently, Ochatt et al. (2009) obtained a few haploid pea plantlets confirmed by flow
cytometry. However, these plantlets were weak and did not survive glasshouse
transfer. It is notable that after more than forty years of international research effort,
no robust DH protocol has been developed in pea or any other legume species and
116
little is known about the fundamental mechanisms involved in androgenesis
elicitation.
In the first part of this research thesis (Chapter 2), a number of key factors
known to be involved in androgenesis elicitation were identified and optimised in
order to develop a robust and efficient anther culture protocol for pea. In this study,
the combined application of multiple stress treatments, including the novel stress
agent sonication, applied at the appropriate time (uni-nucleate microspore
developmental stage) and the optimisation of key culture factors (i.e. light intensity
and use of plant growth regulators) led to the development of an efficient protocol for
the routine induction of androgenesis and advanced-stage embryo production from
extracted anthers of genetically diverse pea genotypes.
The requirement of hormones for the induction of haploid divisions in pea is still
unclear. Ochatt et al. (2009) indicated that the hormonal composition of the basal
medium had little influence on either initial or further proliferation of isolated
microspore culture for pea. In contrast, Bobkov (2010) obtained callus production
from extracted pea anthers by culturing in medium supplemented with 2,4-D. The
flow cytometry analysis presented in Chapter 3 showed that the application of
multiple stress treatments, with no exogenous hormones, permitted the induction of
androgenesis from cultured anthers of pea. From these results it can be inferred that
stress is indeed the trigger diverting microspores from their normal development in
pea and that hormones are not required for the induction of androgenesis from
cultured anthers. In the present research, the addition of hormones to the culture
medium promoted callus initiation after stress induction of microspores.
117
It was clear from this study that it was the combined application of multiple
stress treatments, including a cold pre-treatment, electroporation, centrifugation,
osmotic shock and the novel stress sonication, which triggered androgenesis
elicitation from cultured anthers of pea. The benefit of pyramiding multiple stress
treatments on the induction of haploid divisions has also been reported in isolated
microspore culture for pea (Ochatt et al., 2009) and anther culture for chickpea (Cicer
arietinum L.) (Grewal et al., 2009). Little is known about the exact mechanisms
underlying elicitation of androgenesis by stress treatments. While many attempts
have been made to explain the possible effects of some of these stress factors
(reviewed by Shariatpanahi et al., 2006) it has been difficult to evaluate the effect of
these stress treatments on microspore DNA synthesis. Thus, in Chapter 3, a flow
cytometry study was undertaken to further understand the effect of individual and
combined stress treatments on androgenesis elicitation. Analysis of the flow
cytometry results revealed clear differences in the relative nuclear DNA content of
microspores within anthers after stress treatments. Based on these results, stress
treatments could, for the first time, be differentiated between those that elicit
androgenesis (cold, electroporation and osmolarity) and those that enhance DNA
synthesis following elicitation (centrifugation and sonication). The publication of this
finding has established flow cytometry as a method to quickly assess the effect of
individual and combined stress treatments, based on the relative nuclear DNA content
(Ribalta et al., 2012). This technique will be particularly useful in assisting researchers
to rapidly identify effective stress treatments during DH protocol development, not
only for legumes but also for other plant species.
118
On the basis of the research presented in Chapters 2 and 3, the following
protocol for the elicitation of androgenesis and haploid embryo production from
extracted anthers of pea is proposed:
Harvest buds 6-7 mm in length (when microspores are at the uni-nucleate stage).
Microscopic analysis of stage of microsporogenesis is recommended when
working with new genotypes.
Apply a cold pre-treatment to buds in the dark at 4°C for a minimum of 48 h (but
no longer than 14 d).
Sterilise buds for 1 min in 70% v/v ethanol, followed by 10 min 1% v/v in
commercial sodium hypochlorite. Rinse buds three times in sterile water.
Remove anthers from buds, ensuring complete removal of filament and apply the
following stress treatments prior to culture: electroporation (three successive
pulses at 10 s intervals of 1000-1500 V/cm with a capacitance of 100 µF and a
resistance of 50 Ω), centrifugation (170 g for 10 min at 4°C, brakes off) and
sonication (38 kHz for 30 s at room temperature).
Place 10 anthers per treatment in a 10 x 35 mm Petri dish containing 2 ml of liquid
medium containing 17% (w/v) sucrose and incubate at 24°C in the dark. After 1
week replace culture medium with fresh medium containing 10% (w/v) sucrose.
