Indian Journal of Biotechnology Vol 15, January 2016, pp 17-24

Genetic transformation of chilli (Capsicum annuum L.) with Dreb1A transcription factor known to impart drought tolerance Manamohan Maligeppagol*, R Manjula, Prakash M Navale, K Prasad Babu, Bhimanagoud M Kumbar and R H Laxman Division of Biotechnology, Indian Institute of Horticultural Research (IIHR), Hessaraghatta Lake Post, Bangalore 560 089, India Received 21 September 2014; revised 29 January 2015; accepted 13 March 2015 Chilli, an important commercial crop, is recalcitrant to in vitro regeneration. In the present study, an attempt has been made to optimize the in vitro regeneration of two chilli local cultivars, viz., G4 and LCA334, and transform G4 with a transcription factor dreb1A under the control of a desiccation inducible promoter rd29A, known to impart desiccation tolerance, using binary vector pCAMBIA 2301. Different phytohormones and their concentrations in MS medium were evaluated for in vitro response; 0.25 mg L-1 zeatin and 2 mg L-1 phenyl acetic acid (PAA) supported the highest regeneration rate (37.86%). Among the explants, cotyledonary leaf exhibited a higher (51%) regeneration response compared to that of hypocotyls (24.73%), and genotype G4 had better (33.80%) regeneration rate compared to LCA334. Rooting of the shoots was the highest in MS medium with 2 mg L-1 indole butyric acid (IBA) compared to other hormones. The presence of transgene was confirmed by polymerase chain reaction (PCR) and Southern blotting. The acclimatized transformants were grown in pots and screened for drought tolerance. Some of the transformants showed improved tolerance to drought by lower wilting compared to the control plants. The transformation and regeneration protocol described in the present study may help in optimization of transformation of chilli for agronomic traits of interest. Keywords: Capsicum annuum, dreb1A, drought tolerance, rd29A promoter, transformation

Introduction Chilli (Capsicum annuum L.) (Family: Solanaceae) is cultivated throughout world as a spice and vegetable. Its extract is used in food, cosmetics and pharmaceutical industries. India is the largest producer and consumer of the chilli in the world, with a production of 13 lakh metric tons from a cultivable area of 7.9 lakh ha (Indian Agriculture Database, 2012-13). The commercial importance of chilli fuelled considerable progress in crop improvement and efforts are underway through conventional breeding as well as biotechnological interventions. The biotechnological approach needs to be utilized for hastened and focused genetic enhancement of the crop. However, the members of the genus Capsicum are known to be recalcitrant for in vitro manipulations and lag far behind other economically important members of Solanaceae, such as, tobacco, tomato and potato, in developments in the area of in vitro regeneration and genetic transformation1,2. It is mainly because of the lack of reliable, efficient and reproducible regeneration methods3. —————— *Author for correspondence: Te: +91-80-28466420; Fax: +91-80-28466291 [email protected]

During the past decade, researchers have made great progress worldwide towards chilli regeneration and transformation4. Transgenic chilli plants expressing the cucumber mosaic virus (CMV) coat protein gene and CMV satellite RNA5 were obtained but with low regeneration and transformation efficiencies and the published protocols were hard to reproduce3. The in vitro response of chilli is poor and problems in regeneration are due to the severe recalcitrance, morphogenic nature, formation of rosette shoots or abnormal shoot buds, genotype dependent in vitro response etc., which have hindered the whole tissue culture efforts. A few reports with a relatively lower regeneration rate in chilli have been reported6,7. For the first time, Agrobacterium-mediated transformation in chilli, using hypocotyls, cotyledons and leaves, was reported by Liu et al8 with the wild tumorigenic strains A281 and C58. After that a number of transformation reports were published in different cultivars of chilli4-7. Using callus induced transformation (CIT) method, a transformation efficiency of 0.43-0.66% was obtained, but CIT method required a highly skillful manpower9. Regeneration and transformation of chilli for genetic improvement has been limited because of the difficulties in

