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IN VITRO DIRECT AND INDIRECT
ORGANOGENESIS AND PLANT REGENERATION OF KENAF (Hibiscus cannabinus L) VAR. KB6
ZETI ERMIENA SURYA BT MAT HUSSIN
MASTER OF SCIENCE
2016
In Vitro Direct And Indirect Organogenesis And Plant
Regeneration Of Kenaf (Hibiscus cannabinus L) Var. KB6
by
Zeti Ermiena Surya Bt Mat Hussin
A thesis submitted in fulfilment of the requirements for the degree of
Master of Science
Faculty of Agro Based Industry
UNIVERSITI MALAYSIA KELANTAN
2016
THESIS DECLARATION
I hereby certify that the work embodied in this thesis is the result of the original
research and has not been submitted for a higher degree to any other University or
Institution.
OPEN ACCESS I agree that my thesis is to be made immediately
available as hardcopy or on-line open access (full text).
EMBARGOES I agree that my thesis is to be made available as
hardcopy or on-line open access (full text) for a period
approved by the Post Graduate Committee.
CONFIDENTIAL (Contains confidential information under the Official
Secret Act 1972)*
RESTRICTED (Contains restricted information as specified by the
organization where research was done)*
I acknowledge that Universiti Malaysia Kelantan reserves the right as follows.
1. The thesis is the property of University Malaysia Kelantan.
2. The library of Universiti Malaysia Kelantan has the right to make copies for the
purpose of research only.
3. The library has the right to make copies of the thesis for academic exchange.
SIGNATURE SIGNATURE OF SUPERVISOR
I/C/PASSPORT NO. NAME OF SUPERVISOR
Date: Date:
ACKNOWLEDGEMENTS
In the name of Allah Taala, The Most Gracious, with God’s help and His
amazing grace, I’m finally completed this dissertation after going through the four
years of challenging and stressful period. Nevertheless, this period is so meaningful
because it gives me an opportunity to step foot into the realm of true research work. I
would like to take this opportunity to express my appreciation to their efforts and
kindness.
First and foremost, I thank my chairperson supervisory committee, Dr. Dwi
Susanto for his invaluable guidance, suggestions, and help throughout the course of
this study. I also wish to express my deep sense of gratitude and sincere thanks to Dr
Mohammed Arifullah and Dr. Fatimah Kayat who kindly provided me with their
knowledge, guidance, and advice in carrying out this study as well as completion of
this thesis. They also struggled to setup new laboratory for tissue culture in Jeli
Kampus to give the best tissue culture laboratory to us.
I also would like to million thank to all staff members at tissue culture
laboratory, especially to En. Suhaimi from Faculty of Agro Based Industry, Universiti
Malaysia Kelantan, which is person I considered as a good helper and very supportive.
I also want to thank my labmates, Mr. Ahmed, Husna, Ilfah Husna and Vikram, I owe
a special word of thanks for their ready help and timely courtesy. Not forgetten my
“Jeli Penthouse group” which act as a spirits around me and also their endless care,
help and moral support given to me, I’d really appreciate our memorable moments.
Last but not least, I would like to express my deepest gratitude to my beloved
family especially to my parents, my lovely husband Mohd Hamka and my dear uncle
Dato’ Pahamin A. Rajab for their unstinting love, endless encouragement, concern,
patience and sacrifices which had helped me in undertaking and completing this study.
I could not have asked for better without them, My study would not have been
possible. ALHAMDULILLAH!