After 4 weeks of culture in the dark transferred calli to the Loiseau et al. (1995)
media sequence for embryo germination and maturation.
5.1.1 Future directions
In this research, key factors involved in haploid embryogenesis were identified
and optimised in pea. However, additional research is still required before this
technology will be routinely available within a pea breeding program. Further
119
improvement in the development of DH protocols and the expansion to recalcitrant
species can be expected with a more thorough understanding of the fundamental
mechanisms involved in androgenesis. Doubled haploid research still lacks a legume
model species that would enable us to gain a better fundamental understanding of
the haploid embryogenesis process in legumes and lead to improved protocols.
Despite these limitations, in recent times regeneration via overexpression of
genes, such as WUSCHEL (WUS) or LEAFY COTYLEDON (LEC), in Arabidopsis thaliana
(L.) Heynh. has started to provide a basis for understanding the genes involved in
somatic embryogenesis (Rose and Nolan, 2006). The LEC genes LEC1, LEC2 as well as
FUSCA3, have major effects on embryo development (Harada, 2001). LEC transcription
factors establish environments that promote cellular processes characteristic of the
maturation phase and the initiation of somatic embryogenesis formation (Braybrook
and Harada, 2008). Ectopic expression of LEC1 and LEC2 induces somatic
embryogenesis in the absence of exogenous auxin or stress treatments (Lotan et al.,
1998; Stone et al., 2001), while the ectopic expression of FUSCA3 results in elevated
abscisic acid (ABA) levels (Braybrook and Harada, 2008). Interestingly, the presence or
absence of these two groups of growth regulators was recently studied to unravel the
progression of mitosis and the onset of endoreduplication (indicative of the transition
from the cell division to the storage product accumulation phase) in immature seeds
and excised embryos of Medicago truncatula Gaernt. (Ochatt, 2011; Atif et al., 2013).
These studies showed that the establishment of such transition and the onset of
storage product accumulation were required for early embryo development and
subsequent germination. The ectopic expression of the transcription factors WUS and
BABY BOOM (BBM) have also been implicated in the induction of somatic embryos in
120
Arabidopsis (Rose and Nolan, 2006). Future research should concentrate on the
identification and isolation of selected candidate genes involved in somatic
embryogenesis in model species like A. thaliana and M. truncatula during the
acquisition of androgenesis competence by microspores. These studies will provide
valuable information on how the androgenesis process is controlled in different
species and under different culture conditions.
There is now a growing body of evidence that it is the application of multiple
stress agents that will overcome recalcitrance in legumes to androgenic induction. As
discussed in Chapter 2, this is likely to be due to the increasing hormone levels in
stressed anthers. Androgenesis has been shown to be mediated through ABA levels in
a number of species (Imamura and Harada, 1980; Van Berger et al., 1999; Wang et al.,
2000; Żur et al., 2008); however, other hormones, such as gibberellins, auxins and
cytokinins, have also been associated (Brugière et al., 2003; Maraschin et al., 2005).
Recently, Lülsdorf et al. (2012) showed a possible link between androgenesis induction
and auxin levels, particularly IAA-asparagine (IAA-Asp), in stress-treated anthers of pea
and chickpea. More research is now required to further elucidate the effect of
endogenous hormone levels on androgenesis elicitation. The improved knowledge on
the role of endogenous hormones on the induction of haploid divisions, together with
a more thorough understanding of the genetic basis of androgenesis will have a
significant impact in the improvement of available DH protocols.
Success in the development of DH technology has been proportional to the
number of laboratories involved and the availability of research funding. In the most
frequently studied species, progress in DH protocol development has been the result
of the sustained, combined effort across research institutes worldwide. In comparison,
121
a relatively small number of research groups have undertaken DH research in
legumes. Further progress in DH protocol development in legume species will require
collaboration among scientists from different research institutes. It is reasonable to
expect that given enough resources robust DH protocols will be developed for non-
amenable species such as the large-seeded legumes.
Although significant progress has been achieved over the past few years in the
development of DH protocols for the legumes pea, chickpea and Lathyrus, a more
thorough understanding of the fundamental mechanisms involved in microspore
embryogenesis is still required. The advent of modern biotechnological tools for high-
throughput functional genomics will greatly facilitate the identification and isolation
of genes involved in androgenesis elicitation. A more comprehensive understanding of
the genetic basis of androgenesis together with improved in vitro culture techniques
will facilitate the development of more efficient DH protocols across a range of
species, particularly for intractable species such as the large-seeded legumes.