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INDIAN J BIOTECHNOL, JANUARY 2016

efficient development of induced multiple shoots into complete plants. Chilli is mostly grown under rainfed cultivation and the crops are often affected by low moisture stress leading to inconsistent yield and quality. Thus, enhancement of drought tolerance is an important objective in chilli crop improvement. dreb1A is known to impart multiple stress tolerance to drought, cold and salinity stress10. In this study dreb1A transcription factor, driven by a desiccation inducible promoter rd29A was introgressed in to chilli. DREB1A protein binds to the dehydration responsive element (DRE) sequence “TACCGACAT” in the promoter regions of the downstream target genes and upregulates their expression. The target genes coding for stress responsive proteins, such as, heat shock protein 70/HSP70 (HSC70-7), stress-responsive protein (KIN2)/stress-induced protein (KIN2)/coldresponsive protein (COR6.6)/cold-regulated protein (COR6.6), low-temperature-responsive protein 78, Glycosyl transferase family 8 protein, Galactinol synthase, Cold-acclimation protein etc., and imparts the stress tolerance11,12. In view of this, the present study was undertaken to develop a reliable protocol for plant transformation to enhance the drought tolerance of the chilli by transformation with dreb1A transcription factor known to impart desiccation tolerance in a number of crops. Material and Methods Preparation of Gene Construct

Dreb1A was isolated from Arabidopsis thaliana ecotype Columbia and was placed under the control of a desiccation inducible promoter, rd29A (NCBI acc. no. DQ 018385). The promoter and genes were cloned directionally in pBluescript II (Stratagene) using the restriction enzymes KpnI-SalI for the rd29A and SalI-HindIII for dreb1A gene. Then synthetic construct containing the rd29A:dreb1A was placed in the binary vector pCAMBIA 2301 (Fig. 1). Mobilization of the gene construct from pBluescript II to pCAMBIA 2301 and was performed by restriction digestion of the gene construct by HindIII and blunting was carried out using T4 DNA polymerase, followed by restriction digestion with KpnI. The gel eluted 1.14 Kb insert was inserted into the pCAMBIA 2301 and restriction digested by KpnI and PmlI restriction enzymes by replacing the CaMV 35S promoter and gusA gene in front of the nos terminator. The primer sequences used were: rd29

forward primer, 5′-gaccggtaccAAAGAGCCACA CGACGTAAACGTA-3′ and rd29 reverse primer, 5′-acgcgtcgacTGAGTAAAACAGAGGAGGGTC TCA-3′; Dreb1A forward primer, 5′-acgccgtcgac CCTGAACTAGAACAGAAAGAGAGA-3′ and Dreb1A reverse primer, 5′-gccggaagcttGAGTTTTAA TAACTCCATAACGA-3′ (in the primer, sequence in bold italics lower case depict the respective restriction sequences and letters in lower case the anchor sequences for efficient restriction digestion). All the primers used in the experiment were from Eurofins India Pvt. Ltd. The construct was sequenced and was mobilized into A. tumefaciens LBA4404 by electroporation, which was used for transformation of chilli. Hormones and Antibiotics Used for Chilli Transformation

Phytohormones, such as, zeatin, 1-naphthaleneacetic acid (NAA), thidiazuron (TDZ), indole-3-acetic acid (IAA) and indole butyric acid (lBA), were procured from Sigma-Aldrich (India). All the phytohormones were prepared using small quantity of either 0.1 N NaOH or ethanol for dissolving and then sterilized distilled water was used as diluent to make up the required volume. The stocks were filter sterilized and stored at –20°C. The concentrations of the stocks were: 0.5 mg L-1 zeatin, 1 mg L1 IBA, 1 mg L-1 IAA, 1 mg L-1 TDZ and 1 mg L-1 NAA. Similarly antibiotics were prepared in the concentration of 100 mg mL-1 and 300 mg mL-1 for kanamycin (Macleods Pharma Ltd. India) and augmentin™ (amoxicillin and potassium clavulanate) (Glaxo SmithKline, India), respectively and were added after the medium was cooled down to approx 50°C after autoclaving (Table 1). Different concentration of zeatin, such as, 0.1, 0.5 and 1 mg L-1 were added to the MS medium to select the best hormone concentration for shoot regeneration. Chilli Genotypes and Explants