Zeti Ermiena Surya Bt Mat Hussin
TABLE OF CONTENTS
PAGE
THESIS DECLARATION i
ACKNOWLEDGMENT ii
TABLE OF CONTENTS iii
LIST OF TABLES vi
LIST OF FIGURES ix
LIST OF ABBREVIATIONS xii
ABSTRACT xiv
ABSTRAK xv
CHAPTER 1 INTRODUCTION
1.1 Uses of kenaf (Hibiscus cannabinus L) 1
1.2 Objectives of the study 5
CHAPTER 2 LITERATURE REVIEW
2.1 Plant tissue culture 6
2.2 Micropropagation 7
2.3 Kenaf 10
2.3.1 Plant describtion 12
2.3.1.1 Stem (Stalk) 12
2.3.1.2 Leaf 13
2.3.1.3 Inflorescence 15
2.3.1.4 Fruit & seed 16
2.4 Kenaf variety KB6 18
2.5 A brief review on in vitro studies of kenaf 19
CHAPTER 3 MATERIALS AND METHODS
3.1 Materials 22
3.1.1 Plant collection 22
3.1.2 Chemical, glassware and equipment list 23
3.2 In vitro propagation method 24
3.2.1 Washing and storage of glassware 24
3.2.2 Preparation & composition of nutrient media 24
3.2.3 Preparing the sterile transfer hood 27
3.2.4 Culture condition and subculture 27
3.3 Experimental protocol for kenaf (Hibiscus cannabinus L) 28
3.3.1 Direct shoot organogenesis 28
3.3.2 Indirect shoot organogenesis 30
3.3.3 Acclimitization procedure 32
3.4 Statistical analysis 32
CHAPTER 4 RESULTS AND DISCUSSION
4.1 Shoot tip culture 34
4.1.1 Direct shoot organogenesis from shoot tip explant 34
4.1.2 Indirect shoot organogenesis from shoot tip explant 39
4.2 Node culture 46
4.2.1 Direct shoot organogenesis from node explant 46
4.2.2 Indirect shoot organogenesis from node explant 50
4.3 Leaf culture 56
4.3.1 Direct shoot organogenesis from leaf explant 56
4.3.2 Indirect shoot organogenesis from leaf explant 60
4.4 Petiole culture 67
4.4.1 Direct shoot organogenesis from petiole explant 67
4.4.2 Indirect shoot organogenesis from petiole explant 68
4.5 Acclimitization 76
4.5.1 The effect of humidity 76
CHAPTER 5 CONCLUSION
5.1 Summary & Conclusion 79
5.2 Recomendation for future research 81
5.3 Schematic sketch of micropropagation protocol for kenaf var. KB6 82
REFERENCES 83
APPENDIX 92
Appendix A-Table A.1 92
Appendix B-Figure B.1 93
-Figure B.2 94
NO.
LIST OF TABLES
PAGE
2.1 Previous study on kenaf propagate through tissue culture 21
technique
3.1 Distribution of plant growth regulator (PGR) BAP and IAA 29
(mg/1) in MS medium for direct organogenesis shoot induction
3.2 Distribution of PGR NAA and IAA (mg/1) MS medium for root 30
induduction
3.3 Distribution of PGR 2,4-D and Kinetin (mg/l) in MS medium to induce
callus in indirect organogenesis
31
3.4 Distribution of PGR BAP and IAA (mg/l) in MS medium for shoot 31
induction from callus
4.1 The percentage multiple shoot, average number of shoot 35
induction average of shoot length and average number of leaves
for shoot tip culture using MS medium with different
concentration of BAP and IAA (mg/l)
4.2 Percentage and average of root induction, root length, root number 37
from regenerated shoot using NAA (mg/l) verses IAA (mg/l)
4.3 The percentage pf explants forming callus and cullusing morphology 41
from shoot tip induction of callus using MS medium different
concentration of KN (mg/1) and different concentration of
2,4-D (mg/1)
4.4 Frequency of callus generate into multiple shoot, average of shoot
induction per explants and average of shoot length from shoot tip
42
callus using MS medium with different concentration of BAP (mg/l)
and IAA (mg/l)
4.5 Rooting induction of regenerated shoot from shoot tip callus on 43
MS medium with various concentration of NAA and IAA (mg/1)
4.6 The percentage of multiple shoot, average number of shoot induction 47
average of shoot length and average number of leaves for node
cultureusing MS medium with different concentration of BAP (mg/l)
and different concentration of IAA (mg/l)
