GENETIC TRANSFORMATION OF RICE (Oryza sativa L.) USING DREB1A GENE BY PARTICLE BOMBARDMENT
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Date
2013
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Publisher
Punjab Agricultural University
Abstract
The present investigation entitled “Genetic transformation of rice (Oryza sativa L.) using
DREB1A gene by particle bombardment” was undertaken in indica rice variety PR 116. In
rice, several biotic and abiotic stresses continue to threaten productivity. Among abiotic
stresses, water deficit is increasingly becoming frequent in irrigated areas. Keeping above in
view, 821 mature seeds of rice variety PR 116 were cultured on MS medium supplemented
with 2, 4-D (2.5 mgL-1) + Kinetin (0.5 mgL-1) + Proline (560 mgL-1), out of which
441 calli were formed with percent callus induction of 53.8%. The percent
regeneration from embryogenic calli cultured on MS medium supplemented with
BAP (2.5 mgL-1) + NAA (1 mgL-1) + Kinetin (0.5 mgL-1) was 55.56%. After
standardization of regeneration protocol, the calli were bombarded with stress
tolerant DREB1A gene. Two antibiotics viz. kanamycin (40 mgL-1) and
paromomycin (100 mgL-1) were used for selection of bombarded calli. Use of
kanamycin as selective agent leads to albinism in plants. Therefore, paromomycin
was found to be better selective agent for selection of bombarded calli. Rooted and
hardened plants were transplanted in pots. Molecular characterization of putative
transgenic lines was carried out through polymerase chain reaction (PCR) by
amplification of DREB1A gene. Out of 80 plants, 11 were found PCR positive with
percent transformation of 1.5%. Expression of transgene DREB1A was determined
by reverse transcription polymerase chain reaction (RT-PCR). Total RNA was
isolated from leaves of PCR positive plants and cDNA was synthesized. Thereafter,
cDNA was amplified using DREB1A gene specific primers which showed the
expression of DREB1A gene in all the four PCR positive transgenic lines.
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Keywords
polymerase chain, cDNA