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Plant Regeneration Through Multiple Shoot Formation in Blackgram

S. K. Roy
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

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

M. K. Siddiqua
Department of Horticulture, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Abstract/ Executive Summary:

Blackgram (Vigna mungo L. Hepper, syn. Phaseolus mungo L.) is one of the four major pulse crops. So far, no reports of tissue culture studies aiming at genetic improvement of this crop are available in Bangladesh. The present study comprised of experiments for direct shoot regeneration and plantlet formation in blackgram. Shoot tip explants were cultured on different concentrations of BAP (1.0, 5.0 and 10.0 mg L^-1) and Kinetin (1.0, 5.0 and 10.0 mg L^-1). The highest percentage (85.00%) of multiple shoot initiation was observed in the MS medium supplemented with 5.0 mg L^-1 kinetin. The regenerated shoots were transferred to rooting medium supplemented with different concentrations of NAA. A high frequency of 85% rooting was observed with 0.2 mg L^-1 NAA. The rooted plants were transferred to pots for hardening.

Keywords: Blackgram, Multiple shoot, Regeneration, Shoot tip culture

Location: Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Black gram

Objectives:

To determine the optimum concentrations of growth regulators for in vitro callus induction,
To sort out suitable explant for regeneration, and
To develop a protocol of plantlet regeneration.

Methodology:

Seeds of the variety BINA Mash-1 were collected from Bangladesh Institute of Nuclear Agriculture. 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 for germination in sterile vials. Later the vials were wrapped with Para film. Seven to ten days after germination, shoot tips of seedlings were dissected and cultured on MS medium supplemented with with three levels of BAP (0, 5, 10 mg/l) and three levels of Kinetin (0, 5, 10 mg/l) for direct regeneration. For successful rooting, 3-4 cm usable shoots excised from multiple shoots were implanted to rooting medium consisting of MS medium supplemented with IBA (2 mg L-1), IAA (5 mg L^-1) and NAA (0.2 mg L^-1). MS medium supplemented with different concentrations and combinations of phytohormones as per treatments was used for shoot induction, shoot multiplication and rooting of shoots. Hormones were added separately to different media according to their requirements. For the preparation of media, stock solutions were prepared at the beginning and stored at 4 ± 1ºC temperature. The respective media were prepared from the stock solutions. pH of the media was adjusted to 5.8 and then agar @ 9.0 g was added to solidify the medium. The medium was then gently heated without boiling with continuous stirring till complete dissolution of agar. Required volume of hot medium was dispensed into culture vessels or conical flasks. After dispensing the medium the culture vessels were plugged with cork and marked with different codes with the help of glass marker to indicate specific hormonal combination. To ensure aseptic condition under in vitro, all instruments, glass wares and culture media were sterilized by autoclaving at 1.16 kg/cm2 pressure at 12ºC for 30 minutes. The culture vials were transferred to growth room and were allowed to grow in controlled environment. The temperature of the growth room was maintained with in 25 ± 2ºC by an air conditioner. A 16 hours light period was maintained with light intensity of 2000 lux. 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. These plantlets were transplanted in small pots contained compost, soil and sand (1 : 2 : 1) and were covered with transparent sheets. They were nurtured under room temperature in presence of sufficient light. An average days nursing under room condition enabled them to adapt in outdoor conditions. In that conditions the plantlet were watered every alternate day. The data for the parameters recorded in the present study were statistically analyzed by the programmed MSTATC and Microsoft Excel wherever applicable. The experiment was arranged in Completely Randomized Design (CRD). The analysis of variances for different parameters was performed and the means were compared by Duncan’s Multiple Range Test (DMRT).

Source of Information: Progress. Agric. 18(2) : 11-16, 2007 ISSN 1017-8139

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

In vitro plant regeneration capability BINA Mash-1 cultivar of blackgram was studied using shoot tips as explants. Shoot tip explants were cultured on different concentrations of BAP and Kinetin for shoot proliferation. The percentage of shoot initiation was significantly influenced by the concentration of the growth regulators used in the experiment. The highest shoot proliferation (85.0%) was observed with the supplementation of 5.0 mg L^-1 Kinetin and the lowest shoot proliferation (17.5%) was observed in the addition of 10.0 mg L^-1 BAP to the medium. Cultured explants were carefully observed in regular basis for data collection on days required for shoot regeneration. The growth regulators significantly affected the days required for shoot initiation. The 5.0 mg L^-1 Kinetin performed better and needed least number of days (14.75) for shoot regeneration which was statistically similar to that in 10.0 mg L^-1 Kinetin. Maximum number of days 18.25 was required in 1.0 mg L^-1 BAP. The root elongation was also examined. It was observed that the concentration of 0.2 mg L^-1 NAA showed the highest elongation of roots (6.12 cm) and the shortest roots (2.0 cm) were observed in the growth regulator free medium. It was clear from the above discussion that 0.2 mg L^-1 NAA was better than any other treatments for root formation. Healthy plantlets of 7-8 cm in height were planted in a mixture of garden soil, sand and cowdung at the ratio of 2 : 1 : 1. Immediately after transplantation, the plants along with pots were kept in diffused sunlight in the controlled environment of the growth room. Survival rate of the transplanted plantlets was 80%. A method of direct regeneration from shoot tips has been developed. However, further studies are necessary to regenerate plants via callus formation.

Knowledge Management: Journal