4.7 Percentage and average of root induction, root length, root 48
number from node culture regenerated shoots using MS medium and
different level of NAA (mg/l) and MS medium and different level
of IAA (mg/l)
4.8 The percentage of explants forming callus and callusing morphology 51
from node induction of callus using MS medium different
concentration of KN (mg/l) and different concentration of
2,4-D (mg/l)
4.9 Frequency of callus generate into multiple shoot, average of 52
shoot induction per explants and average of shoot length from
node callus using MS medium with different concentration of
BAP (mg/l) and different concentration of IAA (mg/l)
4.10 Rooting induction of regenarated shoot for node callus using 54
MS medium with different level of NAA (mg/l) and MS
medium and different level of IAA (mg/l)
4.11 Frequency of shoot induction, percentage of root form, average 57
number of root per explants, average of root length in induction of
shoot stage from leaves culture using MS medium with different
concentration of BAP and IAA (mg/l)
4.12 The frequency explants forming callus and callus morphology, 62
in indirect organogenesis from leaves culture using MS medium
with different concentration of KN and 2, 4-D (mg/l)
4.13 The frequency of callus generate into multiple shoot (%), average 63
of shoot induction per explants and average of shoot length
from leaves culture callus using MS medium with different
concentration of BAP and IAA (mg/l)
4.14 Rooting induction of regenerated shoot for leaves callus using 64
MS medium with different level of NAA (mg/l) and MS medium
and different level of IAA (mg/l)
4.15 Percentage of explants forming callus and callus morphology 70
from petiole culture induction of callus using MS medium
with different concentration of KN and 2, 4-D (mg/1)
4.16 The frequency of callus generate into multiple shoot (%) average
of shoot induction per explants and average of shoot length (cm)
from petiole culture callus using MS medium with different
concentration of BAP and IAA (mg/l)
73
4.17 Rooting induction of regenerated shoot for petiole callus using 71
MS medium with different level of NAA (mg/l) and MS medium
and different level of IAA (mg/l)
4.18 Different high of plantlets, the percentage of explants survive
and the different number of explants leaves for acclimitization
process from effect of humidity
74
LIST OF FIGURES
NO.
2.1a
Leaves of kenaf was picked fresh from farm
PAGE
14
2.1b Canabis sativa or Marijuana leaf identical to kenaf's
leaf (Source from Wikipedia)
14
2.2a Flower of the kenaf freshly picked from farm 16
2.2b Center of the flower, red in colour 16
2.2c Pollen grain under the microscope (source from Ahmed et al, 2014) 16
2.3a The wedges-shaped seed of kenaf, seed sample collected from
MARDI Telong
17
3.1 Preparation of stock solutions and volumes taken from the
stocks to prepare 1 liter of MS medium. Gamborg and shyluk (1981),
Gamborg (1982), 1991) and Dodds and Roberts (1982) in
(Aurifullah, 2006)
26
4.1a Single initiation of shoot tip explants on MS medium on 1st
day of inoculation, rapid observation was done on first 10 days
to observe the contamination
38
4.1b Single shoot initiation of shoot tip explants on MS medium with
0.05 mg/l BAP and 0.01 mg/l IAA after 2 weeks
38
4.1c Shoot proliferation on MS medium with 0.05 mg/l BAP and
0.01 mg/l IAA after 3 week
38
4.1d Multiple shoot production MS medium with 0.05 mg/l BAP 38
and 0.01 mg/l IAA after 5 weeks
4.1e Induction and elongation of multiple shoot on MS medium
with 0.05 mg/l BAP and 0.01 mg/l IAA after 6 weeks
38
4.1f Elongation of single plantlet and rooting of in vitro raised
