Taylor et al. - Unknown - Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology Official Journal of the Societa Botanica Italiana Establishment o-annotated.pdf

7/29/2019 Taylor et al. - Unknown - Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology Offici

1/7

This article was downloaded by: [Universidad De Concepcion]On: 01 June 2012, At: 13:12Publisher: Taylor & FrancisInforma Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House37-41 Mortimer Street, London W1T 3JH, UK

Plant Biosystems - An International Journal Dealing

with all Aspects of Plant Biology: Official Journal of th

Societa Botanica ItalianaPublication details, including instructions for authors and subscription information:http://www.tandfonline.com/loi/tplb20

Establishment of Aster sedifolius and Aster caucasicus

callus cultures as a potential source of antioxidantsMaria Minutolo

a, Immacolata Caruso

a, Gianluca Caruso

a, Pasquale Chiaiese

a& Angela

Erricoa

aDepartment of Soil, Plant, Environmental and Animal Production Sciences, University of

Naples Federico II, Via Universit 100, 80055, Portici, NA, Italy

Available online: 01 Dec 2011

To cite this article: Maria Minutolo, Immacolata Caruso, Gianluca Caruso, Pasquale Chiaiese & Angela Errico (2012):Establishment of Aster sedifolius and Aster caucasicus callus cultures as a potential source of antioxidants, Plant Biosystem- An International Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica Italiana, 146:1,41-46

To link to this article: http://dx.doi.org/10.1080/11263504.2011.601333

PLEASE SCROLL DOWN FOR ARTICLE

Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions

This article may be used for research, teaching, and private study purposes. Any substantial or systematicreproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form toanyone is expressly forbidden.

The publisher does not give any warranty express or implied or make any representation that the contentswill be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses shouldbe independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims,proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly inconnection with or arising out of the use of this material.
http://dx.doi.org/10.1080/11263504.2011.601333http://www.tandfonline.com/page/terms-and-conditionshttp://dx.doi.org/10.1080/11263504.2011.601333http://www.tandfonline.com/loi/tplb20


2/7

Establishment ofAster sedifolius and Aster caucasicus callus cultures as

a potential source of antioxidants

MARIA MINUTOLO, IMMACOLATA CARUSO, GIANLUCA CARUSO,

PASQUALE CHIAIESE, & ANGELA ERRICO

Department of Soil, Plant, Environmental and Animal Production Sciences, University of Naples Federico II, Via

Universita 100, 80055 Portici (NA), Italy

Abstract

Callus cultures were established for Aster sedifolius and Aster caucasicus, two Aster species used in natural medicine for theiranticancer, antibacterial and antiviral activities attributed to the high content of antioxidant compounds such as polyphenolsand ascorbate. The effects of growth medium and light condition on the induction and growth rate of callus from leaf, petioleand root explants are reported. Callus induction and proliferation depended on the genotype and the experimentalconditions. In particular, a profuse callus culture was obtained from leaf explants grown in the light on mediumsupplemented with 2,4-D (0.1 mg l71) for A. caucasicus and on medium supplemented with 2,4-D (0.44 mg l71) plus6-benzil-ammino-purine (BAP) (0.22 mg l71) for A. sedifolius. The content of total polyphenol and ascorbic acid wasestimated in leaf and petiole explants of in vivo plants and in the relative derived calli. In calli, polyphenol content was lowerthan in the corresponding in vivo organs. Furthermore, the total ascorbic acid content decreased in calli while the reducedascorbic acid pool increased. These findings demonstrate that Aster callus cultures produce antioxidant compounds and assuch might be a model system to investigate the regulation and production of these important metabolites.

Keywords: Ascorbic acid, in vitro culture, polyphenols

Introduction

The genus Aster (Asteraceae) is widely distributed in

nature, including about 600 species adapted to

various niches. A large number of species and

interspecific hybrids are grown as ornamentals plants

or for cut flowers; however, theyre predominantly

used for their therapeutic properties known since

ancient times. In traditional Chinese medicine, Aster

species have been used for the treatment of cough,

fever, and tonsillitis. In recent years, diuretic, anti-

tumor, antibacterial, antiviral and anti-ulcer activ-

ities have been attributed to these plants (Ng et al.

