OPTIMIZATION OF AGROBACTERIUM MEDIATED TRANSFORMATION PROTOCOLS FOR SELECTED ELITE INDICA RICE VARIETIES
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Date
2004
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ACHARYA N. G. RANGA AGRICULTURAL UNIVERSITY RAJENDRANAGAR, HYDERABAD
Abstract
In the present investigation, in vitro studies were carried out to determine the
effect of different hormonal concentrations in MS and N6 basal media for callus
induction, growth and regeneration in two rice genotypes viz., BPT-5204 and Swarna.
Studies were conducted to optimize various parameters for stable transformation in
rice through Agrobacterium mediated gene transfer.
Irrespective of the genotype, callus induction frequency in N6 medium in
light was higher than the frequency observed in dark. Among different levels of
2,4-D in N6 and MS basal media, the maximum callus induction was observed
in N62L medium (N6 basal medium + 2.5 mgL-1 2,4-D) in Swarna. In BPT-
5204, highest percent of callus was recorded in N63L medium (N6 basal
medium +3.0 mgL-1 2,4-D + 0.5 mgL-1 kinetin). Callus induced on MS3
medium in light gave higher frequency of regeneration in both the varieties.
Transformation of calli derived from mature seeds of two genotypes
was attempted using Agrobacterium. Agrobacterium EHA105/1304 plasmid
containing cry1AC gene and gus reporter gene was used for transformation
studies. Gus expression was observed in two genotypes. The success in plant
transformation using Agrobacterium depend on several factors including (i)
concentration of Acetosyringone (ii) concentration of Agrobacterium cells in
culture (iii) infiltration time and (iv) hygromycin in selection medium.
Concentration of Acetosyringone, 100 μM and concentration of Agrobacterium
cells, 2 x 108 cells/ml (OD=2) with infiltration time of 15 minutes showed
higher transformation rate. The present study thus indicates that genetic
transformation in rice can be successfully achieved by optimizing various
parameters for Agrobacterium infection.
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Keywords
OPTIMIZATION, AGROBACTERIUM, MEDIATED, TRANSFORMATION, PROTOCOLS, SELECTED, ELITE INDICA, RICE, VARIETIES