5.2 In vitro based Single-Seed-Descent
In vitro based modified single-seed-descent (SSD) systems have been proposed
as an effective method to accelerate generation turnover across a number of species
(Franklin et al., 2000; Ochatt et al., 2002; Asawaphan et al., 2005; Zhang, 2007; Ochatt
and Sangwan, 2008). In pea, only three in vitro flowering protocols have been
published (Fujioka et al., 1999; Franklin et al., 2000; Ochatt et al., 2002) and these
have been developed for a limited number of early-flowering cultivars. While a
considerable number of publications exist in the area of in vitro SSD and proof of
concept has been established across a number of species, there is no evidence of this
122
technology being integrated into a breeding program. This is likely due to genotype-
specificity, but this is not recorded in the existing publications.
As stated earlier, one of the aims of this research was to develop a robust
protocol that would enable the integration of this technology on a broader scale
within a pea improvement program. In Chapter 4, an optimised in vitro based SSD
system was developed which enabled rapid in vitro flowering and seed-set across a
range of pea genotypes including, for the first time, mid to late flowering types
(Ribalta et al., 2014). In the proposed protocol, the antigibberellin Flurprimidol was
used to control in vitro plant size, and plants with the meristem removed or excised
shoot-tip explants were cultured into glass tubes under white fluorescent light. With
this strategy more than five generations per year were obtained with mid to late
flowering genotypes and over six generations per year for early to mid flowering
genotypes.
The use of the antigibberellin Flurprimidol to control plant growth under
glasshouse conditions has been previously reported in a number of species (Hamid
and Williams, 1997; Pobudkiewicz and Treder, 2006; Burton et al., 2007), including
pea (Ochatt et al., 2002). In Chapter 4, the use of Flurprimidol in an in vitro flowering
protocol is reported for the first time (Ribalta et al., 2014). In the present study, the
addition of Flurprimidol to the culture medium resulted in the smaller plants required
for in vitro growth, thus permitting in vitro flowering across a range of pea cultivars.
This is particularly important for in vitro culture of tall genotypes. Interestingly,
differential responses were observed between the different tissues tested (intact
plants, plants with the meristem removed and shoot tip explants) and also between
the conventional (Frisson and Victor) and semi-leafless (Bundi and Excell) cultivars.
123
These differences were attributed to the variation in endogenous content of active
GAs within the tissues in combination with the antigibberellin effect of the
exogenously applied Flurprimidol.
The effect of environmental factors such as light quality, photoperiod, and
growth temperature, on the transition to flowering has been extensively explored
(Reid et al., 1996; Cerdan and Chory, 2003; Ausín et al., 2005; Nelson et al., 2010).
While the effect of most of these factors is fairly well understood, the effect of light
quality on flower initiation in pea is still unclear (Runkle and Heins, 2001; Cummings et
al., 2007). In the present study, the hypothesis that the addition of far-red filtered
light would help reduce the red: far-red ratio and thus stimulate in vitro flowering and
seed development in pea was rejected. The addition of far-red filtered light probably
did not correct the red: far-red ratio sufficiently to mimic natural light, as discussed by
Runkle and Heins (2001) and Cummings et al. (2007). It was then concluded that white
fluorescent light was the best of the light sources tested in the proposed in vitro
flowering protocol.
Other key findings from the present study were the important effect of the type
of culture vessel (glass tubes vs. plastic containers) and its closure (use of gas-
permeable membrane or lids) on in vitro plant growth and development, likely
associated with better gas exchange in glass tubes compared to containers as
reported by Jackson et al. (1991) and Lentini et al. (1988). In this study, a higher
percentage of seed-setting plants were observed when culturing in tubes covered with
polypropylene lids compared to containers sealed with screw caps covered in a
breathable membrane. Interestingly, a lower number of flowers were observed in
plants grown in tubes compared to those grown in containers, probably due to the
124
reduced amount of culture medium in tubes. This decrease in flower number was
linked to lower early pod abortion due to reduced competition between flowers. From
these results, glass tubes were considered the best culture vessel in the proposed in
vitro flowering protocol irrespective of the pea genotype studied.
