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In Vitro Regeneration of Chickpea (cicer Arietinum L.) (2016-2017)

M. M. Khatun
Biotechnology Division, Bangladesh Agricultural Research Institute, Joydebpur, Gazipur-1701, Bangladesh

M. R. Kabir
Biotechnology Division, Bangladesh Agricultural Research Institute, Joydebpur, Gazipur-1701, Bangladesh

D. Khanam
Biotechnology Division, Bangladesh Agricultural Research Institute, Joydebpur, Gazipur-1701, Bangladesh

M. A. Y. Akhond
Biotechnology Division, Bangladesh Agricultural Research Institute, Joydebpur, Gazipur-1701, Bangladesh

Abstract/ Executive Summary:

Embryonal axis of BARI chola 7 was used for in vitro regeneration. Six different treatments (T_1, T_2, T_3, T_4,T₅& T₆) were used which consist of TDZ alone and in combination with 2ip and Kinetin. The maximum number of shoot (4.45) was obtained from T₂treatment. The highest shoot length (5.72 cm) was obtained from T₄treatment. When in vitro grown shoots were induced for rooting in four root induction medium, the maximum root (3.50) was obtained from T₂treatment. The highest root length (3.62 cm) was also recorded from T₂treatment. Well rooted plantlets were transferred to potting media containing sand, soil and compost at the ratio of 1:1:1 for establishment.

Keywords: Pulses, Chickpea, Healthy seeds, Sand, Soil, Compost

Location: Biotechnology Division, Bangladesh Agricultural Research Institute, Joydebpur, Gazipur-1701, Bangladesh

Start Date: 00-00-2016

End Date: 00-00-2017

Program Area: Crop-Soil-Water Management

Commodity: Chickpea

Objectives:

To develop an efficient regeneration protocol for Barichola which will be used for future for the development of Chickpea transgenic with desired traits.

Methodology:

Chickpea seeds were collected from Pulse Research Centre (PRC) Joydebpur, Gazipur. Healthy seeds were surface sterilized with detergent for 10 min and washed with running tap water for 15 min. Then the seeds were sterilized with 0.1% HgCl₂with 1-2 drops of Tween 20 for 10 min. and washed three to four times with sterile distilled water. After that the seeds were soaked overnight in sterile distilled water. Overnight soaked seeds were decoated and germinated on shoot induction medium (SIM), consisting of six different treatments (T₁, T₂, T₃, T₄, T₅ & T₆) which consist of TDZ alone and in combination with 2ip and Kn for 6d. Explants consisting of single cotyledon or without cotyledon with half embryonal axis with plumular and radicular ends were exeised from resulting 6d old seedlings and inoculated on SIM for further duration of 6d. The explants were subsequently transferred to MS basal medium (i.e. devoid of growth regulators) for 10-15d.

Elongation of shoots

The adventitious buds/multiple shoot induced from the explants were excised from the bunch without any callus or globular structures and cultured on the shoot elongation medium (SEM) for 10d. They were then routinely subcultured at an interval of 10-15d on SEM.

Rooting

When shoots were 3 to 8 cm long, they were transferred to MS medium supplemented with 4 different concentrations of IBA for root induction. All media had 3% sucrose and 0.8% agar. The pH of all media was adjusted to 5.8 before autoclaving. The media were autoclaved at 121⁰C and 1.06 kg/cm² pressure for 20 minutes. All cultures were incubated at 25 ± 1⁰C and kept in 16/8 hours light/dark photoperiod with a light intensity of 2000 lux under cool white fluorescent tubes.

Source of Information: Annual Research Report 2016-2017, Biotechnology Division, BARI, Gazipur

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

Embryonal axis was cultured in six different treatments which consist of TDZ alone and in combination with 2ip and kn for shoot regeneration. Results of different treatments are presented. Embryonal axis showed significant swelling and exhibited initiation of shoot induction within 6-8d. The maximum shoot number was obtained from T₂ treatment (4.45) followed by T₃ (3.85). The minimum number of shoot was obtained from T₁ treatment (2.35). The highest shoot length (5.72 cm) was observed fromT₄ treatment followed by T₅(5.10 cm). TDZ has been noted to be the most promising cytokinin for shoot induction in chickpea by number of research teams (Barna and Wakhlu, 1993; Murthy et al., 1996; Rizvi and Singh, 2000; Jayanand et. al., 2003; Senthi et. al., 2004; Karim et al., 2005). TDZ was most effective in inducing a large number of healthier shoots when used in lower concentrations (10μM) as compared to higher concentration (Malik and Saxena, 1992; Murthy et al., 1998; Jiang et. al., 2005). Use of TDZ with purine ring containing cytokinins such as kinetin, BAP and 2ip has been shown to promote the formation of a large number of healthy shoots (Radhika et al., 2006). In our investigation, it was noticed that TDZ in combination with 2ip and Kinetin was found suitable for expansion of meristematic zone followed by shoot induction. Subsequent to the transfer of cultures onto MS basal medium i.e. growth regulator free medium emergence of a large number of adventitious shoots/ expant from all over the surface of the swollen embryonal axis was recorded with 7-15 d. The induced multiple shoots were excised from the bunch, without any callus or globular structures. Shoots were cultured on the shoot elongation medium (SEM) for 10d. They were then routinely subcultured at an interval of 10-15 d on SEM.

Knowledge Management: Report/Proceedings