2003).The Aster therapeutic features have been widely

ascribed to the high content of antioxidant com-

pounds such as polyphenols and ascorbic acid which

exhibit antibacterial, antiviral, anti-inflammatory,

anti-allergic, antithrombotic and vasodilator actions

and are useful in the treatment of arteriosclerosis,

cancer, diabetes, neurodegenerative diseases, and

arthritis, as well as for the prevention of otherdiseases (Tiwari 2001; Pourmorad et al. 2006).

Antioxidant content is variable in plants because

their synthesis and storage is a direct consequence of

plantenvironment interaction.

Although much research has been carried out

on the isolation and determination of antioxidants

from plants, little is known about their regulation

and production in plants growing in vitro or under

tissue culture conditions. Callus and cell suspension

cultures are an attractive model system and represent

an interesting tool to increase the knowledge of

antioxidant production and accumulation (Bauer

et al. 2004; Keskin & Kunter 2008). Cell cultureshave been established for many plant genera but have

been reported only for a few species of the Aster

genus (Cammareri et al. 2001, 2002; Uno et al.

2009). Therefore, the aims of the present study were

to develop an efficient protocol for callus culture

from A. sedifolius and A. caucasicus by finding the

most suitable explant source and the best callus

Correspondence: Dr. Maria Minutolo, Dipartimento di Scienze del Suolo, della Pianta, dellAmbiente e delle Produzioni Animali, Universita di Napoli

Federico II, Via Universita 100 , 80055 Portici (NA), Italia. Tel: 39 0812539430. Fax: 39 0812539186. Email: [email protected]

The first two authors contributed equally to this work.

Plant Biosystems, Vol. 146, No. 1, March 2012, pp. 4146

ISSN 1126-3504 print/ISSN 1724-5575 online 2012 Societa Botanica Italiana

http://dx.doi.org/10.1080/11263504.2011.601333


3/7

growth conditions, and to evaluate the production of

antioxidant metabolites.

Materials and methods

Plant material

A. sedifolius, accession number no. 151, andA. caucasicus, accession no. 81, were kindly provided

by the Botanical Garden of Stuttgart (Germany)

and the Botanical Garden of Bayreuth (Germany),

respectively. A set of three plants per species were

grown in vivo, in a non-conditioned greenhouse, and

in vitro on hormone free medium containing Mura-

shige and Skoog (1962) mineral salt and vitamins

formulation supplemented with sucrose 3% (w/v),

agar 0.9% (w/v), and adjusted to pH 5.8 (MS0).

Plant tissues culture

In A. sedifolius, leaf explants of three different

developmental stages, namely rosette (L), initial stem

elongation phases (LIS) and final stem elongation

phases (LFS), were used while for A. Caucasicus,

leaves were taken only at rosette stage (L) (Figure 1).

For both species, petiole (P) and leaf explants were

collected from in vitro and in vivo grown plants, while

root (R) explants were collected just from in vitro

plants. Explants from in vivo plants were surface-

sterilized with a solution of ethanol 70% (v/v) for

5 min, and then left for 15 min in a solution of

sodium hypochlorite 2% (v/v) plus 0.1% (v/v) of

Tween-20. Finally, they were rinsed three times in

sterile distilled water. For each tissue (P, L, LSF

and so on) and tested experimental condition, 30

explants were collected from in vivo and 30 from

in vitro growing plants. They were cut into rectan-gular small pieces (ca. 1 cm2) and cultivated on

Petri dishes containing MS0 medium supplemented

with 2,4-dichlorophenoxyaceti acid (2,4-D) and

6-benzil-ammino-purine (BAP) at various concen-

trations: 0.1 mg l71 of 2,4-D (medium A); 1 mg l71

of 2,4-D (medium B); 10 mg l71 of 2,4-D (medium

C); 0.94 mg l71 of 2,4-D and 0.18 mg l71 of BAP

(medium D); 0.44 mg l71 of 2,4-D, and 0.22 mg l71

of BAP (medium E). All cultures were incubated at

258C+2 under a 14/10 (light/dark) photoperiod at

125 mEm72 s71 irradiance provided by cool white

fluorescent tube (Philips) or in complete darkness.

Subcultures were performed monthly. Callus growth

(CG), was recorded after 15, 30, and 45 days of

in vitro growth, and estimated as explants area

showing callus. A score was assigned at each explant

according to the following rank: 0, no visible callus; 1,

appearance of callus only at cut edges; 2, callus on

20% of explant area; 3, callus on 50% of explant

area and 4, callus on 70% and more of explant area

(Figure 2).