The reliability of any tissue culture system is one of main requirements for the
efficient application of this technology in a plant breeding program. A key aspect of
the in vitro flowering protocol proposed herein is the removal and separate culture of
the meristem of each plant, permitting the production of a second cloned plant and
the provision of a ‘back-up’ plant in case of loss. This ‘back-up system’ has not been
proposed previously and will be of particular importance when working with rare and
valuable genotypes and when using the protocols to rapidly produce a population of
recombinant inbred lines.
5.2.1 Future directions
Some late and very-late flowering genotypes of pea remain unresponsive to
the in vitro flowering technology and further research is required to expand this
protocol to these genotypes. A combined in vivo/ in vitro strategy has been used in
legumes like pea and bambara groundnut (Vigna subterranea L.) (Ochatt et al., 2002),
as well as in A. thaliana (Ochatt and Sangwan, 2008) to reduce the length of each
generation cycle. This approach consists of alternating a first step in vitro for
germination and a second step in vivo for full development. In the early flowering pea
cv. Frisson more than five generations per year have been obtained with this approach
(Ochatt et al., 2002). For late and very late flowering pea genotypes, the development
of a combined in vivo/ in vitro strategy may offer a valuable alternative for the
shortening of generation cycle.
125
In vitro selection has been used as a tool for the screening of a number of
different abiotic stresses across a range of species (Samantaray et al., 1999; Zair et al.,
2003; Flowers, 2004). However, this technology has not been adopted on a broad
scale to legumes due to their general difficulty to culture in vitro (Dita et al., 2006).
The in vitro flowering technology developed in this study offers the capacity to add an
in vitro screen for key abiotic stresses into the culture protocol. For example, in pea
boron toxicity is an important disorder that can limit plant growth (Bagheri et al.,
1992). With the in vitro flowering technology a screening step for boron tolerance
could be incorporated into the protocol at the immature embryo stage leading to an
efficient integration of tolerant/ resistant germplasm into the breeding program of
pea. The development of novel in vitro breeding tools for accelerating legume crop
improvement will permit a timely response to emerging biotic and abiotic threats.
Another potential application for the in vitro flowering strategy is the
development of genetically modified organisms (GMOs) for the fixation of transgenes
in a riskless in vitro environment. Further, it can be applied in marker-assisted
selection in breeding programs. Additionally, the in vitro flowering study presented
herein provides the possibility to answer fundamental questions related to in vitro
plant growth and development via the exploration of the endogenous changes in
phytohormone levels under different growth conditions. Thus, the in vitro flowering
technology is a fascinating area of research and has potential not only for the rapid
fixation of new traits, but also for fundamental studies on flower regulation, mineral
nutrition, hormonal manipulation, plant physiology, plant transformation and
functional genomics.
126
5.3 Final conclusions
The potential of doubled haploidy in plant breeding is clearly evident. Doubled
haploid systems are broadly applied in plant breeding, genetic mapping, mutation and
transformation studies and in many areas of fundamental biology in a number of
species, particularly Brassica spp. and cereals. However, legumes are still considered
unresponsive to this technology. The broad application of the DH technology in
legumes species will require the development of robust, genotype-independent
protocols. In this research, key factors involved in androgenesis elicitation were
identified and optimised in order to develop a reliable pea anther culture protocol.
The application of multiple stress treatments, including the novel stress sonication and
the optimisation of culture conditions, led to the routine induction of haploid division
and the production of advanced-stage embryos from cultured anthers of pea. A flow
cytometry study permitted, for the first time, the assessment and differentiation of
the effect of stress on the elicitation of androgenesis based on relative nuclear DNA
content of microspores. The results presented in this research form a solid platform
for further efforts designed to enhance androgenic response in pea and to extend this
technology to other legumes.
In fulfilment of the second objective of this research, an optimised in vitro based
SSD system was developed which enabled rapid in vitro flowering and seed-set across
a range of pea genotypes including, for the first time, mid to late-flowering types. This
research resulted in a strategy to obtain more than five generations per year with mid
to late-flowering genotypes and over six generations per year for early to mid-
flowering genotypes. In the absence of a robust DH protocol, the in vitro based SSD
system reported herein will offer a valuable alternative method for the rapid
127
achievement of homozygosity by shortening each generation cycle. In addition, this
technology has excellent potential as a breeding tool for the screening of key abiotic
stresses in the development of tolerant/ resistant germplasm; as well as in
transformation studies and fundamental studies related to in vitro plant growth and
development. The studies undertaken within this thesis have contributed valuable
knowledge to the doubled haploidy and in vitro flowering research areas and are
expected to have a significant impact on the acceleration of the breeding process in
pea.