shoot on MS basal medium on 8 weeks
38
4.2a Shootip explants were inoculated in MS medium with KN
and 2, 4-D on first day
45
4.2b Callus induction for shoootip explants in MS medium
with 0.2 mg/l 2,4-D after 10 days
45
4.2c Shoot induction from callus in MS medium with 0.1 mg/l BAP 45
and 0.03 mg/l IAA after 20 days
4.2d Shoot elongation and multiplication on MS medium with 0.1 mg/l BAP 45 and 0.03 mg/l IAA after 5 weeks
4.2e Single shoot elongation & rooting in MS basal medium on 45
2nd week in rooting medium
4.3a Direct organogenesis of node explants in MS medium with 0.1 mg/l 49 BAP after 6 days
4.3b Shoot proliferation & petiole fall down from node explants 49 in MS medium with 0.5 mg/l BAP and 0.05 mg/l IAA after 14 days
4.3c Multiplication of shoot in MS medium with 0.5 mg/l BAP 49 and 0.05 mg/l IAA after 5 weeks
4.3d Elongation of single plantlet and rooting of in vitro raised shoot 49 on MS medium basal on 8 weeks
4.4a Indirect organogenesis of node explants in MS medium 55 with 0.2 mg/l 2, 4-D on 1st 7 days start to swollen at the bottom
4.4b Callus induction from nodal explants on MS medium 55
with 0.2 mg/l 2,4-D after 12 days
4.4c Shoot induction from pieces of callus on MS medium 55 with 0.1 mg/l BAP after 3 weeks
4.4d Shoot induction from nodal callus on MS medium 55
with 0.1 mg/l BAP after 5 weeks
4.5a Direct organogenesis of leaves on MS medium with BAP and IAA in first week
59
4.5b Root form from leaves direct organogenesis on MS medium 59
with 0.05 mg/l BAP and 0.01 mg/l IAA after 9 days
4.5c Abudant of root from leaves culture on MS medium 0.1 mg/l BAP 59
with 0.05 mg/l IAA after 4th weeks
4.6a Indirect organogenesis of leaves on MS medium
with KN and 2, 4-D first week of inoculation
66
4.6b Callus induction of leaves indirect organogenesis on MS medium 66
with 0.2 mg/l 2, 4 D after 14 days
4.6c Callus induction of leaves indirect organogenesis on MS medium 66
with 0.2 mg/l 2, 4 D after 25 days
4.6d Shoot induction from callus in MS Medium with 0.3 mg/l BAP 66
and 0.03 mg/l IAA after 20 days
4.7a Indirect organogenesis of petiole in MS medium 75
with KN and 2, 4-D on 1st day of inoculation
4.7b Callus induction after 15 days on MS medium with 0.2 mg/l 2,4-D 75
4.7c Callus induction after 10 days on MS medium 1.0 mg/l KN 75 with 0.1 mg/l 2,4-D
4.7d Initiation of multiple shoot from callus after 25 days on MS medium 75 with 0.1 mg/l BAP and 0.02 mg/l IAA
4.7e Elongation and rooting of multiple shoot after 47 days on MS medium 75 with 0.1 mg/l BAP and 0.02mg/l IAA
4.8a Combination of sand, coco pit and vermiculite with 3:2:2 ratios 78
4.8b Plantlets were covered with the hole plastic containers on 78 1
st day of acclimatization
4.8c The plantlet survive after 9 days of acclimatization under shaded roof 78
4.8d Field plant of kenaf plantlet was successfully tested after 15 days in 78
shaded roof. The plantlets were 40 days
LIST OF ABBREVIATIONS
µm Micron
μg Microgram
μm Micrometer
µl Microlitre
% Percent
°C Degree Celsius
2,4-D 2,4-Dichloro-phenoxy acetic acid
BAP Benzyl-6-aminopurine
C2H5OH Ethyl Alcohol
cm Centimeter
Fig. Figure
g Gram
h Hour
HgCl2 Mercuric Chloride
IAA Indole-3-acetic acid
KIN Kinetin
KNO3 Potassium Nitrate
m Meter
M Molarity
min Minute
ml Millilitre
mg Miligram
mg/l Miligram/litre
mm Millimetre
MS Murashige and Skoog’s Medium
n Sample size
NAA Naphthalene-3-acetic acid
PGR Plant growth regulator/s
s Second
Sp Species
Direct and indirect organogenesis and plant regeneration of an industrial plant
kenaf (Hibiscus cannabinus L) var. KB6
ABSTRACT
As an important fiber crop, many potential applications of kenaf are being
