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<strong>Agrobacterium</strong> <strong>mediated</strong> <strong>transformation</strong> <strong>and</strong> <strong>regeneration</strong> <strong>of</strong><br />

Cauliflower (Brassica oleraceae var. botrytis)<br />

Ashwini Kumar Ray 1 , Ashish Kumar Choudhary 1 ,<br />

S<strong>and</strong>hya Jha 2 , Indranath Mishra 1<br />

1 Department <strong>of</strong> Biotechnology, LN Mithila University, Darbhanga, Bihar 846004,<br />

India; Email: indralnmu@rediffmail.com; 2 Department <strong>of</strong> Botany, C.M. Science<br />

College, Darbhanga, Bihar 846004, India<br />

ABSTRACT<br />

Cotyledons <strong>and</strong> hypocotyls as an explants <strong>of</strong> in vitro aseptically grown seedlings <strong>of</strong> cauliflower were taken <strong>and</strong><br />

inoculated with A. tumefaciens strain LBA 4404 harboring a binary vector pbin Bt3 <strong>of</strong> size 12.60 Kb.<br />

The T-DNA <strong>of</strong> plasmid pbin Bt3 contains a Neomycin Phosphotransferase-Il gene (nptll) for<br />

kanamycin resistance, under Nopaline Synthase gene (NOS) promoter, <strong>and</strong> a Cry-1A(c) gene which is<br />

for developing insect resistant. Explants were transformed <strong>and</strong> regenerated on MS basal Media. The<br />

different concentration <strong>of</strong> BAP (benzyl aminopurine) was used with MS basal media for the <strong>regeneration</strong> <strong>of</strong><br />

transformed explant. The concentrations <strong>of</strong> BAP used were 0.2, 0.5 <strong>and</strong> 1.0 mg/L. Although a little bit<br />

callusing was observed on all the three BAP concentrations, but better <strong>regeneration</strong> was found from explants<br />

cultured on the media supplemented with 0.5 mg/L.<br />

Keywords: <strong>Agrobacterium</strong>, <strong>regeneration</strong>, <strong>transformation</strong>, Cry-1A(c) gene, explant<br />

INTRODUCTION<br />

Brassica oleraceae is a diverse group which includes many important vegetable crops; cauliflower<br />

is one <strong>of</strong> its important members. Due to its large nutritional value it is grown world wide in many<br />

countries like US, Italy, France, China <strong>and</strong> India (www.prota.org). Annual world production <strong>of</strong><br />

cauliflower was over 12.7 million metric tons in 1996, with China leading with more than 30% <strong>of</strong><br />

world production. But due to insect pest invasion the estimated loss is about 20-30% [1]. It is easily<br />

attacked by Lepidoptera pest such as Plutella xylostella <strong>and</strong> Pieris rapae. Chemical insecticide can<br />

reduce loss but residual chemical are harmful to human <strong>and</strong> ecosystem. Massive quantities <strong>of</strong><br />

insecticides are used especially in mid <strong>and</strong> late-season crop in the north Indian plains where<br />

incidence <strong>of</strong> pests is high.<br />

Traditional breeding methods have significantly enhanced productivity <strong>and</strong> quality <strong>of</strong> the crop,<br />

but insect-pests, for which no source <strong>of</strong> resistance is available, continue to cause severe damage.<br />

Environment <strong>and</strong> health concerns associated with the use <strong>of</strong> pesticides call for alternative methods<br />

<strong>of</strong> pest control. Hence genetic engineering experiments have to be undertaken so as to produce more<br />

ecologically <strong>and</strong> economically viable traits. Currently the most widely used method for transferring<br />

genes into the plants are A. tumefaciens <strong>mediated</strong> transfer [2] <strong>and</strong> the particle bombardment mainly<br />

for monocots [3], which involves a device called as gene gun, particle gun or microprojectile in<br />

which the gene to be introduced into the plant cells is coated as to tiny metal particle like gold <strong>and</strong><br />

tungsten <strong>and</strong> then physically shot on to target cells.<br />

Research Article, Biotechnol. Bioinf. Bioeng. 2012, 2(1):580-583<br />

© 2012 Society for Applied Biotechnology; pISSN 2249-9075, eISSN 2249-9938


Biotechnol. Bioinf. Bioeng. 2012, 2(1):580-583<br />

MATERIALS AND METHODS<br />

In vitro grown explant<br />

Cauliflower seeds were surface sterilized with sodium hypochlorite along with 5% cetrimide for 5-<br />

10 min after treatment with fungicide (Bavistin), rinsed with distilled water <strong>and</strong> placed over<br />

germination media <strong>of</strong> half strength MS [4]. The pH <strong>of</strong> media was set to 5.9 before autoclaving at<br />

