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|Agrobacterium tumefaciens-mediated Transformation of Chickpea (Cicer arietinum L.) using Mature Embryonic Axes and Cotyledonary Nodes
Singh, A K
Amla, D V
|Chickpea;Agrobacterium tumefaciens;-glucuronidase;Transformants;Cicer arietinum L.;Southern hybridization
|The mature embryo axes and cotyledonary nodes of chickpea (Cicer arietinum L.) were evaluated for Agrobacterium-mediated transformation and the production of stable transgenic plants expressing the reporter gene GUS was documented. The major limitation in delivering the T-DNA in these explants of grain legume has been the low frequency and inconsistency. With manipulation of co-cultivation conditions and preparation of excised explants with exposed regenerative cells in L2 and L3 layers followed by the stringent screening on selection pressure of 100-150 mg C1, kanamycin exhibited significantly higher transformation frequency as indicated in the transient GUS expression. Amongst various strains of Agrobacteria, the strain, LBA 4404 was found to be most suitable for maximal transfer of T-DNA and minimum induction of hypersensitive response into the excised explants of chickpea. Kanamycin resistant chickpea shoots selected after 3-4 cycles of kanamycin screening were grafted onto 8-day-old seedlings. Mature plants recovered thereafter showed integration of T-DNA in PCR and Southern analysis and expression of GUS gene in histochemical assay. Screening of putative transformants of chickpea harbouring modified gfp gene (pCAMBIA 1303) at an early stage was possible on the basis of green fluorescence but the transformants failed to grow further. The quantitative evaluation of GUS activity in primary transformants showed expression levels ranging from 580-1950 p mol methyl umbelliferone (MU) per mg protein per min. Out of 22 primary transformants, only six showed setting of Fl seeds while others failed to produce mature seeds.
|0975-0967 (Online); 0972-5849 (Print)
|Appears in Collections:
|IJBT Vol.02(4) [October 2003]
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