Generation and Characterization of Transformation Events in Tomato Carrying Sclerotium rolfsii Lectin Gene (srl1) for Root Knot Nematode and White Fly Resistance
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Date
2011
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UAS, Dharwad
Abstract
Transgenic tomato plants carrying Sclerotium rolfsii lectin gene (srl1) were
generated by Agrobacterium-mediated transformation. Of the 90 independent
regenerants, 40 could amplify 429 bp product with srl1-specific PCR. Segregation
analysis in three selected events indicated single copy insertion in SRL1-T0(7) and
multi-copy integration in SRL1-T0(10) and SRL1-T0(21). In SRL1-T0(7), the TDNA/
srl1 insertion was in a non-genic region between 85,375 and 85,376 bp of the
clone C11_HBa0054I23 (GenBank Acc. No. AC212431.2) of chromosome 11. Crude
extract of SRL1-T0(7), SRL1-T0(10) and SRL1-T0(21) showed an extra band of 17
kDa on SDS-PAGE. This protein along with their partially purified forms could
agglutinate rabbit erythrocytes, indicating lectin activity. Specific activity of the lectin
was more in the partially purified samples compared to protein from the crude
extracts.
Bioassay with second stage juveniles (J2s) of Meloidogyne incognita, the root
knot nematode showed that on 3rd day of inoculation the infection rate was 66.66,
83.33, 91.66 percent in SRL1-T0(7), SRL1-T0(10), SRL1-T0(21), respectively
compared to 100 per cent infection in the non-transgenic plant. On 6th day, the
percentage of vermiform J2s developed into sausage-form was higher in nontransgenic
plants compared to transgenic lines. After 70 days of inoculation, nontransgenic
plants showed an average gall number of 59.6 per plant whereas, it was
reduced to 33.6, 43.2 and 40.8 galls/plant, respectively in SRL1-T0(7), SRL1-T0(10),
SRL1-T0(21). In general, non-transgenic plants showed a gall index of 7, hence they
were rated as “highly susceptible”, whereas transgenic lines with a gall index of 3
were classified as “moderately resistant”. Nematode infection also resulted in
significantly reduced average shoot and root length, and biomass in non-transgenic
plants compared to transgenic plants. Transgenic plants were evaluated for white fly
(Bemisia tabaci) mortality. It was 24%, 22% and 16% in SRL1-T0(7), SRL1-T0(10),
SRL1-T0(21), respectively, which were higher than that found in non-transgenic plant
(10%).
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Keywords
Plant Biotechnology