128
5.4 References
Asawaphan P, Mangkita W, Kachonpadungkitti Y, Matsuyama S, Satake T, Hisajima S (2005) Efficient flower induction from peanut (Arachis hypogaea L.) seedling in vitro. SABRAO J. Breed. Genet. 37: 131-140
Atif RM, Boulisset F, Conreux C, Thompson R, Ochatt SJ (2013) In vitro auxin treatment promotes cell division and delays endoreduplication in developing seeds of the model legume species Medicago truncatula. Physiol. Plant.: Published online DOI 10.1111/j.1399-3054.2012.01719.x
Ausín I, Alonso-Blanco C, Martinez-Zapater JM (2005) Environmental regulation of flowering. Int. J. Dev. Biol. 49: 689-705
Bagheri A, Paull JG, Rathjen AJ, Ali SM, Moody DB (1992) Genetic variation in the response of pea (Pisum sativum L.) to high soil concentrations of boron. Plant Soil 146: 261-269
Bobkov SV (2010) Isolated pea anther culture. Russian Agric. Sci. 36: 413-416 Braybrook SA, Harada JJ (2008) LECs go crazy in embryo development. Trends Plant Sci. 13:
624-630 Brugière N, Jiao S, Hantke S, Zinselmeier C, Roessler JA, Niu X, Jones RJ, Habben JE (2003)
Cytokinin oxidase gene expression in maize is localized to the vasculature, and is induced by cytokinins, abscisic acid, and abiotic stress. Plant Physiol. 132: 1228-1240
Burton AL, Pennisi SV, van Iersel MW (2007) Morphology and postharvest performance of Geogenanthus undatus C. Koch & Linden ‘Inca’after application of ancymidol or flurprimidol. HortScience 42: 544-549
Cerdan PD, Chory J (2003) Regulation of flowering time by light quality. Nature 423: 881-885 Croser JS, Lülsdorf MM, Davies PA, Clarke HJ, Bayliss KL, Mallikarjuna N, Siddique KHM
(2006) Toward doubled haploid production in the Fabaceae: Progress, constraints, and opportunities. Crit. Rev. Plant Sci. 25: 139-157
Cummings IG, Reid JB, Koutoulis A (2007) Red to far-red ratio correction in plant growth chambers – growth responses and influence of thermal load on garden pea. Physiol. Plant. 131: 171-179
Dita MA, Rispail N, Prats E, Rubiales D, Singh KB (2006) Biotechnology approaches to overcome biotic and abiotic stress constraints in legumes. Euphytica 147: 1-24
Flowers T (2004) Improving crop salt tolerance. J. Exp. Bot. 55: 307-319 Franklin G, Pius PK, Ignacimuthu S (2000) Factors affecting in vitro flowering and fruiting of
green pea (Pisum sativum L.). Euphytica 115: 65-74 Fujioka T, Fujita M, Miyamoto Y (1999) In vitro flowering and pod setting of non-symbiotically
germinated pea. J. Japan. Soc. Hort. Sci. 68: 117-123 Grewal RK, Lülsdorf M, Croser J, Ochatt S, Vandenberg A, Warkentin TD (2009) Doubled-
haploid production in chickpea (Cicer arietinum L.): role of stress treatments. Plant Cell Rep. 28: 1289-1299
Gupta S, Ghosal KK, Gadgil VN (1972) Haploid tissue culture of Triticum aestivum var. Sonalika and Pisum sativum var. B22. Indian Agric. 16: 277-278
Hamid MM, Williams RR (1997) Effect of different types and concentrations of plant growth retardants on Sturt's desert pea (Swainsona formosa). Scientia Hortic. 71: 79-85
Harada JJ (2001) Role of Arabidopsis LEAFY COTYLEDON genes in seed development. J. Plant Physiol. 158: 405-409
Imamura J, Harada H (1980) Effects of abscisic acid and water stress on the embryo and plantlet production in anther culture of Nicotiana tabacum cv. Samsun. Z. Pflanzen-physiol. 100: 285-289
