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Research Journals >
Indian Journal of Biotechnology (IJBT) >
IJBT Vol.02 [2003] >
IJBT Vol.02(4) [October 2003] >
| Title: | Agrobacterium tumefaciens-mediated Transformation of Chickpea (Cicer arietinum L.) using Mature Embryonic Axes and Cotyledonary Nodes |
| Authors: | Sanyal, Indraneel
Singh, A K
Amla, D V |
| Keywords: | Chickpea
Agrobacterium tumefaciens
 -glucuronidase
Transformants
Cicer arietinum L.
Southern hybridization |
| Issue Date: | Oct-2003 |
| Publisher: | NISCAIR-CSIR, India |
| Abstract: | 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. |
| Page(s): | 524-532 |
| CC License: |  CC Attribution-Noncommercial-No Derivative Works 2.5 India |
| ISSN: | 0975-0967 (Online); 0972-5849 (Print) |
| Source: | IJBT Vol.02(4) [October 2003]
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