identified and developed in 21 century, especially in developed countries such as
America, Japan, and France and Malaysia as well. The present study report a protocol
for the efficient in vitro propagation of kenaf (H.cannabinus L) to initiate multiple
shoot from mother plant part (shoot, petiole, node and leaf) through direct and indirect
organogenesis using MS medium + BAP + IAA for direct organogenesis and MS
medium + KN+2,4-D for indirect organogenesis to get callus and after 8 weeks, the
calli were put in the MS medium + BAP +IAA for shoot induction. The highest
number of shoots produced from node explants part via direct organogenesis
(16.33/explants) in MS medium + 0.5 mg/l BAP +0.05 mg/l IAA. The highest
percentage explants forming callus and callus generate into shoot also from node
explants part which was induced 75% callus from explants and 73.33% of callus turn
into shoot in MS medium + 0.1 mg/l BAP. Several subcultures were drived in order to
enhance the multiplication rate. The treatments have their significant different with
others. The shoots then were transferred to the root induction medium. Shoots showed
the vigorous roots in the MS basal medium. The in vitro rooted plantlets were
acclimatized in sand+ coco pit + vermiculite with ratio 3:2:2 and were covered with
hole container for 0-15 days to test the effect of humidity to plantlets. The 9th-15
th days
explants covered with container showed 100% of survive. Survive well rooted
plantlets were transferred to the field. Plants grew well into maturity without any
remarkable morphological variations within the treatments.
Regenerasi Tumbuhan Secara Langsung & Secara Tidak Langsung Tumbuhan
Industri, Kenaf (Hibiscus cannabinus L) var. KB6
ABSTRAK
Sebagai salah satu tanaman yang penting, banyak aplikasi potensi kenaf sedang
dikenalpasti dan dibangunkan di abad 21, terutama di negara-negara maju
membangun seperti Amerika, Jepun, dan Perancis dan juga Malaysia. Laporan kajian
ini adalah untuk mengkaji protokol yang terbaik dalam pembiakan in vitro kenaf
(H.cannabinus L) dengan menggunakan eksplan dari pelbagai bahagian pada induk
kenaf variasi KB6 (pucuk, daun, nod dan daun nod) melalui organogenesis langsung
dan tidak langsung dengan menggunakan MS medium + BAP + IAA untuk
organogenesis langsung dan MS sederhana + KN + 2,4-D untuk organogenesis tidak
langsung untuk mendapatkan kalus dan selepas 8 minggu, kalus telah dimasukkan ke
dalam medium MS + BAP + IAA untuk mendapatkan pucuk. Bilangan tertinggi pucuk
dihasilkan dari nod eksplan bahagian melalui organogenesis langsung (16.33 /
eksplan) dalam MS medium + 0.5 mg / l BAP 0,05 mg / l IAA. Peratusan tertinggi
eksplan yang membentuk kalus dan kalus berpotensi mengeluarkan pucuk juga dari
nod eksplan sebahagian yang disebabkan 75% kalus daripada eksplan dan 73.33%
daripada kalus bertukar menjadi pucuk di MS medium + 0.1 mg / l BAP. Beberapa
subkultur telah dilakukan bagi meningkatkan kadar pengeluaran pucuk. Setiap rawatan
mempunyai perbezaan yang signifikan dengan rawatan lain. Pucuk kemudian
dipindahkan ke medium induksi akar. Pucuk mengeluarkan akar yang baik dalam
medium asas MS. In vitro anak pokok berakar telah dipindahkan ke medium pasir +
coco pit + vermikulit dengan nisbah 3: 2: 2 dan ditutup dengan bekas berlubang untuk
0-15 hari untuk menguji kesan kelembapan ke atas anak pokok. Anak pokok yang
ditutup dengan bekas selama 9-15 hari menunjukkan 100% daripada bermandiri. Anak
pokok yang berakar dipindahkan ke tanah lapang di luar kawasan rumah hijau. Anak
pokok tumbuh dengan baik matang tanpa apa-apa perubahan morfologi yang luar biasa
dalam rawatan.