Two open pollinated cultivars commercially cultivated in South India, viz., G4 and LCA334, were chosen for the study. Chilli seeds were obtained from the Division of Vegetable crops, Indian institute of

Fig. 1—Schematic representation of T-DNA region of pCAMBIA 2301, containing rd29A:dreb1A gene construct and neomycin phosphotransferase II (nptII) as selection marker, linked to the CaMV 35S promoter and nos terminator.

MALIGEPPAGOL et al: DEVELOPMENT OF DROUGHT TOLERANT TRANSGENIC CHILLI

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Table 1—Chilli regeneration media components used to develop transgenic chilli Components Media Seed germination medium Pre-culture Medium Co-culture medium Agrobacterium elimination medium (AEM) Selective regeneration medium (SRM) Shoot elongation medium Rooting medium

MS salts

Sucrose (g L-1)

Agar (g L-1)

Gelrite (g L-1)

0.5× 1× 1×

0 30 30

7.0 0 0

0 3.0 3.0

0 0.25 0.25

0 2 2

0 0 0

0 0 0

0 0 0

0 0 0

1×

30

0

3.0

0.25

2

0

0

0

300

1×

30

0

3.0

0.25

2

0

0

100

300

1× 0.5×

30 15

0 0

3.0 1.5

0 0

0.05 0

0 0.5

0.05 0

100 0

300 300

Horticultural Research, Bangalore. Seeds were surface sterilized sequentially with 70% ethanol and 4% sodium hypochlorite for 5 and 10 min, respectively and rinsed with sterile water 3 to 4 times. The washed seeds were blot dried on sterile tissue towels before sowing. Half strength MS medium devoid of vitamins was used. After adjusting the medium at pH 5.8, 0.7% of agar was added and it was steam sterilized at 121°C for 20 min. About 35 to 40 seeds were sown in the about 250 mL capacity bottles. Initially the seeds were incubated in dark at 25°C for 2 d, then shifted to light/dark cycle of 16/8 h with a light intensity of 40-60 µmol m-2 s-1. The in vitro grown seedlings were used as source of explants (cotyledonary leaves & hypocotyls) for transformation. Standardization of Transformation Protocol in Chilli Agrobacterium Strain and Plasmid Vector

In the present study, Agrobacterium-mediated in vitro transformation method was employed for gene transfer. The disarmed Agrobacterium strain LBA4404 harboring pCAMBIA 2301 was used for in vitro transformation. pCAMBIA 2301 contained rd29:dreb1A gene construct and neomycin phosphotransferase II (npt II) as selection marker linked to the CaMV 35S promoter and nos terminator (Fig. 1). The Agrobacterium carrying dreb1A gene was maintained on the solid yeast extract mannitol agar (YEMA) medium containing 100 mg L-1 kanamycin with occasional subculture on fresh medium. For transformation, a single Agrobacterium colony was taken from the plate and inoculated in 100 mL of YEMA broth containing selective antibiotic and was incubated with shaking for 48 h at 28°C. The culture was adjusted to OD 0.8 at 600 nm by diluting with fresh sterile YEMA broth and was used for cocultivation.