Figure 1. Tissues sampled from A. sedifolius and A. caucasicus plants.

Figure 2. Callus development of A. sedifolius petiole explants. Score 0 (a), score 1 (b), score 2 (c), score 3 (d), and score 4 (e).

42 M. Minutolo et al.


4/7

Antioxidant extraction and quantification

Antioxidants extraction was performed only from 45

days old calli developed in medium E for A. sedifolius

and in medium A for A. caucasicus, both under light

condition. Three extraction experiments were per-

formed for each sample; for each extraction, anti-

oxidants quantification was performed in triplicate.

Total polyphenols were extracted and assayedaccording to FolinCiocalteus procedure (Singleton

and Rossi 1965). Total ascorbic acid (total-AsA), as

sum of reduced and oxidized forms, and reduced

ascorbic acid (AsA) were extracted and quantified

using the procedure described by Kampfenkel et al.

(1995).

Statistical analysis

Statistical analysis was performed by using the

Statistical Package for Social Sciences Package

version 14 (SPSS Inc Chicago, Illinois). The effects

of experimental factors on callus growth were

estimated by the ANOVA procedure and the

Duncan test was used for mean separation referring

to p 0.01 and p 0.05 probability levels.

Results and discussion

Callus growth rate was highly variable for both

species and for all explants assayed (Figure 3a

and b). In the first 15 days of culture, the highest

CG rate was detected in A. sedifolius from leaf

explants at initial (LIS) and at final stem elongation

stage (LFS) followed by leaves at rosette stage (L),petioles (P) and finally roots (R). Root explants

turned brown and died within 3 weeks. At day 45, a

CG rate of 2.6 was detected for leaf explants (L, LIS

and LFS) while the lowest rate (1.5) was calculated

for petiole explants (Figure 3a). On the contrary, in

A. caucasicus, root explants showed a high CG rate

after 15 and 30 days. Furthermore, at day 45 of

culture, a CG rate of 3.84.0 was detected for all

explant types (Figure 3b) suggesting that callus

production in Aster is species and genotype depen-

dent (Cammareri et al. 2001). In plant species such

as Aster tripolium L. (Uno et al. 2009), Saussurea

obvallata (Dhar & Joshi 2005) and Actinidia deliciosa(Akbas et al. 2009) profuse callus production and

proliferation were obtained using leaf explants. On

the contrary, for Changium smyrnioides, petioles were

the best explants for obtaining callus culture (Jiang

et al. 2009).

On medium B (2,4-D 1 mg/l), no callus develop-

ment was observed in both species (Figure 3c and d).

This is in contrast with the observations of Uno et al.

(2009) reporting that friable white callus was

obtained after cultivation of A. tripolium leaf explants

on medium containing 1 mg l71 of 2,4-D.

The herbicide 2,4-D has been commonly used for

obtaining friable callus culture; however, in our

experiments, an increase of its concentration in

the growth medium negatively affected callus induc-

tion and development. All explants in medium C

(2,4-D 10 mg l71) died after 3 weeks of culture. On

medium A, in which 2,4-D concentration was ten-

fold less, explants have a higher CG, comparableto that obtained by Cammareri et al. (2001). In

contrast, in other Asteraceae such as Inula racemosa,

rapid browning of callus was reported for explants

cultured on medium containing 0.8 mg l71 of 2,4-D

and further increase in 2,4-D concentration led to

death within 20 days (Jabeen et al. 2007). In our

condition, after 45 days of in vitro culture the best

CG rate was observed using medium D (0.94 mg l71

of 2,4-D and 0.18 mg l71 of BAP) and medium

E (0.44 mg l71 of 2,4-D and 0.22 mg l71 of BAP)

(Figure 3c). Also, an increase in callus induction rate

and callus proliferation was observed in A. tripolium

when using 2,4-D plus citochinins rather than auxin

alone (Uno et al. 2009). A similar trend was

observed in A. caucasicus cultures, in which no

induction was observed on explants cultured on

medium B (1 mg/l 2,4-D), whereas callus grown on

medium C (2,4-D 10 mg/l) turned brown and died.

The best CG rate was recorded on medium A

(Figure 3d). These data suggest that Aster cells are

susceptible to high 2,4-D concentrations.

