IN VITRO PLANT REGENERATION STUDIES IN BRINJAL

Journal of Cell and Tissue Research Vol. 12(2) 3213-3218 (2012) ISSN: 0974-0910 (Available online at www.tcrjournals.com) Original Article IN VITRO PLANT REGENERATION STUDIES IN BRINJAL BARDHAN, S. K., SHARMA, C. 1 AND SRIVASTAVA, D. K. Department of Biotechnology, Dr Y.S. Parmar University of Horticulture and Forestry, Nauni, Solan 173230; 1 School of Agricultural Biotechnology, Punjab Agricultural University, Ludhiana 141 001 (Punjab) E. mail: chhaya_uhf@yahoo.co.in Received: March 10, 2012; Accepted: April 6, 2012 Abstract: The aim of this study was to develop an efficient protocol for establishment of in vitro plant regeneration through hypocotyl and cotyledon explants of Eggplant (Solanum melongena L.). The cotyledon explants showed high frequency of shoot regeneration (77.46%) as compared to hypocotyl explants on modified Murashige and Skoog s medium supplemented with 2.5 mg/l Kn + 0.4 mg/l IAA. Shoot elongation was carried out on same medium used for shoot regeneration. MS supplemented with 0.10 mg/l IAA was found best for root regeneration from in vitro raised shoots. The plantlets were able to regenerate within 8-10 weeks. The regenerated plantlets were acclimatized in pre-sterilized mixture of cocopeat. The protocol in the study might be useful for the production of disease free, healthy plant materials and also it would be useful for genetic transformation of eggplant using biotechnological approach. Key words: Plant regeneration, Brinjal,? INTRODUCTION Establishment of an efficient tissue culture protocol is an essential prerequisite in harnessing the advantage of cell and tissue culture for genetic improvement. It is an important tool, which can be used to improve productivity of crop via rapid availability of superior planting stock [1]. Brinjal or eggplant (Solanum melongena L.) is an agronomically important non-tuberous solanaceous crop grown primarily for its large oval fruit. In India total production of brinjal is 10,378 000 tonnes. More than 4 million acres (2,043,788 hectares) are devoted to the cultivation of brinjal in the world [2]. Biotechnology is a novel approach, which includes a range of techniques, that improved numerous economically important plants and food crops during the last 20 years [3]. The ability to regenerate plant is crucial to the successful application of in vitro methods. A number of reports have been published describing the plant regeneration of a variety of brinjal cutivars [3-5]. In this paper, we described a protocol for plant regeneration in brinjal using hypocotyl and cotyledon explants. The aim of developing an efficient plant regeneration system for brinjal to provide a useful system for producing genetically modified plants through genetic transformation. MATERIALS AND METHODS Plant material: Hypocotyl and cotyledon explants of brinjal were obtained from the 10-12 days old in vitro grown seedlings. The explants were sterilized with 0.1 per cent mercuric chloride for 60 seconds under laminar flow. They were then washed 3-4 times with distilled water to remove traces of mercuric chloride. The explants cut into 0.5-1.0 cm size and cultured on MS basal medium containing different concentration of auxins and cytokinins, MS salts (macro and micro), vitamins supplemented with 100 mg/l meso-inositol, 3% sucrose and 0.8% agaragar were used as basal medium [6]. After inoculation (plant tissues), the cultures were kept in culture room at 26 ± 2 0 C with 16 h photoperiod. Shoot regeneration from hypocotyl and cotyledon explants: Hypocotyl and cotyledon explants were cultured on MS basal medium supplemented 3213