120 0 C for 20 min. Cotyledons <strong>and</strong> hypocotyl were cut from in vitro grown 4, 6, 10, 15, 20 days old<br />

seedlings.<br />

Strains <strong>and</strong> plasmid<br />

<strong>Agrobacterium</strong> strain LBA 4404 harboring a binary vector pbin Bt3 <strong>of</strong> size 12.60 Kb. The T-<br />

DNA <strong>of</strong> plasmid pbin Bt3 contains a Neomycin Phosphotransferase-Il gene (nptll) for<br />

kanamycin resistance, under Nopaline Synthase gene (NOS) promoter, <strong>and</strong> a Cry 1A(c) gene<br />

which is for developing insect resistance, was used.<br />

Transformation<br />

500 ml <strong>of</strong> the overnight grown log phase culture <strong>of</strong> A. tumefaciens was used. The flask was<br />

agitated at 65 rpm, at 28°C, for either 30 min or 1 hr. The explant was co-cultured with<br />

overnight grown log phase culture <strong>Agrobacterium</strong>. After the period <strong>of</strong> incubation, the<br />

bacterial suspension was drained out. The explants were then blotted dry using a sterile tissue<br />

paper <strong>and</strong> co-cultivated on co-cultivation medium for 4-6 days. After co-cultivation, explants<br />

were washed three times with sterile distilled water. After this the explants were subjected to MS<br />

medium containing carbenicillin (500mg l -1 ) to prevent overgrowth <strong>of</strong> <strong>Agrobacterium</strong> for 6-7 days.<br />

If the carbenicillin alone was not sufficient to control bacterial growth then carbenicillin (500 mg l -<br />

1 ) was used in combination with cephotaxime (250 mg l -1 ).<br />

RESULTS AND DISCUSSION<br />

In vitro grown explants<br />

Seedling explants <strong>of</strong> Cauliflower are best for in vitro <strong>regeneration</strong>. Usually these explants are<br />

obtained from sterile in vitro germinated seedlings. Before germination, the seeds were surface<br />

sterilized <strong>and</strong> the different treatments used for the surface sterilization <strong>and</strong> their results are given in<br />

the table 1. Sprouting <strong>of</strong> the seeds usually starts after 3 days <strong>of</strong> inoculation. Contamination usually<br />

appears on an average after 5 to 7 days, if any. Among all the protocols followed the best protocol<br />

was A 5 followed by A 1 in terms <strong>of</strong> germination as well as sterilization (Table 1). After proper<br />

sterilization seed were grown on half strength MS medium [4].<br />

Regeneration from hypocotyls <strong>and</strong> cotyledons<br />

For the <strong>regeneration</strong>, hypocotyls <strong>and</strong> cotyledons explants from in vitro grown seedlings used. The<br />

different concentration <strong>of</strong> BAP (benzyl aminopurine) was used with MS basal media for the<br />

<strong>regeneration</strong>. The concentrations <strong>of</strong> BAP used were 0.2 mg/L, 0.5 mg/L <strong>and</strong> 1.0 mg/L. The explants<br />

were sub-cultured after 15 days. Although a little bit callusing was observed on all the three BAP<br />

concentrations, better <strong>regeneration</strong> was found from hypocotyls explants cultured on the media<br />

supplemented with 0.5 mg/L. This result is comparable to the result <strong>of</strong> Bhalla <strong>and</strong> Smith, who found<br />

that hypocotyl explants expressed the highest adventitious bud <strong>regeneration</strong> capacity [5]. Bhalla <strong>and</strong><br />

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Biotechnol. Bioinf. Bioeng. 2012, 2(1):580-583<br />

Smith tested hypocotyls, cotyledon <strong>and</strong> root explants <strong>of</strong> the eleven Australian cauliflower genotypes<br />

<strong>and</strong> found that the root <strong>and</strong> hypocotyl explants were more responsive to shoot <strong>regeneration</strong> than that<br />

<strong>of</strong> cotyledon explants [6]. Cytokinin is the most critical phytohormone group for stimulating bud<br />

organogenesis from cauliflower explants. The optimum cytokinin concentration varies with<br />

genotype <strong>and</strong> explant types. On cytokinin containing shoot induction (<strong>regeneration</strong>) medium, callus<br />

develop from the explants. Adventitious buds subsequently develop from the callus. Shoot<br />

elongation <strong>and</strong> rooting <strong>of</strong> cauliflower adventitious buds can occur on basal medium without<br />

hormonal supplement. However, some workers used specific media for shoot elongation/rooting<br />

stages (Figure 1).<br />

(a)<br />

(b)<br />

(c)<br />

Figure 1. (a) In vitro germination <strong>of</strong> Cauliflower seed on half strength MS medium; (b) Cotyledon<br />

explants in <strong>regeneration</strong> medium with 50 mg/ml kanamycin; <strong>and</strong> (c) Hypocotyl explant in <strong>regeneration</strong><br />

medium with 50mg/ml kanamycin.<br />

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Biotechnol. Bioinf. Bioeng. 2012, 2(1):580-583<br />

