In vitro manipulation of pollen for transformation in cotton and Tomato
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Date
2009
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UAS Dharwad
Abstract
The laboratory and field experiments on pollen transformation in cotton and tomato
were carried out during 2004-20007 at the Department of GPB, UAS, Dharwad. The main
objective of the study was to assess the potentiality of production of transgenic cotton and
tomato plants consistently using pollen as vector.
In the present study, in vitro pollen germination medium for cotton variety Sahana
and tomato variety Pusa ruby were standardized. The co-cultivation of pollen with
Agrobacterium tumefaciens strain LBA4404 containing plasmid vector pCAMBIA1305.1
significantly reduced the germination per cent and tube growth compared to control both in
cotton and tomato. The co-cultivation of cotton pollen with Agrobacterium tumefaciens
containing GUS as a reporter gene in cavity slides resulted in 3.11 percentage of pollen grains
showing transient GUS expression.
The co-cultivated pollen grains were used for pollination. To favour the transformed
pollen grains in fertilization, the style and stigma of the emasculated flowers were treated
with various concentrations of hygromycin (250, 500 and 1000 ppm in cotton and
250,500,1000 and 1500 ppm in tomato). The increased concentration of hygromycin to
stigma and style has significantly reduced the boll set in cotton and fruit set in tomato. The
number of seeds obtained from each treatment also varied. Totally, 223 cotton plants and 390
tomato plants were screened for presence and expression of transgene. In cotton, 32 plants
(14.34%) showed resistance to hygromycin application, 14 plants (6.27%) were positive for
PCR amplification and 12 plants (5.38%) were positive for GUS assay in leaves in T0
generation. In case of tomato, 13 plants (3.3%) were resistant to hygromycin application.
Only two plants (0.15%) were positive for PCR amplification and one plant (0.25%) showed
positive for GUS staining in leaves in T0 generation. Further, the positive plants were
advanced to T1 generation. The T1 plants showed resistant to hygromycin application and
positive for PCR amplification, suggesting stable integration of DNA introduced through
pollen grains.
In order to increase the frequency of pollen grains with transgene, different pretreatments
to pollen grains were given. The pre- hydration of pollen for 30 minutes, pretreating
cotton pollen with 40 and 45% PEG and tomato pollen with 12% PEG for 2 hours,
pre germination of pollen grains for 45 minutes. The pre-treatments increased the frequency
of transgene insert into the pollen significantly. Further, parameters of particle bombardment
were optimized for transformation of cotton pollen grains. Two particle size (0.7& 1.1μ), two
shooting distance (6 cm &9 cm) and three pollen treatments (pre- hydration, PEG treatment
and control) were used. The bombardment of PEG pre- treated pollen grains with 1.1μ
particles at a target distance of 9 cm produced the highest frequency (29.04%) of transient
GUS expressing pollens.