J. Cell Tissue Research with various combinations and concentrations of plant growth regulators such as BAP-IAA and Kn-IAA. Explants were evaluated for average number of shoots per explants and percent shoot regeneration. After culturing the culture vessels were kept in culture room at 26 ± 2 0 C for 4 weeks. The regenerated shoots sub cultured on fresh medium for shoot multiplication and elongation medium. Multiplication and elongation of shoots: Multiplication and elongation of shoots were obtained on same shoot regeneration medium containing different concentration and combinations of growth regulators. Root regeneration and hardening of in vitro developed shoots: The regenerated shoots which were obtained from cotyledon and hypocotyl explants were transferred to root regeneration medium having MS basal medium with various concentrations of different auxins (IAA, NAA and IBA) to get complete plantlets. In vitro developed plantlets were taken out and washed gently under running tap water to remove adhering agar media. The plantlets were transplanted to the pots containing potting mixture (sterilized cocopeat by autoclaving at 15lbs/inch 2 pressure for one hour at 121 0 C). The plantlets were watered with 0.5 percent bavistin solution and kept under varying conditions of humidity and light intensity and observed for growth/survival. RESULTS Sterilization of explants: For the regeneration of shoots, hypocotyl and cotyledon explants of Brinjal cv. Pusa purple long were used as the explants. The explants were treated with 0.1% HgCl 2 for 60 second. It was observed that with increase in sterilization time and concentration the percentage of explant survival decreased and with reduction in sterilization time and concentration the percentage contamination of explants increased. Shoot regeneration from hypocotyl explants: After surface sterilization the hypocotyl explants (0.5-1.0 cm) were inoculated on various regeneration media supplemented with different concentrations and combinations of BAP-IAA and Kn-IAA. i) Effect of BAP and IAA on shoot regeneration from hypocotyl explants: Two different concentrations of BAP-IAA were used for shoot regeneration. Callus initiation was observed after 12-14 days on both media. Shoot initiation started after 26 days at cut ends of explant through callus formation. The maximum shoot regeneration (57.17%) and average number of shoots per explants (0.6) were observed on MS basal medium containing 2.5 mg/l BAP + 0.5 mg /l IAA (Table 1; Fig. 1). ii) Effect of Kn and IAA on adventitious shoot regeneration from hypocotyl explants: Three different concentrations and combinations of Kn-IAA were used for shoot regeneration. The callus initiation was observed after 14 days and shoot initiation started after 22-24 days at cut ends of explant through callus. Out of three media, the maximum shoot regeneration (51.26%) and average number of shoots per explants (0.3) were observed on MS basal medium containing 2.0 mg/l Kn + 0.3 mg/l IAA (Table 2) Shoot regeneration from cotyledon explants: After surface sterilization the cotyledon explants (0.5-1.0 cm) were inoculated on various regeneration media supplemented with different concentrations and combinations of BAP-IAA and Kn-IAA. i) Effect of BAP and IAA on shoot regeneration from cotyledon explants: Two different concentrations of BAP-IAA were used for shoot regeneration from cotyledon explants. The callus induction was reported in both media and callus initiation was observed after 10-12 days of culturing. Shoot regeneration was observed after 21 days in combination of BAP-IAA. The higher shoot regeneration (42.7%) and average number of shoots per explants (0.6) were observed on MS medium containing 2.0mg/l BAP and 0.3mg/l IAA (Table 3). ii) Effect of Kn and IAA on shoot regeneration from cotyledon explants: Three different concentrations of Kn-IAA were