Analysis of Transgenic Tomato Carrying Remusatia vivipara Lectin Gene (rvl1)
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Date
2012
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UAS, Dharwad
Abstract
Transgenic tomato plants carrying Remusatia vivipara lectin (rvl1) gene were
generated, characterized and analyzed for resistance to root knot nematode
(Meloidogyne incognita) and white fly (Bemisia tabaci). Co-cultivation of 1000
cotyledonary leaf-disc explants from Pusa Ruby variety of tomato with
Agrobacterium tumefaciens LBA 4404 carrying pNR73 binary vector with rvl1
resulted in 100 independent regenerants. The transgene-specific PCR amplified 771
bp product from 32 plants. Segregation analysis in T1 generation of two selected
events indicated single copy integration of T-DNA/rvl1 in RVL1-T0(28) and
multicopy integration in RVL1-T0(11). TAIL-PCR analysis in RVL1-T0(28) showed
T-DNA integration between 1,07,295 bp and 1,07,296 bp on the clone
C05_HBa0078I05 of chromosome 5. Proteins from both the events agglutinated
rabbit erythrocytes. The total lectin activity for RVL1-T0(11) was 1.6 × 103 and the
specific activity was 2.6 × 102. Likewise, for RVL1-T0(28), the total lectin activity
was 3.2 × 103 and the specific activity was 4.2 × 102 which was relatively higher than
that of RVL1-T0(11). Both the transgenic lines showed an expected band of 26.2 kDa
on SDS-PAGE.
Bioassay with M. incognita showed infection of juveniles (J2) in RVL1-
T0(11), RVL1-T0(28) and the control plants on 3rd day of inoculation. On 6th day,
55.55 per cent of the juveniles developed into sausage form in control plants, but it
was reduced to 47.05 per cent and 41.66 per cent in RVL1-T0(11) and RVL1-T0(28),
respectively. On 70th day, the average number of galls on control plants was 62.85 per
plant; and it was reduced by 36.36% and 45.46% in RVL1-T0(11) and RVL1-T0(28),
respectively over the control. In general, non-transgenic plants showed the gall index
of 7, and were rated as “highly susceptible”. Transgenic lines with the gall index of 3
were classified as “moderately resistant”. Nematode infection also resulted in
significantly reduced average shoot and root length, and biomass in control plants
compared to transgenic plants. Transgenic plants also caused white fly mortality.
Control plants showed the mortality rate of 18.6%, and RVL1-T0(11) and RVL1-
T0(28) exhibited an increased mortality of 69.35% and 92.47%, respectively over the
control.
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Plant Biotechnology