The effect of light on callus induction and growth

depends on the species. In A. caucasicus, light

positively affected callus cultures obtained from all

media assayed (Figure 3e). On the other hand, in A.sedifoilius a positive effect was recorded only during

the first 15 days of in vitro culture (Figure 3f). In

barley, no differences were observed in callus

induction of leaf explants under light or dark

conditions (Zapata et al. 2004) while Lemna gibba

callus induction and growth required light (Moon

and Stomp 1997).

The physiological stage of the mother plant affects

callus development and growth. In A. sedifolius, at 15

days, the CG of all explants collected from in vivo

grown plants was higher than that from in vitro grown

plants (Figure 3g) although after 45 days of culture

no differences were detected. On the contrary, for A.caucasicus a higher and faster CG was recorded on

explants collected from in vitro tissues rather than on

explants collected from in vivo plants (Figure 3h). A.

sedifolius calli were dark green brownish and generally

compact; they became partially friable after 5 months

of culture (Figure 4a, b, and c) on medium E. Calli

developed on A. caucasicus explants, on the other

hand, were generally white yellowish and friable

(Figure 4d, e, and f).

Evaluation of total polyphenols (Phe), reduced

ascorbic acid (AsA), and total ascorbic acid

Aster sedifolius and Aster caucasicus callus cultures 43


5/7

(total-AsA) contents was performed on in vivo LIS

and LFS of A. sedifolius, and leaf and petiole of A.

caucasicus and the relative in vitro callus cultures. In

A. sedifolius, Phe accumulation in callus cultures

obtained from LIS and LFS was lower than thatfound in their original tissues. The lower content of

Phe in callus culture is in accordance with that

observed in other species and might be due to the

negative effect of these compounds on cell division.

In cell culture, an increase of Phe content is generally

observed at the end of the exponential or steady

growth phase of the growth curve (Phillips &

Henshaw 1977). Gurney et al. (1999) reported that

caffeine and theobromine production in Theobroma

cacao callus obtained from leaf explants were 10%

lower than those found in vivo. In Vitis cell cultures,

anthocyanins were produced and accumulated just

after the end of cell division, when an increase of

phenylalanine ammonia-lyase (PAL) and chalcone

synthase (CHS) activities were also recorded (Kake-

gawa et al. 1995).For Aster, total-AsA and the percentage of

reduced AsA on total-AsA (AsA%) in LIS were

not statistically different from those recorded in LFS

(Table I). These results are in contrast with studies

in other species; in Arabidopsis thaliana, Medicago

sativa, and Impatiens walleriana, ascorbic acid con-

tent was higher in mature leaves than in younger

leaves (Franceschi & Tarlyn 2002). The reduced

AsA% found in callus culture from LIS and LFS was

doubled compared to those of differentiated tissues.

This is not surprising because an increase of the

Figure 3. Effect of tissue type (panels a and b), media (panels c and d), light conditions (panels e and f) and explant source (panels g and h)

on callus growth rate in A. sedifolius and A. caucasicus. For each culture day, different letters represent statistical differences (p50.05) among

treatments. Callus growth (CG) at 15, 30, and 45 days of in vitro culture is reported. L, leaf at rosette stage; LIS, leaf at initial stem

elongation stage; LFS, leaf at final stem elongation stage; P, petiole; R, root.



6/7

endogenous reduced ascorbic acid level and of the

relative redox status was generally recorded during

cell division, suggesting that an intracellular decrease

in the amount of dehydroascorbate could be a signal

for the cell to proceed from G1 into S phase (Kato

and Esaka 1999). On the other hand, root quiescent

cells (mitotically inactive cells) have generally low orundetectable levels of reduced ascorbic acid (Kerk

and Feldman 1995). These findings are in agree-

ment with our observations on Phe accumulation

confirming that A. sedifolius calli were still dividing

and growing under the optimized in vitro culture

conditions.

For A. caucasicus, no differences in metabolite

amounts were observed between leaf and petiole

(collected from in vivo plants) (Table I). These

findings are in contrast with data reported in other

species where polyphenol and ascorbic acid levels are

generally higher in leaves than in petioles (Baba et al.

1956; Zainol et al. 2003; van der Rest et al. 2006;

Zadeh et al. 2007). These discrepancies could be

due to A. caucasicus leaf morphology; the separation

between leaf and petiole is not well delineated

and explants could have been arbitrarily collected

(Figure 1). On the other hand, polyphenol contentwas similar in different tissues of Pisum sativum(Singh et al. 2002). In any case, although polyphenols

are generally a constitutive component of plant

tissues, their presence is also influenced by environ-

mental conditions and plant phenological stages.