Zeatin PAA IBA BAP Kanamycin Augmentin (mg L-1) (mg L-1) (mg L-1) (mg L-1) (mg L-1) (mg L-1)

Agrobacterium-mediated Transformation

Cotyledonary leaves and hypocotyls of 15-d-old seedlings were used as explants. Media components used for chilli transformation are listed in the Table 1. Cotyledonary leaves were trimmed at the tip and base, similarly hypocotyls were excised below the apical bud and above the roots. About two to three explants of hypocotyl approx 1 cm in length could be obtained per seedling and these were cultured on the pre-conditioning medium for 48 h. Then these explants were incubated in Agrobacterium cell suspension (OD 0.8 at 600 nm) for 15 min with 3 intermittent shakings in a clean-air bench. After incubation, explants were blot dried on sterilized tissue towel and explants were co-cultivated in Petri dish containing preculture medium and incubated for 48 h. Later to eliminate Agrobacterium from the explants, these were shifted on to the same medium containing antibiotic, augmentin 300 mg L-1 for 48 h. Then the explants were transferred onto shoot regeneration selection medium (SRM) containing kanamycin 100 mg Ll-1 and augmentin 300 mg L-1. The Petri dishes containing explants were incubated at around 25°C under 16/8 h light/dark cycle. Explants exhibiting regeneration response on SRM were subcultured onto fresh SRM medium every 3-4 wk (Fig. 2A & B). The regenerated shoots of about 1 cm length were excised and shifted to shoot elongation medium containing 0.05 mg L-1 BAP and 0.05 mg L-1 PAA (Fig. 2C). The elongated shoots were then transferred to rooting medium (Fig. 2D). The well rooted-shoots were acclimatized by transferring to polythene bags (12×6.5 cm2) containing steam sterilized mix of coco-peat and perlite (Soilrite™ Keltech Energies Ltd. Bangalore, India) supplemented with 5 mL of 0.25 strength MS salts (Fig. 2E & F). The top open-end of the polythene bag was stapled.

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INDIAN J BIOTECHNOL, JANUARY 2016

transformants were screened by PCR and the products were resolved on 1.2% agarose TAE (Tris acetate EDTA) gel and documented. Southern Blot Hybridization Analysis

Fig. 2 (A-I)—Transgenic chilli plants expressing dreb1A gene: (A & B) Shoot regeneration from cotyledonary leaves and hypocotyls; (C) Chilli shoots under elongation medium; (D) Elongated chilli shoots under rooting medium; (E & F) Complete regenerated plant; (G) Potted transgenic plants; (H) Drought tolerant chilli plants (indicated by arrow); & (I) Chilli transgenic plant produced profuse fruits with fleshy pulp and fleshiness was retained for longer periods compared to the control plants.

After 7 d, 10 mL of 0.25× MS salts was applied (Figs 2E & F). After about 2-3 wk, the acclimatized plants were transferred into pots containing potting mix and kept in a net house. The plants were irrigated (Fig. 2G), fertigated and plant protection measures were carried out as required. The flowers were bagged to minimize cross-pollination and fruits were harvested at red ripe stage for seed extraction. Molecular Analysis of Transformants DNA Isolation and PCR Screening

Genomic DNA was isolated from the T0 young leaves12. To screen the presence of transgene, PCR amplification of genomic DNA was performed using the rd29:Dreb1A specific primers, i.e., rd29 forward primer: 5′-AAAGAGCCACACGACGTAAACGTA-3′ and Dreb1A reverse primer: 5′-GAGTTT TAATAA CTCCATAACGA-3′. PCR reaction was performed in a 25 µL reaction volume containing: 100 ng DNA, 1.5 mM MgCl2, 200 µM dNTP’s, 10× buffer, 2 units of Taq DNA polymerase (Bangalore Genei Pvt. Ltd.) and 1 µM of each primer. The cycling parameter used for PCR were: initial denaturation at 95°C for 4 min, denaturation at 94°C for 30 sec, annealing at 52°C for 35 sec, extension at 72°C for 40 sec, and final extension was carried out at 72°C for 10 min. All the