Table 1. Cauliflower seeds treated with different concentration <strong>of</strong> antimicrobial substances at<br />

various time periods.<br />

Experiment Bavistin Sodium hypochlorite 0.1% DS SI SG Germination<br />

number<br />

% Time<br />

(min.)<br />

5%<br />

Cetrimide<br />

HgCl 2<br />

(min.)<br />

%<br />

A 1 + 5.0 10 + - 3 405 308 76.00<br />

A 5 + 20.0 5 + - 3 198 155 78.00<br />

A 9 + 7.5 5 + - 3 105 - -<br />

A 2 - 10.0 30 - - 7 98 54 55.00<br />

A 3 - 20.0 15 - - 6 92 56 60.46<br />

A 4 - 20.0 30 - - - 65 - -<br />

A 10 + 5.0 5 + 3 3 108 - -<br />

A 6 + 20.0 5 + 3 3 195 120 61.53<br />

A 7 - 10.0 5 + 5 7 68 - -<br />

A 8 - 10.0 5 + 10 3 69 - -<br />

DS: Days <strong>of</strong> sprouting; SI: Seeds inoculated; SG: Seeds germinated.<br />

Transformation<br />

Agrobactrium-<strong>mediated</strong> <strong>transformation</strong> <strong>of</strong> hypocotyl <strong>and</strong> cotyledon explants was carried out using<br />

the <strong>Agrobacterium</strong> strain LBA 4404. This Agrobacerium harbours the binary vector pbin Bt3 <strong>of</strong><br />

12.60kb containing the Cry 1A(c) gene. For the <strong>transformation</strong>, explants first kept on a preculturing<br />

medium for 2-5 days. The preculturing medium was supplemented with BAP 1 mg/L <strong>and</strong> Kinetin<br />

0.5 mg/L. The explants were co-cultured with the <strong>Agrobacterium</strong> strain for 2 days, <strong>and</strong> then they<br />

were washed with the washing media (MS media without sucrose <strong>and</strong> agar). After washing the<br />

explants were cultured on selection media containing Kanamycin (50 mg/L) for selection <strong>of</strong><br />

transformed explants. During the process <strong>of</strong> selection the successfully transformed explants were<br />

able to grow whereas the non-transformed explants failed to grow. Most <strong>of</strong> the successful<br />

cauliflower <strong>transformation</strong> reports were based on <strong>Agrobacterium</strong>-<strong>mediated</strong> <strong>transformation</strong> <strong>of</strong><br />

seedling explants using binary plasmid [5,7]. It was found that explants from 4-day old seedlings<br />

were better than from 7- to 14-day old seedlings, as very few green shoots recovered when older<br />

seedlings were used [6]. They used hypocotyls <strong>and</strong> cotyledon explants for the <strong>transformation</strong>.<br />

Although the hypocotyls produced as many as twice the number <strong>of</strong> shoots per explant compared to<br />

cotyledon explants, higher numbers <strong>of</strong> transformed plants were obtained from cotyledon explants<br />

(Figure 1). They have concluded that cotyledon from young seedling was the best target for<br />

<strong>transformation</strong> <strong>of</strong> cauliflower; presumably such explants are more amenable to genetic<br />

<strong>transformation</strong>. Of course, such claim has to be confirmed by other workers too.<br />

REFERENCES<br />

[1] Estruch JJ, Carozzi NB, Desai N, et al. Nat. Biotech. 1997, 15:137-141.<br />

[2] Chilton MD, Drummond MH, Merlo DJ, et al. Cell 1997, 11:263-271.<br />

[3] Klein TM, Wolf ED, Wu R, Sanford JC. Nature 1987, 327:70-73.<br />

[4] Murashige T, Skoog F. Physiol. Plant. 1962, 15:473-498.<br />

[5] Ding LC, Hu CY, Yeh KW, Wang PJ. Plant Cell Rep. 1998, 7:854-860.<br />

[6] Bhalla PL, Smith N. Mol. Breed. 1998. 4:531-541.<br />

[7] De Block M, Brouwer D, De Tenning P. Plant Physiol. 1989, 91:694-701.<br />

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