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In Vitro Callus Initiation of Chickpea (cicer Arietinum L.)

Md. Forhadul Miraz
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Md. Abdul Mozid
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Md. Shahidul Haque
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

M.A. Latif
Department of Biotechnology, Bangladesh Agricultural University, Mymensingh-2202, Bangladesh.

Abstract/ Executive Summary:

The present study was undertaken to develop a reproducible protocol for efficient in vitro callus initiation and plantlet regeneration of chickpea (Cicer arietinum L.). Different concentrations and combinations of growth regulators were used in the MS medium to observe their relative efficiency for callus induction and plantlet regeneration from the cotyledonary node, epicotyl, and hypocotyl as explants. Among these explants, the cotyledonary node produced the highest percentage (56.25%) of callus while the epicotyls showed the lowest frequency (51.04%) of callus. Among the growth regulator combinations, the highest rate of callus induction (91.11%) was observed in MS medium containing 0.2 mg/L NAA, 3 mg/L BAP and 2 mg/L Kn. Variety BARIchola-1 showed a higher percentage ( 56.11% ) of callus formation than BARIchola-2 which produced 49.72 % of callus. Further studies were needed to evaluate the performance of the shoot regeneration from initiated callus in order to establish the in vitro methods.

Keywords: Callus initiation, Plantlet regeneration, Epicotyl, Cotyledonary node, Hypocotyl, Growth regulators, Chickpea.

Location: Department of Biotechnology, Bangladesh Agricultural University, Mymensingh.

Start Date: 00-07-2008

End Date: 00-05-2009

Program Area: Variety and Species

Commodity: Chickpea

Objectives:

The present study was to establish a suitable in vitro method for callus initiation and regeneration of chickpea genotypes and to identify the explants suitable for regeneration.

Methodology:

The experiment was conducted at the USDA Biotech Laboratory of the Department of Biotechnology, Bangladesh Agricultural University, Mymensingh, during the period from July 2008 to May 2009. Two Chickpea (Cicer arietinum L.) genotypes viz BARI Chola-1 and BARI Chola-2 were used for conducting the study. The seed materials of these Chickpea genotypes were collected from Bangladesh Agricultural Research Institute (BARI), Gazipur. Seeds were soaked with 70% ethyl alcohol for 30 seconds and then washed 2-3 times with sterile distilled water. The seeds were then surface sterilized with the mixed solution of 0.1% (w/v) aqueous sodium hypochlorite and 3 drops of Tween-20 per 100 ml solution for 20 minutes, followed by 3 or 4 rinses in sterile distilled water to remove all the reagents. After sterilization required amount of sterilized seeds were germinated aseptically in seed germination vials. In each vial, 3 seeds were inoculated and then incubated in the incubation room till the germination of seeds. Then 5 to7 days old seedlings were ready for the source of contamination-free explants. Tissue culture techniques include axenic culture, explant culture, subculture or transfer, roots were used in this study. The seedlings raised in axenic culture were the source of different kinds of explants such as epicotyls, cotyledonary nodes, and hypocotyls. Different culture media with specific hormones were prepared for callus induction and plant development from different explants to find out the most suitable hormonal combination for callus induction and plantlet regeneration. For callus induction and plantlet regeneration of chickpea MS medium (Murashige and Skoog, 1962) was used. For MS medium preparation, sucrose and Difco-Bacto agar was added at a rate of 30 g/L and 8 g/L respectively and the ph of the medium was adjusted at 5.8+-1. Growth regulators were added to the media prior to autoclaving at 121°C with 15 psi. After autoclaving, 20 ml of the sterile molten medium was poured into the vial. After inoculation of sterilized seeds, vials were sealed with parafilm. Four different callus induction medium used in this study were: (T₀) Murashige & Skoog (MS) medium (control), (T₁) MS medium + 0.1 mg/L NAA (Naphthaleneacetic acid) + 2.0 mg/L BAP (Benzylamino purine) + 2.0 mg/L Kn (Furfuralamino purine), (T₂) 0.2 mg/L NAA + 3.0 mg/L BAP + 2.0 mg/L Kn, (T₃) 0.3 mg/L NAA +4.0 mg/l BAP + 2.0 mg/L Kn. The explants were cultured at 25+1C temperature in the incubation chamber and 10 days later callus induction rate was measured. Each treatment replication comprised 5 vials and each vial consisted of 4 explants. After callus initiation, the calli were cultured on four different regeneration medium to determine the regeneration response of the used genotypes and to determine suitable medium for this purpose Regeneration medium used in this study were : (T₀) Murashige & Skoog (MS) medium (control) (T₁) MS + 1 mg/L IAA + 2 mg/L BAP + 3 mg/L Kn (T₂) MS + 2 mg/L IAA + 3 mg/L BAP + 4 mg/L Kn (T₃) MS + 3 mg/L IAA + 4 mg/L BAP + 5 mg/L Kn. The experiment was conducted in a growth room and arranged in a Completely Randomized Design. The data for the parameters recorded in the present study were statistically analyzed by the statistical package MSTATC and Microsoft Excel wherever applicable. The analysis of variances for the different parameters was performed and means were compared by the Duncan,s Multiple Range Test (DMRT ).

Source of Information: International Journal of Natural and Social Sciences, 2015, 2(5): 44-51

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

From the above experiment, it can be mentioned that MS medium supplemented with 3 mg/L NAA, 4 mg/L BAP & 2 mg/L Kn was effective for callus induction. Further studies were needed to evaluate the performance of the shoot regeneration from initiated callus in order to establish the in vitro methods.

Knowledge Management: Journal