Selected T1 plants were analyzed for the determination of copy number and transgene integration in the genome using the AlkPhos Direct Labeling and Detection System (GE Healthcare) with CDP-Star according to the manufacturer’s recommendations. Briefly, about 10 µg of DNA was restriction digested with BamHI and the reaction was separated on 0.8% agarose TAE gel and transferred to positively charged nylon membrane. For the preparation of DNA probe, PCR products were labelled with alkaline phosphatase (AlkPhos Direct Labeling and Detection System from GE Healthcare Life Sciences, India). CDP-Star was used for chemiluminescence detection and documented using X-ray film employing standard developing procedure. Phenotypic Screening for Improved Drought Tolerance

The progenies of the two T0 lines were screened for drought stress tolerance by withholding watering for 12-15 d and visual observation for wilting of leaf was recorded on daily basis. Statistical Analysis

For each experiment three replicates were maintained and statistical analyses were carried out by one-way ANOVA and Student’s ‘t’ test using Graphpad Prism version 5.0 statistical analysis software. The evaluation of difference between the treatments was considered at P>0.05 significance level. Results Shoot Regeneration of Chilli Transformants Regeneration via Callus

During the in vitro regeneration, all type of explants gave a regeneration response by forming callus at cut ends within 12 to 23 d. The calli formed varied in colour and composition; white friable, green compact and glossy, in some browning was noticed. Formation of shoot buds was more frequent in dark green callus (Figs 2A & B). The frequency of callus formation in both, cotyledonary leaf and hypocotyl explants was similar, but organogenesis was higher in the calli derived from cotyledonary leaves (Data not shown). Of the two cultivars, G4 exhibited a significantly higher callusing and the concomitant shoot regeneration compared to the LCA334.

MALIGEPPAGOL et al: DEVELOPMENT OF DROUGHT TOLERANT TRANSGENIC CHILLI

Effect of Hormones

The highest (37.8%) shoot regeneration response of transformants was observed in the combination of 0.25 mg L-l zeatin with PAA 2 mg L-1. However, only 8.0 and 3.0% shoot regeneration was achieved in 0.5 and 0.1 mg L-1 zeatin, respectively (Fig. 3A). The explants cultured in shoot regeneration medium exhibited callus formation at the cut ends, from where 5 to 6 shoots emerged (Figs 2A & B), which were like rosette and grew slowly. These shoots did not elongate further in the shoot regeneration medium and, therefore, were carefully cut and shifted to the elongation medium. In the elongation medium, these shoots elongated 1.5-3 cm within a period of 2-3 wk (Fig. 2C).

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compared to LCA334 with only 12.95% shoot regeneration efficiency (Fig. 3D). Rooting and Hardening of Putative Transformants

Eleven different hormonal combinations were screened for the selection of medium supporting highest rooting response (Table 2; Fig. 2D). It was observed that full strength MS medium supplemented with 2 mg L-1 IBA resulted in the highest (60.68%) rooting of the transformed shoots. Further higher or lower IBA concentrations supported poor rooting response. Other treatments either gave poor rooting response or no response. In general, root initiation in 2 mg L-1 IBA was observed after c 2 wk of incubation, while root formation was completed by 5th-6th wk.

Effect of Explant

Molecular Characterization of Putative Transformants

In both the genotypes, cotyledonary leaf explants yielded more shoots compared to the hypocotyls explants. In case of G4, cotyledonary leaf explants gave nearly double number of shoots (51% regeneration efficiency) as compared to hypocotyls explants, which had shown 24.7% regeneration efficiency (Fig. 3B). While in case of LCA334, shoot regeneration efficiency in cotyledonary leaves and hypocotyls was only 13.7 and 10.4%, respectively (Fig. 3C). Overall, cotyledonary leaf explants were significantly better in comparison to hypocotyls in terms of shoot regeneration efficiency (p>0.05). Genotype dependent regeneration response was noticed in chilli, as the genotype G4 exhibited a significantly higher regeneration efficiency of 33.80%

