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Establishment of Cell Suspension Culture and Plantlet Regeneration of Brinjal (solanum Melongena L.)

M. J. Hossain
Department of Genetics Engineering and Biotechnology, University of Rajshahi, Rajshshi 6205, Bangladesh.

M. Rahman
Department of Genetics Engineering and Biotechnology, University of Rajshahi, Rajshshi 6205, Bangladesh.

M. A. Bari
Biotechnology Laboratory, Institute of Biological Sciences, University of Rajshahi, Rajshshi 6205, Bangladesh.

Abstract/ Executive Summary:

The aim of this study was to show, an efficient protocol for establishment of cell suspension culture and plantlet regeneration through cell culture from the cotyledonary explants of Brinjal (Solanum melongena L.). In this investigation, three varieties of Brinjal cv. Loda, China and Jhotika were used. In first step, the somatic embryogenic calli formation was done using MS medium supplemented with different concentrations of auxin and cytokinin singly or in combination. Cells of the three varieties were isolated from the rapidly growing embryogenic and friable calli using orbital shaker. For callus induction the isolated cells were transferred to MS liquid medium containing different hormonal concentrations and after 37-63 days of incubation the micro-calli were appeared. The Loda and China varieties showed the best result (8.0 and 8.2%, respectively) in 2 mg L^-1 NAA+0.05 mg L^-1 BAP and 2 mg L^-1 2,4-D+0.05 mg L^-1 BAP. For embryo formation, micro-calli were subcultured on MS solid medium and the Loda variety showed the best result (21%) in the medium containing 1.0 mg L^-1 BAP+0.05 mg L^-1 GA₃. The bipolar embryos were selected and cultured in MS medium with different combinations and concentrations of auxin (NAA) and cytokinin (BAP and IBA) for shoot and root formation. Optimum shoot and root formations were recorded in MS medium supplemented with 0.75 mg L^-1 NAA+1.5 mg L^-1 BAP and 2.0 mg L^-1 NAA+0.5 mg L^-1 IBA, respectively. The plant lets appeared in the embryo mass were cultured and acclimatized.

Keywords: Brinjal, Cotyledon, Embryogenic calli, Cell Suspension, Somatic embryogenesis, Regeneration

Location: Bangladesh Agricultural Development Corporation (BADC), Rajshahi, Bangladesh.

Start Date: 00-00-2006

End Date: 00-00-2006

Program Area: Variety and Species

Commodity: Brinjal

Objectives:

To describe an easy procedure for cell culture of Brinjal and plantlet regeneration through cell suspension culture of Brinjal.

Methodology:

The cotyledonary explants of three varieties of Brinjal cv. Loda, China and Jhotika were used as experimental materials in this investigation. Seeds of these varieties were collected from Bangladesh Agricultural Development Corporation (BADC), Rajshahi, Bangladesh. Seeds were washed thoroughly under running tap water and after then treated with 1% savlon supplied by ACI and 2-3 drops of Tween-80 for about 10 min. This was followed by successive three washing with distilled water to make free the seeds from savlon and Tween-80, surface sterilization was carried out with 0.1% HgCl₂ for 6-7 min followed by gentle shaking. After this treatment, the seeds were rinsed 4-5 times in sterile distilled water to make free the seeds from HgCl₂. Sterilized seeds were aseptically germinated in glass bottle containing 50 mL of autoclaved (121°C temperature and 15 psi for 15 min) half strength MS medium and double autoclaved soil separately. Germinating seeds were maintained at 25±2°C temperature and 60° RH in darkness. After germination, seedlings were maintained under 16/8 h light and dark region. The experiment was conducted in the Institute of Biological Sciences, Rajshahi University, Bangladesh in 2006. Cotyledons from 8 days old aseptically grown seedlings were used as explants. Explants (20 pieces) were cultured in 9 cm petridish and placed horizontally in the callus induction medium. The MS medium supplemented with 3% sucrose and different concentrations of 2,4-D (2, 4-Dichlorophenoxy acetic acid) NAA (α-Naphthalene acetic acid), IAA (Indole-3-acetic acid) and BAP (6-Benzyl aminopurin) singly or in combination were compared for the induction of embryogenic calli. The medium was adjusted to pH 5.8 and autoclaved (as described earlier). All the cultures were maintained as described earlier for aseptic seed germination. The data for callus initiation were scored after 28-30 days of culture. Induction frequencies for all types of callus were calculated as the percentage of cultured pieces of cotyledons. Embryogenic and rapidly proliferating friable calli subcultured for 18 days, were transferred to MS liquid medium supplemented with 1.0 mg L^-1 NAA+0.05 mg L^-1 BAP. The culture bottles, wrapped with brown paper, were placed on a rotary shaker (100 rpm). After 6-7 days, the liquid medium containing cells and micro calli were filtered through a 500 μm sieve. The cells collected in liquid medium were kept in a stationary position for 20-25 min for sedimentation. The supernatant was discarded keeping the cells settled at the bottom. The cells were distributed to petridishes (4 cm) containing the fresh liquid medium to observe the growth efficiency of cells and to obtain calli. To observe the growth efficiency of cells, some petridishes were kept on a orbitary shaker and the weight of cells in 5 mL liquid medium was taken in every other two days. On the other hand, to obtain callus some of the cultured petridishes were kept at stationary position at 25°C in dark and after 37-63 days of incubation, micro calli were appeared in the plates initiating induction of callus. For embryo formation the calli derived from isolated cells, were subcultured on the agarifide MS medium supplemented with different concentration of NAA+BAP, BAP+GA₃ and KIN+GA₃. After 20-21 days the embryos were appeared in the solid MS medium. The embryos showed various polarities like unipolar and bipolar. The number of embryos per callus was calculated under microscope from the beginning of embryogenesis and their average number were calculated. For shoot and root formation, the bipolar embryos were transferred in MS medium containing different concentrations of NAA with BAP, NAA with IBA and IBA singly.

Source of Information: Journal of Plant Sciences, 2(4): 407-415, 2007, ISSN 1816-4951.

Article Link (Online Journal): DOI: 10.3923/jps.2007.407.415; URL: http://scialert.net/abstract/?doi=jps.2007.407.415

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The regenerated shoots were transferred to rooting medium containing different concentrations of IBA singly and with NAA in combination for root induction. No shoots of any variety produced any root in the media containing IBA only. But when the different concentrations of IBA were used with NAA, the shoots of three varieties (Loda, China and Jhotika) produced roots and the highest rooting percentages were 85, 82 and 76, respectively. After the sufficient development of root the plant lets were taken and transplanted to small plastic pots containing sterilized soil. Plant lets were successfully acclimated with natural condition through gradual increase of duration of exposure to sunlight. The in vitro regeneration protocol described here is easily reproducible, requires minimum hormonal supplements. Present protocol can effectively be used for genetic manipulation of brinjal cells by culturing and transferring the genes in cells and protoplasts.

Knowledge Management: Journal