Similarly to what observed for A. sedifolius, a decrease

of Phe and total-AsA and an increase of reduced

AsA% in callus tissues compared with the original

tissues were also found for A. caucasicus (Table I).

In conclusion, we established a valuable callus

culture procedure for two Aster species: for

Figure 4. Leaf derived calli of A. sedifolius in vivo grown plants cultured (under light conditions) on media A (a), D (b) and E (c), and of A.

caucasicus in vitro grown plants cultured on medium A (light conditions) (d) and medium E (light or dark conditions, respectively) (e, f).

Table I. Content of total polyphenol, total-ASA and percentage of reduced ASA on total-ASA (AsA%), in in vivo grown tissues and derived

calli of A. sedifolius and A. caucasicus.

Polyphenols (mg/g) Total AsA (umol/g) AsA%

Aster sedifolius Callus from LIS* 0.22 (0.08) c 0.33 (0.05) b 24.1 (4.07) a

Callus from LFS** 0.15 (0.01) c 0.17 (0.05) c 22.3 (6.76) b

LIS* 6.13 (0.05) b 0.83 (0.15) a 9.6 (1.47) c

LFS** 7.80 (0.15) a 0.84 (0.10) a 10.7 (1.86) c

Aster caucasicus Callus from leaf 0.17 (0.02) B 0.18 (0.02) B 22.7 (2.59) A

Callus from petiole 0.28 (0.06) B 0.17 (0.02) B 21.3 (2.85) A

Leaf 6.4 (0.20) A 0.39 (0.01) A 7.7 (1.38) B

Petiole 6.2 (0.23) A 0.61 (0.03) A 10.1 (1.01) B

Mean and standard error values are reported. Different letters represent statistical differences (p 5 0.05) among tissues. *LIS, leaf at initial

stem elongation stage; **LFS, leaf at final stem elongation stage.

Aster sedifolius and Aster caucasicus callus cultures 45


7/7

A. Sedifolius, the best result was obtained with leaf

explants collected from in vivo grown plants and

cultivated on medium containing 0.44 mg l71 of 2,4-

D and 0.22 mgl71 of BAP under light conditions; for

A. Caucasicus, the best callus was obtained from

in vitro root explants growing on medium containing

0.1 mg l71 of 2,4-D. Furthermore, our experiments

demonstrated that Aster callus cultures produceantioxidants and might be used as a model system

to investigate the regulation and production of these

metabolites.

Acknowledgments

The authors thank Dr. Di Matteo Antonio for

assistance in metabolites analysis and Mrs. Lotti

Elvira for her technical assistance. This work was

supported by a grant from the Ministry of Agricul-

tural, Food and Forestry Policies (MiPAAF), Italy,

as part of the BIOPEST project and contribution

from DISSPAPA n. 218 is also acknowledged.

References

Akbas F, Isikalan C, Namli S. 2009. Callus induction and plant

regeneration from different explants of Actinidia deliciosa. Appl

Biochem Biotechnol 158: 470475.

Baba I, Inada K. 1956. On the oxidationreduction potential of

tissue fluid of rice plant. Nippon Sakumotsu Gakkai Kiji 25:

7577.

Bauer N, Leljak-Levanic D, Jelaska S. 2004. Rosmarinic acid

synthesis in transformed callus culture of Coleus blumei Benth.

Z Naturforsch 59c: 554560.

Cammareri M, Errico A, Filippone E, Conicella C. 2001.

Screening of Aster wild germplasm for in vitro response toregeneration. J Genet Breed 55:255260.

Cammareri M, Errico A, Filippone E, Esposito S, Conicella C.

2002. Induction of variability in chimeric Aster cordifolius

White Elegans through somaclonal variation. Euphytica

128: 1925.

Dhar, U, Joshi, M. 2005. Efficient plant regeneration protocol

through callus for Saussurea obvallata (DC) Edgew (Aster-

aceae): Effect of explant type, age and plant growth regulators.

Plant Cell Rep 24: 195200.

Franceschi VR, Tarlyn NM. 2002. L-ascorbic acid is accumulated

in source leaf phloem and transported to sink tissues in plants.

Plant Physiol 130: 649656.

Gurney KA, Evans LV, Robinson DS. 1999. Purine alkaloid

production and accumulation in cocoa callus and suspension

cultures. J Exp Bot 43: 769775.Jabeen N, Shawl AS, Dar GH, Jan A, Sultan P. 2007.