Jackson MB, Abbot AJ, Belcher AR, Hall KC, Butler R, Cameron J (1991) Ventilation in plant tissue cultures and effects of poor aeration on ethylene and carbon dioxide accumulation, oxygen depletion and explant development. Ann. Bot. 67: 229-237
129
Lentini Z, Mussell H, Mutschler MA, Earle ED (1988) Ethylene generation and reversal of ethylene effects during development in vitro of rapid-cycling Brassica campestris L. Plant Sci. 54: 75-81
Loiseau J, Marche C, Deunff Y (1995) Effects of auxins, cytokinins, carbohydrates and amino acids on somatic embryogenesis induction from shoot apices of pea. Plant Cell Tiss. Organ Cult. 41: 267-275
Lotan T, Ohto M-a, Yee KM, West MAL, Lo R, Kwong RW, Yamagishi K, Fischer RL, Goldberg RB, Harada JJ (1998) Arabidopsis LEAFY COTYLEDON1 is sufficient to induce embryo development in vegetative cells. Cell 93: 1195-1205
Lülsdorf M, Yuan HY, Slater S, Vandenberg A, Han X, Zaharia LI (2012) Androgenesis-inducing treatments change phytohormone levels in anthers of three legume species (Fabaceae). Plant Cell Rep. 31: 1255-1267
Lülsdorf MM, Croser JS, Ochatt S (2011) Androgenesis and doubled-haploid production in food legumes. In A Pratap, J Kumar, eds, Biology and Breeding of Food Legumes. CABI Publishing, pp 159-177
Maraschin SF, de Priester W, Spaink HP, Wang M (2005) Androgenic switch: an example of plant embryogenesis from the male gametophyte perspective. J. Exp. Bot. 56: 1711-1726
Nelson MN, Berger JD, Erskine W (2010) Flowering time control in annual legumes: Prospects in a changing global climate. CAB Rev: Perspect. Agric. Vet. Sci. Nutr. Nat. Resour. 5: 49-62
Ochatt S, Pech C, Grewal R, Conreux C, Lülsdorf M, Jacas L (2009) Abiotic stress enhances androgenesis from isolated microspores of some legume species (Fabaceae). J. Plant Physiol. 166: 1314-1328
Ochatt SJ (2011) Immature Seeds and Embryos of Medicago truncatula Cultured In Vitro. In TA Thorpe, EC Yeung, eds, Plant Embryo Culture: Methods and Protocols, Vol 710. Springer Protocols, Berlin, pp 39-52
Ochatt SJ, Sangwan RS (2008) In vitro shortening of generation time in Arabidopsis thaliana. Plant Cell Tiss. Organ Cult. 93: 133-137
Ochatt SJ, Sangwan RS, Marget P, Ndong YA, Rancillac M, Perney P (2002) New approaches towards the shortening of generation cycles for faster breeding of protein legumes. Plant Breed. 121: 436-440
Pobudkiewicz A, Treder J (2006) Effects of flurprimidol and daminozide on growth and flowering of oriental lily ‘Mona Lisa’. Scientia Hortic. 110: 328-333
Reid JB, Murfet IC, Singer SR, Weller JL, Taylor SA (1996) Physiological-genetics of flowering in Pisum. Semin. Cell Dev. Biol. 7: 455-463
Ribalta FM, Croser JS, Erskine W, Finnegan PM, Lülsdorf MM, Ochatt SJ (2014) Antigibberellin-induced reduction of internode length favors in vitro flowering and seed-set in different pea genotypes. Biol. Plant. 58: 39-43
Ribalta FM, Croser JS, Ochatt SJ (2012) Flow cytometry enables identification of sporophytic eliciting stress treatments in gametic cells. J. Plant Physiol. 169: 104-110
Rose R, Nolan K (2006) Invited review: Genetic regulation of somatic embryogenesis with particular reference to Arabidopsis thaliana and Medicago truncatula. In Vitro Cell Dev. Biol. : Plant 42: 473-481
Runkle ES, Heins RD (2001) Specific functions of red, far red, and blue light in flowering and stem extension of long-day plants. J. Amer. Soc. Hort. Sci. 126: 275-282
