STANDARDIZATION OF AGROBACTERIUM MEDIATED TRANSFORMATION PROTOCOL IN TOMATO (Solanum lycopersicum L.) Cv. PKM-1.
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Date
2008
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ACHARYA N.G. RANGA AGRICULTURAL UNIVERSITY, RAJENDRANAGAR, HYDERABAD.
Abstract
The present investigation was carried out to standardize the Agrobacterium
mediated transformation protocol in tomato and it is carried out at transgenic
laboratory of Dept. of Plant Physiology, RARS, Tirupati and Dept. of Genetics and
Plant Breeding, S.V. Agricultural College, Tirupati.
Seeds treated with 5% NaOCl for 20 min and inoculated on MS medium
with out sucrose and incubation in dark for three days produced healthy and uniform
seedlings without contamination. Explants i.e. cotyledons and hypocotyls isolated
from 10 day old seedlings were found ideal for high frequency of regeneration
compared to younger or older seedlings. Among the two explants i.e. cotyledon and
hypocotyls, cotyledons showed better response compared to hypocotyls. Hence
cotyledonary explants from 10 days old in vitro seedlings of PKM-1 were used for
further studies on regeneration and transgenic protocols.
Among the various plant growth regulators combinations tried the best shoot
regeneration was obtained when MS medium was supplemented with BAP 1.5
mg/L + Kinetin 1.0 mg/L and root regeneration was obtained when MS medium
was supplemented with Kinetin 1.0 mg/L respectively.
Cotyledonary explants excised out from 10 days old seedlings were
incubated for 10 min with over night grown Agrobacterium culture and cocultivated
for 2 days followed by transfer to media containing cefotaxime 500 mg/L
for 4 days before transferring to the medium containing 75 mg/L kanamycin which
was found to be optimum for checking the Agrobacterium growth.
Higher plantlet survival (86%) was obtained in soilrite mixture and 9.6
days has been taken for acclimatization.
The transformation was carried out using the Agrobacterium strain LBA4404
containing the binary vector pCAMBIA 2301 harboring npt II as selectable marker
and GUS as reporter gene.
Confirmation of the transgene integration in the putative transformants was
done by using the histochemical staining and PCR. The transformation efficiency of
44.4% was obtained in the cultivar PKM-1. The transformation frequency was 3.5%
and the GUS gene transient expression level in transformants was 44.4%. Thus, the
present study successfully demonstrated the indirect regeneration of transgenic
plants from cotyledonary explants through Agrobacterium mediated genetic
transformation approach in tomato Cv. PKM-1. The standardized protocols of
present study may be utilized for further transgenics development in PKM-1 cultivar
genetic background.
Seeds treated with 5% NaOCl for 20 min and inoculated on MS
medium without sucrose and dark incubation for three days
produced healthy and uniform seedlings without contamination.
Cotyledons and hypocotyl explants isolated from 10 day old seedlings
were found ideal for high frequency regeneration compared to
younger or older seedlings. Comparatively cotyledons showed better
explant response than hypocotyls. Hence, cotyledonary explants from
10 days old in vitro seedlings were used for further studies on
regeneration and transgenic protocols. Best shoot and regeneration,
was obtained when MS medium is supplemented with BAP 1.5 mg/L
+ Kinetin 1.0 mg/L and root regeneration, was obtained when MS
medium is supplemented with Kinetin 1.0 mg/L respectively and
higher plantlet survival (86%) was obtained in soilrite mixture with in
10 days. The transformation was carried out using the Agrobacterium
strain LBA4404 containing the binary vector pCAMBIA 2301
harboring GUS as reporter gene. Cotyledonary explants excised out
from 10 days old seedlings were incubated for 10 min with over night
grown Agrobacterium culture and co-cultivated for 2 days
followed by transfer to media containing cefotaxime 500 mg/L for 4
days before transferring to the medium containing 75 mg/L
kanamycin was found to be optimum for checking the Agrobacterium
growth. Stable integration of the transgene in the putative
transformants was confirmed by using the histochemical staining
and PCR assay.
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Keywords
STANDARDIZATION, AGROBACTERIUM, MEDIATED, TRANSFORMATION, PROTOCOL, TOMATO