PCR and Southern Blot Hybridization Analysis

PCR amplification of rd29A:dreb1A (1.1kb) cassette was carried out to screen for the presence of the transgene in the transformed plants. Plasmid DNA and DNA from control (untransformed plant) served as positive and negative control, respectively. In primary transformants, PCR amplification confirmed the presence of rd29A promoter and dreb1A gene (Fig. 4A). Moreover, Southern hybridization confirmed the integration of the transgene in five randomly selected primary transgenic plants (Fig. 4B). Phenotypic Screening for Improved Drought Tolerance

The progenies of the two T0 lines were screened for drought stress tolerance by withholding watering for Table 2—Rooting response of chili shoots in different hormone combinations in MS medium Media name (MS basal+ hormone) M1 M2 M3

M4 M5

Fig. 3 (A-D)—(A) Effect of zeatin on shoot regeneration; (B & C) Regeneration efficiency between hypocotyl and cotyledonary leaf of G4 and LCA334; & (D) Shoot regeneration between two genotypes G4 and LCA334. [All the data plotted are the mean percentage of shoot regeneration of 10 replicates. Bar graph indicates % of shoot regeneration±standard error (p> 0.05)].

M6 M7 M8 M9 M10 M11

Hormone conc.

1/2 MS+0.5 mg L-1 IBA 1/2 MS+1 mg L-1 IBA 2 mg L-1 PAA+0.25 mg L-1 Zeatin+2 mg L-1 GA3+ 1 mg L-1 IBA 1/2 MS+2 mg L-1 IBA 1/2 MS+mg L-1 IBA+ 2 mg L-1 NAA 2 mg L-1 NAA 2 mg L-1 BAP+2 mg L-1 IAA 0. mg L-1 IBA+0.5 mg L-1 BAP+0.5 mg L-1 TDZ 2 mg L-1 IBA 5 mg L-1 IBA 10 mg L-1 IBA

Rooting response (%) 0 0 0

0 2.46 1.93 0 0 60.68 17.40 20.26

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INDIAN J BIOTECHNOL, JANUARY 2016

Fig. 4 (A & B)—(A) PCR amplification of primary transgenic chilli plants [M, Mol wt marker (Hyper ladder-I Bioline); Lanes 1-13, Transgenic plants; -Ve, Negative control (non-transformed control plant); & +Ve, Positive control]; & (B) Southern hybridization of the randomly selected transgenic plants.

12-15 d. The drought stressed plants were scored visually for wilting symptoms. Transgenic plants from the line C-2 showed improved tolerance to drought by lower wilting symptoms compared to the control plants (Fig. 2H). It was also observed that the drought tolerant plants were comparatively dwarf and produced profuse fruits with fleshy pulp and fleshiness was retained for longer periods compared to the control plants (Fig. 2I). Discussion Abiotic stresses, especially salinity, drought, temperature and oxidative stress, are the primary causes of crop loss worldwide. Efforts are on to develop crop plants with improved tolerance to abiotic stresses. Genetic transformation promises avenues to rapidly introgress heterologous stress tolerance traits13. This requires availability of efficient and reliable plant transformation protocols. Chilli is considered as a recalcitrant crop for in vitro regeneration. Currently reliable transformation and regeneration protocols are limited to a few crops only and there is a need to extend this to many more crops. The present investigation was carried out to develop suitable regeneration and efficient transformation of local cultivar of chilli, viz., G4 and LCA334, and to transform with dreb1A gene through Agrobacteriummediated transformation for imparting drought tolerance. Plant hormones play an important role in plant growth and development. Type and concentration of cytokinin and auxin determine the type of organogenesis. In the present study, we used zeatin and PAA as the source of exogenous cytokinin and auxin, respectively to supplement the MS medium. Zeatin was reported to be more efficient in eliciting the maximum shoot bud induction compared to BAP (6-benzylaminopurine)14 in recalcitrant species. PAA is a naturally occurring auxin whose supplementation

