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In Vitro Direct Regeneration of Three Indigenous Chickpea (cicer Arietinum L.) Varieties of Bangladesh

R. H. Sarker
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Tarannum Ferdous
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

M. I. Hoque
Department of Botany, University of Dhaka, Dhaka-1000, Bangladesh

Abstract/ Executive Summary:

An in vitro direct plant regeneration protocol was developed for three locally grown chickpea (Cicer arietinum L.) varieties of Barichhola, namely BCh-2, BCh-4 and BCh-5. The best response towards multiple shoot regeneration was achieved from cotyledonary nodal explants on MSB5 containing 4× MS micro-salts, 3.0 mg/l BAP and 0.04 mg/l NAA in all the three varieties of chickpea. In decapitated mature embryo axes explant best shoot regeneration was obtained on MS supplemented with 0.5 mg/l BAP, 0.5 mg/l Kn, 0.2 mg/l NAA, 4 mg/l tyrosine, 880 mg/l CaCl₂ and 3800 mg/l KNO₃ in BCh-4 and BCh-5. Shoots regenerated on 1.0 mg/l Kn supplemented medium rooted efficiently on MS containing 0.2 mg/l BAP and 0.5 mg/ NAA in all the three varieties. Micrografting technique has been found to be a good alternative to in vitro rooting.

Keywords: Chickpea, organogenesis, Explants, Transgenic

Location: Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur, Bangladesh

Start Date:

End Date:

Program Area: Variety and Species

Commodity: Chickpea

Objectives:

To establish an efficient regeneration protocol suitable for Barichhola (BCh) so that the same, with or without modification may be applied to obtain chickpea transgenics of desired traits by Agrobacterium-mediated genetic transformation.

Methodology:

Seeds of three local varieties of chickpea (Cicer arietinum L.), namely Barichhola: BCh-2, BCh-4 and BCh-5 collected from Bangladesh Agricultural Research Institute (BARI), Joydebpur, Gazipur were used in the present investigation. Two types of explants were used: (1) Decapitated mature embryo axes from overnight soaked seeds and (2) cotyledonary nodes taken from six - seven days old seedlings. For regeneration of shoots, all explants were cultured on MS or MSB5/MS with B5 vitamins with various hormonal supplements, namely BAP, Kn, NAA, GA3 and TDZ. In certain combinations, tyrosine was added in shoot regeneration medium and MS micro-salts and certain MS macrosalts, namely CaCl₂, KNO₃and NH₄NO₃ were used in higher concentrations than the normal MS. For rooting, 3 - 5 cm long regenerated shoots were cultured on full or half strength MS solid or liquid medium supplemented with various concentrations and combinations of IBA and NAA. All the cultures were maintained under 16 hours photoperiod at 25 ± 2 ºC. Sometimes regenerated shoots were micrografted on to rootstocks taken from six days old aseptically germinated seedlings. The scion (shoot) base was given a “V” shaped cut and the upper part of the rootstock was split gently with a pointed scalpel. After placing the scion into the split of the stock, they were tied together with sterilized cotton thread.

Source of Information: Plant Tissue Cult. & Biotech. 15(2): 135-144, 2005 (December)

Article Link (Online Journal):

Funding Source:

Total Budget (Tk.) :

Achievement/Findings:

Shoots that failed to develop roots were subjected to micrografting on rootstocks of in vitro germinated seedlings. Most of the recent work on chickpea employed this technique for the production of plantlets when normal rooting was unsuccessful. In the present study, the percentage of shoots survived upon grafting was 30 - 41.67. This experiment demonstrated that micrografting could be applied as an alternative to obtain healthy root system for the development of plants in vitro. The in vitro shoot regeneration procedure described in the present study is rapid, efficient and reproducible and can be used for locally grown chickpea varieties. As rooting in chickpea is extremely difficult, further refinement of the rooting technique is necessary. Also as an alternative method, the technique of in vitro grafting can be applied to develop an efficient rooting system for this crop. In future, the regeneration protocol described here can be used for production of transgenic chickpea plants with desired traits.

Knowledge Management: Journal