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In Vitro Regeneration of Chickpea (cicer Arietinum L.) (2018--2019)

M. M. Khatun,
Biotechnology Division, BARI, Gazipur

D. Khanam
Biotechnology Division, BARI, Gazipur

M. A. Y. Akhond
Biotechnology Division, BARI, Gazipur

Abstract/ Executive Summary:

The embryonal axis of BARI Chola 6, BARI Chola 7, BARI Chola 8 and BARI Chola 9 were used for in vitro regeneration in MS medium which consists of TDZ in combination with 2ip and Kinetin. The maximum number of the shoot (13.50), highest shoot length (5.63 cm) and maximum node number (4.58) were obtained from BARI Chola 7. In vitro grown shoots were induced for rooting in MS medium supplemented with IBA. The maximum root number (6.42) was obtained from BARI Chola 6. The highest root length (1.42 cm) and the highest percentage of rooting (67.75%) were obtained from BARI Chola 8. Well rooted plantlets were transferred to potting media containing soil, saw and cocodust mixture for establishment. Twenty percent plantlets were survived.

Keywords: Chickpea, Transgenic, Plantlets

Location:

Start Date: 00-00-2018

End Date: 00-00-2019

Program Area: Variety and Species

Commodity: Chickpea

Objectives:

The present study has been undertaken to develop an efficient regeneration protocol for BARI chola which will be used in the 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) which consists of TDZ in combination with 2ip and Kn for 6d. Explants consisting of single cotyledon or without cotyledon with the half embryonal axis with plumular and radicular ends were excised from resulting 6d old seedlings were inoculated on SIM for a 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 shoots 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 5 cm long, they were transferred to MS medium supplemented with 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 2018-2019, Biotechnology Division, BARI, Gazipur

Article Link (Online Journal):

Funding Source:

Budget:

Total Budget (Tk.) :

Achievement/Findings:

The embryonal axis of BARI Chola 6, BARI Chola 7, BARI Chola 8 and BARI Chola 9 were cultured with TDZ in a combination of 2ip and kn for shoot regeneration. The embryonal axis showed significant swelling and exhibited initiation of shoot induction within 6-8d. The maximum shoot number was obtained from BARI Chola 7 (13.50) followed by BARI Chola 6 (10.42). The minimum number of the shoot was obtained from BARI Chola 9 (7.25). TDZ has been noted to be the most promising cytokinin for shoot induction in chickpea by the number of research teams. TDZ was most effective in inducing a large number of healthier shoots when used in a lower concentration (10µM) as compared to a higher concentration. 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/explant from all over the surface of the swollen embryonal axis was recorded with 7-15d. 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. The highest shoot length (5.63 cm) was observed in BARI Chola 7 and the lowest was found in BARI Chola 9 (2.96cm). In the case of node number, the maximum node number was obtained from BARI Chola 7 (4.58) and the minimum was from BARI Chola 8 (2.08).

Root formation: In vitro grown shoots were used for root induction. When shoots attained 3 to 5 cm long, they were transferred to root induction IBA medium. The development of a good root system is one of the major hurdles in chickpea regeneration. This compelled several researchers to adopt grafting. However, grafting is time-consuming, requires special skills and the success rates vary significantly. Anwar et al. (2008) also reported in their investigations that shoots could be rooted in MS medium with 5µM IBA, but the roots developed were relatively weaker and required 15-20d. In the present study, results showed that maximum root numbers (6.42) were obtained from BARI Chola 6 followed by BARI Chola 7 (5.33). The highest root length (1.42 cm) was recorded from BARI Chola 8 followed by BARI Chola 7 (1.34 cm). We found 61.25- 67.75% rooting in our study (Table 2). In rooting, the highest responsive variety was BARI Chola 8 (67.75%). A more or less similar observation was found by Polowick et.al. (2004).In their study, the frequency of rooting was only 10-60%. Root induction and development of a strong root system is one of the major hurdles that limit Chickpea regeneration. After 30 days, the rooted plantlets were transferred to potting media containing soil, saw and cocodust mixture. The establishment of in vitro raised plantlets in pots/fields is another major hurdle that limits chickpea regeneration. In the present investigation, we found a 20% survival plant in soil.

Knowledge Management: Report/Proceedings