CHAPTER 1
INTRODUCTION
1.1 Uses of kenaf (Hibiscus cannabinus L)
Kenaf (Hibiscus cannabinus L.) is widely known as an important family of
Hibiscus for modern needs. The reason behind it is apparently because it produces high
biomass, and also able to adapt well with local environment. Kenaf has been adopted in
many countries ever since, including Malaysia. This unique plant is still new in Malaysia
and has been cultivated generally in the eastern part of the country, especially Kelantan.
Initially, kenaf is to replace tobacco industry since it is no longer supported by the
government. Far in Africa, kenaf is used in anaemic therapy (Charles et al., 2002).
This plant can rise up to 60-120cm height as young as 2-3 months. U.S.
Department of Agriculture reported that kenaf can produce up to 10 tons of dry fiber per
acre per year. That is 3 to 5 times greater than the production of Southern pine trees, in
which it would take 7 to 40 years before it can be harvested (McLean et al., 1992). As
known in the botanic field, kenaf is classified in various types in which each of them can
perform at their best in certain locations, conditions and other variables. These groups of
kenaf are known to produce different flowering schedule. Some may be harvested earlier
than the other and some later. The good part of it is the flowering can last up to 3 to 4
weeks or more, with each individual bloom can last only for one whole day (Charles et
al., 2002).
Kenaf’s flowers would eventually bloom at the end of growing season. The
process takes place when the flower drops off and leave a seed pod behind. The seeds,
however, will not get matured in many parts of U.S. Although some of the plant’s
varieties are known to bloom early, its biomass production is still not tangible enough to
provide adequate amount of fiber economically. This is because of their African origin
require an additional 60-90 days of frost free condition for the seeds to germinate.
Because of this particular, kenaf is not suitable for massive plantation across the country
like a normal weed. It also presents some interesting challenges for developers to ensure
a consistent supply of seed for next year’s crop (McLean et al., 1992).
The stem of the plant consists of two recognizable types of fiber. The first layer of
fiber is called ‘bast’. It is made up of roughly 40% of the stem’s total dry weight. The
refined ‘bast’ fiber measures at 2.6mm and is similar to the fiber commonly used to make
paper. The core is the second layer. It is made up of 60% of the stem’s total dry weight.
Its refined fiber measures at approximately 6mm and is comparable to hardwood tree
fiber which is used in wide range of paper products (Rowell and Cook, 1998).
Harvesting kenaf is not difficult as it can be processed in a common mechanical
fiber separator. It is very similar to a cotton gin. While the two fibers are separated
through the mechanical separator, they are allowed to be processed independently, in
which it provides raw materials for a wide range of products, such as paper, particle
board, bioremediation aids and animal bedding (McLean et al., 1992).
According to Duke 1983, kenaf was propagated by seed, but seed availability is
limited (Sullivan, 2003). Seed production strategies are affected by the cultivar, location-
especially latitude, and cultural practices. The first issue to address is the cultivar
photosensitivity, whether the cultivar is an ultra-early, an early to medium, or a late
maturing cultivar (Webber III & Bledsoe, 2002). Researchers and seed producers have
reported that the strategy for maximizing harvestable kenaf seed is very different than the
production approach in maximizing kenaf fiber yields (Mullens, 1998; Webber III &
Bledsoe, 2002). Kenaf seed has high oil content (Mohamed et al., 1995) and seed
viability decreases over time when stored at higher relative humidity and higher
temperatures (Webber III & Bledsoe, 2002). Propagation by seed is not recommended
due to the heterogeneous nature of the seedlings owing to its cross pollination. Thus, any
kenaf variety will decrease in purity, leading to the increment of seed yields and
reduction of fiber yields (Ramesh, 2016).
The application of plant tissue culture as a micropropagation technique has
become an important biotechnological tool which offers additional advantages such as
the rapid multiplication rate (Edwin et al, 2008). Therefore, in vitro propagation methods
would be important to propagate this crop for high and stable cellulosic fiber production,
as large numbers of genetically identical plants and in a relatively short time for the
industrial plantation. Axillary buds are widely used for micropropagation as they have
entire rudimentary vegetative shoot and can be induced to develop into plants easily
which are similar to the parental type (Zapata et al., 1999; Srivatanakul et al., 2000).