Samantaray S, Rout GR, Das P (1999) In vitro selection and regeneration of zinc tolerant calli from Setaria italica L. Plant Sci. 143: 201-209
Shariatpanahi ME, Bal U, Heberle-Bors E, Touraev A (2006) Stresses applied for the reprogramming of plant microspores towards in vitro embryogenesis. Physiol Plant. 127: 519-534
130
Stone SL, Kwong LW, Yee KM, Pelletier J, Lepiniec L, Fischer RL, Goldberg RB, Harada JJ (2001) LEAFY COTYLEDON2 encodes a B3 domain transcription factor that induces embryo development. In Proc. Natl. Acad. Sci., USA, Vol 98, pp 11806-11811
Van Berger S, Kottenhagen MJ, Van Der Meulen RM, Wang M (1999) The role of abscisic acid in induction of androgenesis: a comparative study between Hordeum vulgare L. cvs. Igri and Digger J. Plant Growth Regul. 18: 135-143
Wang M, Van Berger S, Van Duijn B (2000) Insights into a key developmental switch and its importance for efficient plant breeding. Plant Physiol. 124: 523-530
Zair I, Chlyah A, Sabounji K, Tittahsen M, Chlyah H (2003) Salt tolerance improvement in some wheat cultivars after application of in vitro selection pressure. Plant Cell Tiss. Organ Cult. 73: 237-244
Zhang T (2007) In vitro flowering of Perilla frutescens. In Vitro Cell. Dev. Biol.: Plant 43: 91-94 Żur I, Dubas E, Golemiec E, Szechyńska-Hebda M, Janowiak F, Wędzony M (2008) Stress-
induced changes important for effective androgenic induction in isolated microspore culture of triticale (× Triticosecale Wittm.). Plant Cell Tiss. Organ Cult. 94: 319-328
131
Appendix 1
Electroporation buffers
Components Ochatt et al., 2009 Rech et al., 1987
Mannitol 0.5M 0.5M MgSO4 6 mM -- MgCl2 6 mM 6 mM MES 5 mM 5 mM pH 5.5 5.8
Culture media composition
Component (mg/l) NLN (Lichter, 1982) HSO (Ochatt et al., 2009) JFK
KH2PO4 125 61 170
KNO3 125 61 2500
NH4NO3 - - -
MgSO4.7H2O 125 250 370
Ca(NO3)2.4H2O 500 500 -
CaCl2.2H2O - - 600
CuSO4.5H2O 0.025 0.025 0.05
CoCl2.6H2O 0.025 0.025 0.025
H3BO3 10 10 6.2
MnSO4.H2O - 18.95 16.91
MnSO4.4H2O 25 - -
NaFeEDTA - 40 -
Na2EDTA 37.26 - -
FeS04.7H2O 27.8 - -
Na2MoO4.2H2O 0.25 0.25 0.25
ZnSO4.7H2O - 8.6 10
ZnSO4.4H2O 10 - -
KI - 0.83 0.83
D+.Biotine 0.05 0.1 -
Folic Acid 0.5 1.0 -
L-Glutamine 800 800 -
L-Glutathion red 30 30 -
Glycine 2 2 -
Myo-Inositol 100 - 100
Meso-Inositol - 500 -
Nicotinic acid 5 1 5
Pyridoxine HCl 0.5 1 0.5
L-Serine 100 100 --
Thiamine HCl 0.5 0.1 0.5
Casein hydrolisate - - 200
pH 5.5 5.5 5.8
Hormones: 2,4-D (1 mg/L); Picloram (0.25 mg/L); BAP (0.1 mg/L)
132
Appendix 2 – Doubled haploid research
Bud sterilisation and microspore isolation protocol:
Flower buds were placed in a 100 ml beaker (100 buds per treatment) and surface
sterilised for 1 min in 70% v/v ethanol, followed by 10 min in calcium hypochlorite (70
gL-1). Buds were rinsed three times with sterile water. Buds were then transferred to a
sterile mortar and macerated with a pestle in 2ml of Rech et al. (1987) electroporation
buffer. The electroporation buffer was used for the maceration of buds as the
electroporation treatment followed the microspore isolation procedure. Three
successive steps of suspension and centrifugation (100 g, 10°C, 5 min each) were used
to eliminate debris remaining after serial sieving through 80, 60 and 40 µm filters
(Ochatt et al., 2009).
