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Callus Induction and Plantlet Regeneration in Blackgram (vigna Mungo L. Hepper)

S. A. Mony
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

M. S. Haque
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

The present study was undertaken for callus induction and subsequent plantlet regeneration in blackgram. The study comprised of experiments for callus initiation, shoot regeneration and plantlet formation from cotyledon, hypocotyl, root tip and shoot tip explant. The effects of explant and different concentrations and combinations of BAP (0.0, 1.0, 2.5, 5.0 and 10 mg L^-1) and NAA (0.0, 0.5, 1.0, 1.5 and 2.0 mg L^-1) on callus induction were investigated first. Among the explants, hypocotyls showed the best performance in callus formation (92.33%) when cultured on MS medium supplemented with 2.5 mg L^-1 BAP and 1.5 mg L^-1 NAA followed by cotyledon, shoot tip and root tip explant, respectively. The height percentage of shoot regeneration from the calli derived from hypocotyl (56.33%) was achieved in MS medium supplemented with 3.0 mg L^-1 BAP and 0.3 mg L^-1 NAA and 0.5 mg L^-1 GA3. Calli from other explant had no shoot regeneration. The regenerated shoots were transferred to rooting medium supplemented with different concentrations of IBA and NAA. The high frequency (100 %) of rooting was observed with MS medium supplemented with 0.5 mg L^-1 IBA. The rooted plants were transferred to pots for hardening.

Keywords: Blackgram, Callus, Plantlet, Regeneration, Vigna mungo L. Hepper

Location: The Biotechnology Laboratory, Department of Biotechnology, Bangladesh Agricultural University, Mymensingh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Black gram

Objectives:

a. To find out suitable explants for regeneration and to develop a reproducible protocol for in vitro callus formation and plantlet regeneration of Blackgram

Methodology:

The experiment was carried out at the Biotechnology Laboratory, Department of Biotechnology, Bangladesh Agricultural University, Mymensingh. The experimental materials (seeds) of Vigna mungo (2n=22) were collected from Bangladesh Institute of Nuclear Agriculture, for the establishment of culture. Root tips, cotyledons, hypocotyl and shoot tips were used as explants in the present study. Surface sterilization of mature seeds was carried out under Laminar Air Flow Cabinet. Seeds were washed by sterile distilled water for 3 to 5 minutes. Later, they were rinsed in 70% ethyl alcohol followed by washing with sterile distilled water for 3 times. Finally, surface disinfection was done with 0.1% HgCl₂ for five minutes with continuous agitation. The seeds were then washed 5 times with sterile distilled water to remove the sterilant. The mature seeds were placed on a solidified agar medium containing no growth regulators in sterile vials. Later the vials were wrapped with Parafilm. Seven to ten days after germination, root tips, cotyledons, hypocotyl and shoot tips of seedlings were dissected and cultured on MS medium supplemented with different combinations and concentrations of BAP (0, 1.0, 2.5, 5.0 and 10 mg/l) and NAA (0, 0.5, 1.0, 1.5 and 2.0 mg/l) for callus induction. Different concentrations of BAP (0.0, 3.0 and 5.0 mg/l) alone, different combinations of BAP (1.0, 2.0 and 3.0 mg/l) and NAA (0.1, 0.2 and 0.3 mg/l) with or without 0.5 mg/l GA₃ including the control were used for shoot induction from calli. For successful rooting, 3-5 cm usable shoots excised from multiple shoots were implanted to rooting medium consisting of MS medium supplemented with IBA (0.0, 0.25, 0.50, 1.0, 2.0, 3.0, 4.0 and 5.0 mg/L) and NAA (0.20 and 0.50 mg/l). All instruments, glass wares and culture media were sterilized by autoclaving at 1.16 kg/cm2 pressure at 121º C for 30 minutes. The temperature of the growth room was maintained at 25±2ºC by an air conditioner. A 16 hours light period was maintained with light intensity of 2000 lux. At least 16-20 explants were cultured in each experiment and all experiments were repeated thrice. One month old adequately rooted shoots when reached a height of 4-5 cm were brought out from culture room and kept in room temperature for 7 days. Plantlets were then taken out from vial and agar was washed out gently from roots. The data for the parameters recorded in the present study were statistically analyzed by the programmed MSTATC and Microsoft Excel wherever applicable.

Source of Information: Progress. Agric. 19(2) : 27-35, 2008 ISSN 1017-8139

Article Link (Online Journal): http://www.banglajol.info/index.php/PA/article/view/16917/11876

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

The height percentage of shoot regeneration from the calli derived from hypocotyl (56.33%) was achieved in MS medium supplemented with 3.0 mg L^-1 BAP and 0.3 mg L^-1 NAA and 0.5 mg L^-1 GA3. Calli from other explant had no shoot regeneration. The regenerated shoots were transferred to rooting medium supplemented with different concentrations of IBA and NAA. The high frequency (100 %) of rooting was observed with MS medium supplemented with 0.5 mg L^-1 IBA. The rooted plants were transferred to pots for hardening. In conclusion, the callus formation and regeneration ability of the explant have the potential for future biotechnological studies for the improvement of blackgram. However, further study is needed to standardize the protocol of regeneration via callus from different explant other than hypocotyl.

Knowledge Management: Journal