The development of tissue culture and regeneration techniques are the most
important procedures that must be established in order to improve kenaf by genetic
transformation procedure (Banks et al., 1993). There are few reports on the in vitro
propagation of the Hibiscus genus, with kenaf being the most studied species in terms of
organogenesis regeneration. According to McLean et al. (1992), organogenesis of kenaf
via callus culture was not reproducible and resulted in very low regeneration efficiency
and the induction of heritable mutation. Preliminary research on shoot regeneration of
kenaf callus used the shoot apex still unsuccessful though direct shoot regeneration
without a callus phase has been achieved (Zapata et al., 1999; Srivatanakul et al., 2000),
while other authors reported the formation of multiple shoot from young shoot and
cotyledons (Khatun et al., 2003; Herath et al., 2004). A protocol for plant regeneration
from callus using segments of hypocotyls and cotyledons has been established for the H.
siryacus (Jenderek & Olney, 2001). The establishment methods for sterilization from
mother plant also have not been proposed by any researcher due to high contamination in
culture. Using seeds as a means of plant propagation is simple and can be produced by
farmers, but disadvantage are the progeny may be genetically different from the parents
and some plant have the dormancy period. So, in vitro propagation is the one of the
alternative techniques to solve these problems and can be used for mass propagation of
seedlings. That’s why this experiment was proposed.
1.2 Objectives of the present study
Therefore, the aims of this research were to:
(a) To analyses the effect of different plant growth regulator on direct and
indirect organogenesis of kenaf var. KB6
(b) To develop a protocol for kenaf var KB6 (Hibiscus cannabinus L) in vitro
regeneration using different explants like shoot tips, nodes, leaves and
petioles
CHAPTER 2
LITERATURE REVIEW
2.1 Plant tissue culture
Plant tissue culture has gone through a long developmental history to achieve its
current state. The history and milestones of tissue culture development has been
elaborated in many reviews (Gamborg, 2002; Thorpe, 2007; Vasil, 2008). Plant tissue
culture is an essential component of Plant Biotechnology. Plant cell and tissue culture has
already contributed significantly to crop improvement and has great potential for future
(Mehbooba et al., 2011). Research efforts in plant cell and tissue culture have increased
dramatically worldwide in recent years including efforts in developing nations. Plant cell
and tissue culture is defined as the capability to regenerate and propagate plants from
single cells, tissue and organs under sterile and controlled environmental conditions
(Murashige, 1974).
According to George & Debergh (2009), tissue culture techniques are now being
widely applied for improvement of field crop, forest, and horticulture and plantation crop
for increased agricultural and forestry production. Today tissue culture technology is
being exploited mainly for large scale production or micropropagation of elite planting
material with desirable characteristics. This technology has now been commercialized
globally and has contributed significantly towards the enhanced production of high
quality planting material. Recently emphasis has been on genetic transformation,
especially for (1) increased production of secondary metabolite for example, Bourgaud et
al., 2001, was studied about secondary metabolite from hairy roots callus and other organ
culture of Plumbago indica L. The work on in vitro production of secondary metabolites
also has been reviewed by (Ramachandra & Ravishankar, 2002). Many researchers have
reported the production of secondary metabolites by cell cultures where the synthesis and
yield of compounds approached or exceeded the levels found in natural plant resources
(Eran & David, 2000; Nakashima et al., 1997), (2) production of alkaloids
pharmaceutics, nematocidal compounds, and also some novel compounds not found in
the whole plants, regeneration of plant resistant to herbicides, disease, and pests (Leslie &
Johan, 2000) (3) scale up of cultures in bioreactors, Jacqueline & John (1999), was build
a research about hairy root culture by setup the bioreactor culture, (4) plants with
different morphological traits and (5) transgenic crops for production of recombinant
vaccines and anti-microbial antibodies (Peter & Stoger, 2011). These developments have
far-reaching implications in the improvement of medicinal plants as well (Bajaj, 1990).