Electroporation protocol
Isolated microspores were submitted to the electroporation treatment immediately
after the isolation procedure in order the preserve their viability and to avoid
sedimentation in the bottom of the cuvette
Electroporation cuvettes: The size of the cuvettes is determined by the gap width
between electrodes. Cuvettes with electrodes 2 mm apart (model No. 620) were used
in these experiments.
Role of electroporation buffer: For the application of electroporation treatments, two
primary parameters need to be optimised, field strength and pulse length. Field
strength is measured as voltage delivered across an electrode gap and relates to the
potential difference experienced by the cell membrane in the electric field. When the
induced potential reaches a critical value, a reversible breakdown of the cell
membrane occurs resulting in the creation of temporary pores that allow nutrients
from the culture medium to get into the cells (Zimmermann et al., 1976; Kinosita and
Tsong, 1977), thus improving regeneration competence from microspores (Delaitre et
al., 2001; Ochatt et al., 2009) and protoplasts (Rech et al., 1987; Ochatt et al., 1988) of
various species. The electroporation buffer acts as a membrane protector preventing
the creation of irreversible pores that led to cellular death (Ochatt et al., 1988; Delaitre
et al., 2001; Quecini et al., 2002). Also, the conductivity of the electroporation buffer
affects the value of the pulse length. In general, the more conductive the media is, the
lower the pulse length which should be used.
Flow cytometry analysis
Sample preparation: Nuclei were mechanically isolated by gently chopping tissues (30
anthers per treatment and 20 mg of young leaf tissue for the control) for 30 seconds
with a razor blade in 2 mL of single-step isolation + stain buffer (Partec®) containing
4,6 diamidino-2-phenylindole (DAPI). The suspension was then filtered through a 50
µm nylon mesh and immediately analysed using the flow cytometer.
133
Appendix 3 – In vitro flowering experiments
In vivo Flurprimidol experiments: In order to identify the most effective Flurprimidol
treatment, a 5% w/v solution was applied as a drench on the soil at various
concentrations (0, 25, 50 and 75 cm3).
In vitro Flurprimidol experiments: Dry seeds (30 per treatment) were surface-
sterilised by treatment for 5 min in 70% ethanol, followed by 10 min in sodium
hypochlorite (21 g dm-3). Seeds were rinsed three times for one minute with sterile
deionised water and imbibed overnight. The coats of imbibed seeds were removed
and 10 embryos with both cotyledons intact were cultured in a vessel containing 50
cm3 of B5 salts and vitamins (Gamborg et al., 1968) modified by the addition of 10 mM
NH4Cl (Ochatt et al., 2000) and 40 g dm-3 sucrose. The pH of the medium was 5.6.
Vernalisation experiments: A series of experiments was undertaken in order to assess
the effect of a vernalisation treatment on in vitro flowering in the early/ mid flowering
pea cv. Excell and in the late flowering cv. Kaspa. Seeds were sterilised and imbibed
overnight as reported in section 4.2. The coats of imbibed seeds were removed and 10
embryos with both cotyledons intact were placed in a vessel containing 50 cm3 of
modified B5 medium and submitted to a cold treatment (4°C) for 0, 7, 14 and 21 d in
the dark. After this period, the seeds were transferred to a culture room at 24°C with
a light intensity of 145 µmol m-2 s-1 from cool-white-fluorescent tubes (LIFEMAX TL-D
30W/840, Philips Lighting, Thailand) and a photoperiod of 20/4 h light/dark. In the
present experiments, the vernalisation did not have a significant effect on flowering
time, percentage of flowering plants and number of nodes to first flower in the
studied pea cultivars.
134
Appendix 4
Effect of Flurprimidol concentration at day 40 in glasshouse-grown pea landrace 00P016-1: A - Seasonal effect on internode length (cm) (results are expressed as mean ± SD; n = 30). B - Effect on plant size (all bars = 5 cm).
Effect of light quality and culture vessel type on time to flowering in vitro in cv. Frisson (Cont.: containers; W: white light; W+R: white + red light; W+FR: white + far red light).
0
10
20
30
40
50
60
Tubes Cont. Tubes Cont. Tubes Cont.
Day
s to
flo
wer
ing
W W + R W + FR
Base
Shoots
Intact
135
Appendix 5
With the proposed protocol, in vitro flowering and seed-set was achieved across a range of pea genotypes, including cvs. Frisson and Bundi (early flowering), cv. Excell (early/ mid flowering), cv. Kaspa (late flowering) and the landrace 00P016-1 (very late flowering).