Micropropagation of Inula racemosa Hook f. A valuable

medicinal plant. Int J Bot 3: 296301.

Jiang S, Duan J, Chen J, Tao J, Fu K. 2009. Research on callus

induction and cell suspension culture of Changium smyrnioides.

Zhongguo Zhong Yao Za Zhi/China J Chinese Materia Medica

34: 10781081.

Kakegawa K, Suda J, Sugiyama M, Komamine A. 1995. A

biochemical model for the initiation and maintenance of the

quiescent centre: Implications for organization of root mer-

istems. Physiol Plant 94: 661666.

Kampfenkel K, Van Montagu M, Inze D. 1995. Effects of iron

excess on Nicotiana plumbagnifolia plants implications to

oxidative stress. Plant Physiol 107: 725735.

Kato N, Esaka M. 1999. Changes in ascorbate oxidase gene

expression and ascorbate levels in cell division and cellelongation in tobacco cells. Physiol Plant 105: 321329.

Kerk NM, Feldman LJ. 1995. A biochemical model for the

initiation and maintenance of the quiescent centre: Implica-

tions for organization of root meristems. Development 121:

28252833.

Keskin N and Kunter B. 2008. Production of trans-resveratrol in

Cabernet Sauvignon (Vitis vinifera L.) callus culture in

response to ultraviolet-C irradiation. Vitis 47: 193196.

Moon HK, Stomp AM. 1997. Effects of medium components and

light on callus induction, growth, and frond regeneration in

Lemna gibba (Duckweed). In Vitro Cell Dev Biol Plant 33:

2025.

Murashige T, Skoog F. 1962. A revised medium for rapid growth

and bioassays with tobacco cultures. Physiol Plant 15: 473

497.

Ng TB, Liu L, Lu Y, Cheng CHK, Wanget Z. 2003. Antioxidant

activity of compounds from the medicinal herb Aster tataricus.

Comp Biochem Physiol C Pharmacol Toxicol Endocrinol 136:

109115.

Phillips LR and Henshaw GG. 1977. The regulation of synthesis

of phenolics in stationary phase cell cultures of Acer pseudopla-

tanus L. J Exp Bot 28: 785794.

Pourmorad F, Hosseinimehr SJ, Shahabimajd N. 2006. Antiox-

idant activity, phenol and flavonoid contents of some selected

Iranian medicinal plants. African J Biotech 5: 11421145.

Singh UP, Sarma BK, Singh DP, Bahaduret A. 2002. Plant

growth-promoting rhizobacteria-mediated induction of pheno-

lics in pea (Pisum sativum) after infection with Erysiphe pisi.

Curr Microbiol 44: 396400.

Singleton VL, Rossi JA Jr. 1965. Colorimetry of total phenolics

with phosphomolybdic-phosphotungstic acid reagents. Am JEnol Vitic 16: 144158.

Tiwari, AK. 2001. Imbalance in antioxidant defence and human

diseases: Multiple approach of natural antioxidants therapy.

Curr Sci 81: 11791187.

Uno Y, Nakao S, Yamai Y, Koyama R, Kanechi M, Inagaki N.

2009. Callus formation, plant regeneration, and transient

expression in the halophyte sea aster (Aster tripolium L). Plant

Cell Tissue Organ Cult 98: 303309.

van der Rest B, Danoun S, Boudet AM, Rochange SF. 2006.

Down-regulation of cinnamoyl-CoA reductase in tomato

(Solanum lycopersicum L.) induces dramatic changes in soluble

phenolic pools. J Exp Bot 57: 13991411.

Zadeh H, Keulemans RJ, Davey MW. 2007. Expression pattern of

key vitamin c biosynthesis genes in apple. Comm Appl Biol Sci

72: 269273.Zainol MK, Abd-Hamid A, Yusof S, Museet R. 2003. Anti-

oxidative activity and total phenolic compounds of leaf, root

and petiole of four accessions of Centella asiatica (L). Urban

Food Chem 81: 575581.

Zapata JM, Sabater B, Martn M. 2004. Callus induction and

in vitro regeneration from barley mature embryos. Biol

Plantarum 48: 473476.


Documents

Taylor et al. - Unknown - Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology Official Journal of the Societa Botanica Italiana Establishment o-annotated.pdf