used for shoot regeneration. Callus initiation was observed after 8- Table 1: Effect of various combinations and concentrations of BAP and IAA in MS basal medium used for shoot regeneration from hypocotyl explants in brinjal (Solanum melongena L. cv. Pusa Purple long). + Callus formation, ++ Good callus formation Sr. No. Medium composition Callus formation Average number of shoot formed per explants Percent shoot regeneration 1. MS basal + BAP (2.5mg/l) + IAA (0.5mg/l) + 0.67 ± 0.091 57.17 ± 0.117 2. MS basal + BAP (2.5mg/l) + IAA (0.3mg/l) ++ 0.58 ±.095 42.64 ± 0.085 3214

Bardhan et al. E F Fig 1: (A-F) Plant regeneration studies in Brinjal ((Solanum melongena L. cv. Pusa Purple long) from hypocotyl and cotyledon explants. A) Shoot regeneration from hypocotyl explants on shoot regeneration medium [MS (basal medium) +2.5mg/l BAP +0.5mg/l IAA] after 30 days in culture. B) Shoot multiplication and elongation on shoot multiplication [MS (basal medium) +2.5mg/BAP + 0.5mg/l IAA] after 36days in culture. C) Shoot regeneration from cotyledon explants on shoot regeneration medium [MS (basal medium) +2.5mg/l Kn + 0.4mg/l IAA] medium after 30 days in culture. D) Shoot multiplication and elongation on shoot multiplication [MS (basal medium) +2.5mg/l Kn +0.4mg/l IAA] medium after 36 days in culture. E) Regenerated shoots transferred to root regeneration medium [MS (basal medium) + 0.10 mg/l IAA] at 0 day in culture. F) Development of complete plantlet after 22-25 days on root regeneration medium [MS (basal medium) + 0.10 mg/l IAA] 3215

J. Cell Tissue Research Table 2: Effect of different combinations and concentrations of Kn and IAA in MS basal medium used for shoot regeneration from hypocotyl explants in brinjal (Solanum melongena L. cv. Pusa Purple long). + Callus formation, +++ Very good Callus formation Sr. No. Medium composition Callus Average number of shoot Percent shoot formation formed per explants regeneration 1. MS basal + kn (2.0mg/l) + IAA (0.3mg/l) + 0.39 ± 0.102 51.26 ± 0.135 2. MS basal + kn (2.5mg/l) + IAA (0.3mg/l) +++ 1.26 ± 0.101 40.23 ±0.135 3. MS basal + kinetin (2.5mg/l) + IAA (0.4mg/l) +++ 0.29 ± 0.100 40.23 ± 0.130 Table 3: Effect of different combinations and concentrations of BAP and IAA in MS basal medium used for shoot regeneration from cotyledon explants in brinjal (Solanum melongena L. cv. Pusa Purple long). + Callus formation, ++ Good callus formation Sr. No. Medium composition Callus Average number of Percent shoot formation shoot formed per explants regeneration 1. MS basal + BAP (2.0mg/l) + IAA (0.3mg/l) ++ 0.64 ± 0.145 42.70 ± 0.076 2. MS basal + BAP (2.5mg/l) + IAA (0.3mg/l) ++ 0.46 ± 0.101 37.23 ± 0.130 Table 4: Effect of various combinations and concentrations of Kn and IAA in MS basal medium used for shoot regeneration from cotyledon explants in brinjal (Solanum melongena L. cv. Pusa Purple long). + Callus formation, + + Good callus formation, +++ Very good callus formation Sr. No. Medium composition Callus formation Average number of shoot formed per explants Percent shoot regeneration 1. MS basal + kn (2.5mg/l) + IAA (0.3mg/l) ++ 1.25 ± 0.144 68.49 ± 0.153 2. MS basal + kn (2mg/l) + IAA (0.3mg/l) +++ 0.30 ± 0.132 22.75 ± 0.104 3. MS basal + kn(2.5mg/l) + IAA (0.4mg/l) + 2.62 ± 0.111 77.46 ± 0.249 Table 5: Effect of different concentrations of various auxins on per cent root regeneration from in vitro developed shoots of brinjal (Solanum melongena L. cv. Pusa Purple long). * All values in parenthesis are arc transformed values Sr. No. Medium composition Average number of roots Percent root formed per explants regeneration (%) 1. MS basal medium + 0.05 mg/l IAA 8.7 65.95 ( 54.31)* 2. MS basal medium + 0.10 mg/l IAA 10.62 86.43 (68.40) 3. MS basal medium + 0.05 mg/l IBA 6.1 53.58 (47.06) 4. MS basal medium + 0.10 mg/l IBA 7.1 73.73 (59.17) CD0.05 SE± 1.22 1.42 0.37 0.50 10 days on both media. Shoot initiation was observed within 22 days on combination of Kn-IAA. The higher shoot regeneration (77.46%) and average number of shoots per explants (2.6) were observed on MS medium containing 2.5mg/l Kn and 0.4mg/l IAA (Table 4, Fig 1). Multiplication and elongation: The regenerated shoots from both the explants were separated and subcultured on same best shoot regeneration medium containing growth regulators. Elongated shoots were obtained after 55-60 days subculturing. Root regeneration and hardening of in vitro plantlets: The regenerated shoots obtained from both the explants were rooted on the MS basal medium with various concentrations of auxins. Root initiation took place after 20-22 days of inoculation and well developed root system was attained in 4-5 weeks duration in both hypocotyl and cotyledon explants. The maximum percentage of root regeneration was recorded as 86.43 percent on MS medium containing 0.10mg/l IAA (Table 5, Fig 1). After root regeneration plantlets were transferred to the plastic pots containing pre-sterilized cocopeat mixture filled to three fourth of its capacity after 9-10 days. 75% survival of plants was observed during acclimatization. Flowering in tissue culture raised plantlets was observed after 60-65 days under glasshouse conditions (Fig 2). DISCUSSION Plant regeneration is a crucial aspect of plant tissue culture methodology that facilitates the production of genetically engineered plants, somaclonal variants and the rapid of difficult to propagate species. For genetic improvement of brinjal by the techniques of genetic transformation, a reproducible in vitro regeneration system is required. A number of factors 3216

Bardhan et al. F G Fig. 2: (A-G) Hardening of in vitro developed plantlets of Brinjal (Solanum melongena L. cv. Pusa Purple long). A) Regenerated plantlet of strawberry showing healthy root system. B) In vitro regenerated plantlets kept for hardening in pre-sterilized cocopeat mixture at 0 day. C) In vitro regenerated plantlets kept for hardening in pre-sterilized cocopeat mixture covered with transparent polythene bags at 0 day. D) In vitro regenerated plantlets transferred to potting mixture (containing sand + soil + FYM) at 0 day. E) Successful hardened plants showing induction of floral buds after about 40-45 days of transfer to soil. F) Successful hardened plants showing healthy flowers after about 60 days of transfer to soil. G) In vitro grown plants of brinjal showing flowering after 60-65 days in green house 3217

J. Cell Tissue Research such as genotype, culture medium, plant growth regulators and their combinations, physical environment and explant development stage affect adventitious shoot regeneration. The aim of present investigation was to determine high frequency shoot regeneration in the brinjal. Plant growth regulator s balance is the key factor in the regulation of morphogenesis in cultured explants. Furthermore, intrinsic plant growth regulators levels in explants make it respond better on a particular ratio and concentration of plant growth regulator(s) supplemented in culture medium depends upon genotype and crop [7]. Therefore, various combinations of plant growth regulators exhibit wide range of response. Out of the various concentrations and combinations of plant growth regulators tried (in MS medium), high percentage of shoot regeneration (77.46%) in brinjal was achieved from cotyledon explants on MS medium containing 2.5 mg/l Kn and 0.4mg/l IAA after 20-24 days in culture. The regenerated shoots elongated on the same medium has also obtained high frequency shoot regeneration from cotyledon explants [8]. Callus mediated