2.2 Micropropagation
Micropropagation is a rapid multiplication of a selected plant using in vitro
culture techniques and it’s developed in past 35 years (Aneesha, 2015). In vitro clonal
propagation provides true to type plants of a selected genotype using in vitro culture
techniques (Thorpe, 2007). Micropropagation is also used to promote germplasm storage
for maintenance of disease-free stock in controlled environmental condition and in long
term via cryopreservation (Nukari et al, 2009). Micropropagation also gives the rapid
production of high quality, disease-free and uniform planting material (Ahloowalia et al,
2004). The plants can be multiplied under a controlled environment, anywhere,
irrespective of the season and weather, on a year-round basis (Bourgaud et al., 2001).
Production of high quality and healthy planting material of ornamentals, and forest and
fruit trees, propagated from vegetative parts, has created new opportunities in global
trading for producers, farmers, and nursery owners, and for rural employment
(Ahloowalia et al, 2004). One of the most exciting and important aspects of in vitro cell
and tissue culture is the capability to generate and propagate plants from cultured cells
and tissues. The simplest type of in vitro plant propagation is the stimulation of axillary
bud development (Edwin et al., 2008). This technique exploits the normal ontogenetic
route for branch development by lateral (axillary) meristems. The axillary buds are
treated with hormones to break dormancy and produce shoot branches (Zhu et al, 2015).
The shoots are then separated and rooted to produce plants. Alternatively, the shoots are
used as propagules for further propagation (Aneesha, 2015).
Plant regeneration from cultured tissue can also be achieved by culturing tissue
section lacking a preformed meristem (adventitious origin) or from callus and cell
cultures (de novo origin) (De Filippis, 2014). Axillary buds are preformed meristems. In
contrast, adventitious regeneration events occur at unusual sites of a cultured tissue such
as the internode, leaf blade, cotyledon, or root elongation zone, where meristem do not
naturally occur (Edwin et al, 2008). According to Nukari et al. (2009), adventitious plant
regeneration often is dependent upon the presence of organized explants tissue. In
comparison, de novo (literally, “to arise new”) plant regeneration occurs from callus and
cell cultures in the absence of organized explants tissues.
Whether adventitious or de novo in origin, plant regeneration can occur by one of
two processes. Organogenesis is the formation of individual organs, such as shoots or
roots. Somatic embryogenesis is the formation of a bipolar structure containing both
shoot and root meristems, and developing in a manner similar to zygotic embryos (Sara et
al, 2002).
According to Edwin et al (2008), most plant species are capable of plant
regeneration by either organogenesis or somatic embryogenesis, but very few species are
capable of both. Some species are easy to regenerate from callus or cell cultures, while
others regenerate only by an adventitious process. The choice of plant species and the
goal of the research will determine the plant regeneration or propagation procedure
(Vasil, 2008). When available for the same species, the different regeneration approaches
may yield different propagation rates (Chu, 1992). Axillary bud proliferation and culture
of individual nodes are the techniques most widely used in commercial micropropagation
and which show the least variation among the propagated plants (Ngezahayo & Liu,
2014). In contrast, adventitious shoot organogenesis and regeneration of plants from
callus by organogenesis or by somatic embryogenesis show the most variation, as well as
higher propagation rates (Gest, 2004). Direct (adventitious) somatic embryogenesis and
repetitive embryogenesis show the best balance of high propagation rates with relatively
few off-types (Gamborg, 2002).
There are several defined steps in a typical micropropagation system (Murashige,
1974). The first step is the initiation of a sterile culture of the explant (Stage I). The
second step is the multiplication of shoots or other propagules from the explants (Stage
II). Adventitious shoot proliferation is the most frequently used multiplication technique
in micropropagation systems (Chu, 1992). The culture media and growth conditions used
in Stage II systems are optimized for maximum rates of multiplication. The third step is
the development of roots on the shoots to produce plantlets (Stage III). Specialized media