led to enhanced shoot bud formation along with cytokinin. We observed that supplementation of 0.25 mg L-1 zeatin along with 2 mg L-1 PAA to MS medium resulted in the highest (38.7%) shoot regeneration (Fig. 3A). However, use of higher concentrations of zeatin was also reported, while, zeatin was effective in shoot bud induction only in C. frutescence but not in C. annuum15, thus indicating that role of genotype or species of Capsicum in phytohormone action and effect. Similarly, zeatin was also used for shoot bud induction in chilli cv. Pusa Jwala16. Our results are in agreement with the earlier reports14, in which zeatin was used as cytokinin source for the induction of shoots from the callus using cotyledonary leaf explants. There was a problem with shoot elongation in chilli when TDZ was used as a cytokinin source17 but its replacement with 0.05 mg L-1 BAP with low concentration of auxin enhanced the shoot elongation. However, in another experiment, the hormonal combination of 1 mg L-1 GA3 (which is normally employed for elongation) with 1 mg L-1 BAP resulted in excessive callusing and it did not support shoot elongation. In Capsicum, both apical and all nodal segments could be used as primary explants, but cotyledonary leaf and hypocotyls are the most commonly used explants for shoots regeneration18. A comparative study on in vitro regeneration of leaf explants14 revealed that the best regeneration obtained was from cotyledonary leaf explant. In our study, cotyledonary leaf explants were most effective, giving rise to a shoot regeneration of 51% and 13.7 % in cotyledonary leaves compared to 24 % and 10.4 % in hypocotyls in both the chilli cultivars G4 and LCA334, respectively (Figs 3B & C), also indicating genotype dependent regeneration response, which was almost three times higher in G4 than in LCA334 (Fig. 3D). These results are in agreement with previous report16, which reported better regeneration in cotyledonary leaves than in hypocotyls and leaf tissue explants. Cotyledonary leaves were the best source of explants observed18 and responded well to shoot regeneration in two C. annuum and C. frutescence cultivars. It is generally accepted in chilli that genotype or cultivar plays a major role in the in vitro response. Similarly, 17 chilli genotypes were screened for the shoot regeneration ability using different explants and reported that different genotypes exhibited different regeneration rates19. The in vitro transformants obtained in this study were from the explants pre-cultured for 48 h before

MALIGEPPAGOL et al: DEVELOPMENT OF DROUGHT TOLERANT TRANSGENIC CHILLI

co-cultivation with A. tumefaciens. It was opined that presence of actively dividing cells in the shoot apex is important for Agrobacterium-mediated transformation. Shoot elongation and rooting are considered to be the bottleneck in the transformation and regeneration of chilli. Recent reports suggest that, in elongation of the regenerated shoots, a number of inorganic salts, such as, copper sulfate20, silver nitrate and hormones like PAA, epi-brassinosteroids, phloroglucinol etc., have been found to be beneficial in enhancing shoot elongation21. Rooting of the elongated shoots was achieved in a cytokinin rich medium and IBA at 2 mg L-1 resulted in a better rooting rate of over 60 % compared to the MS medium devoid of IBA22. In the present study, in order to improve the drought tolerance in chilli, dreb1A transcription factor was introgressed in a drought inducible manner. The preliminary screening for drought tolerance of T0 and T1 transformants indicated that some of the transgenic lines have exhibited improved tolerance to drought in terms of lower wilting symptoms when irrigation was withheld for 10-15 days compared to the untransformed control and some transgenic lines, indicating that dreb1A transcription may be involved in improving the stress tolerance (Fig. 2H). However, more detailed analysis of the transgenic plants is necessary to conclude its contribution in abiotic stress tolerance. Nevertheless, this study shows that it is possible to genetically enhance chilli for various traits of interest, such as, abiotic and biotic stresses especially the fungal and virus disease for sustaining the crop yield.

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Acknowledgments Authors are grateful to the Director, Indian Institute of Horticultural Research (IIHR), Bangalore for providing the infrastructure facility and encouragement to carry out the research. We would like to thank Dr Madhavi Reddy, Division of Vegetable Crops, IIHR, Bangalore for providing seeds of chilli cultivars.

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