regeneration using various concentrations of cytokinins (BAP) and auxins (IAA or NAA) have been reported in both hypocotyl and cotyledon explants in brinjal [9]. In several other experiments a range of kinetin and TDZ either alone or in combination with NAA or IAA had been used which resulted in callus mediated regeneration response in brinjal [10-13]. Various workers had also used the cotyledon explants for direct and indirect shoot regeneration in brinjal [12,14,15]. Study shows that hypocotyl explants also produce callus along its surface but it failed to produce shoots on some medium compositions and became brown in color. Occasionally some hypocotyl explants initiated the development of shoots at cut ends. High percentage of shoot regeneration (57.17%) from hypocotyl explants was achieved on MS medium containing 2.5 mg/l BAP and 0.5mg/l IAA after 26 days of inoculation. The regenerated shoots elongated on the same medium. In brinjal various workers obtained direct and indirect organogenesis from hypocotyl explants by manipulating the growth regulating substances in culture medium [10, 12,14,16-20]. They also reported low regeneration efficiency with hypocotyl explants. Only, Prakash et al. [4] reported high shoot regeneration frequency from hypocotyl explants. Different auxin i.e. IAA, NAA and IBA of various concentrations were used for root regeneration from in vitro developed shoots of brinjal. Root regeneration was observed after 20-22 days in culture on MS medium containing IAA and IBA. Only callus was formed at the base of shoots on medium supplemented with NAA. High percentage of root regeneration (86.56%) was observed in the medium containing 0.10 mg/l IAA. The regenerated plantlets of brinjal were acclimatized and transferred to glasshouse for further growth and development. 75% plantlets had survived and all the plantlets were morphologically normal. Flowering was observed after about 60 days of transfer to soil. REFERENCES [1] Bhatia, P., Ashwath, N. and Senaranta, T.: In: In vitro culture, transformation and molecular markers for crops improvement (Islam, A.S. eds), Science publishers, Inc. Enfield, NH, USA, pp 17-24 (2004). [2] FAO (Food and Agriculture Organization) FAO Yearbook Production. Food and Agriculture Organization of the United Nations. Rome, Italy (2008). [3] Ferdausi, A., Nath, U.K., Das, B.L. and Alam, M.S.: J. Bangladesh Agri. Uni., 7(2): 253-258 (2009). [4] Prakash, D.P., Deepali, B.S., Asokan, R., Ramachandra, Y.L., Shetti, D.L., Anand, L. and Hanur, V.S. : Indian J. Hort., 65(4): 371-376 (2008). [5] Shivaraj, G. and Rao, S.: Curr. Trends Biotechnol. Phar., 4(3): 156-162 (2010). [6] Murashige, T. and Skoog, F.: Physiol. Plantar., 15: 211-218 (1962). [7] Christianson, M. and Warnick, H.: In Vitro Dev. Biol., 112: 494-497 (1985). [9] Sharma, P. and Rajam, M.V.: J. Exp. Bot., 46 (282): 135-141 (1995). [8] Sarker, R.H., Yesmin, S. and Hoque, M.I.: Plant Tiss. Cult. Biotechnol., 16 (1): 53-61 (2006). [10] Matsuoka, H. and Hinnata, K.: J. Exp. Bot., 30(11): 363-370 (1979). [11] Mukherjee, S.K., Rathinasabapathi, B. and Gupta, N.: Plant Cell Tiss. Org. Cult., 25(1): 13-16 (1991). [12] Magioli, C., Rocha, A.P.M. and Mansur, E.: Plant Cell Rep., 17(8): 661-666 (1998). [13] Gleddie, S.C., Keller, W. and Setterfield, G.: Canadian J. Bot., 61(3): 656-666 (1983). [14] Alicchio, R., Grosso, F., Del, H. and Roschieri, F.: Experientia, 38(4): 449-450 (1982). [16] Kamat, M.G. and Rao, N.A.: Plant Sci. Lett., 13: 57-65 (1978). [17] Picoli, E.A.T., Cecon, P.R., Fan, M.G. and Otoni, W.C.: Inter. J. Hort. Sci., 8(2): 76-82 (2002). [18] Neelima, P. and Reddy, K.J.: Adv. Plant Sci., 18: 457-466 (2005). [19] Khatun, F., Meah, M.B. and Nasiruddin, K.M.: Pakistan J. Biol. Sci., 9(1): 48-53 (2006). 3218