Evaluation of Aphidicidal Properties of PTA (Penellia Ternate Agglutinin) Gene in Lettuce (Lactuca Sativa L.)

Abstract

Lettuce, one of the most widely used freshly consumed leafy vegetable, is very much susceptible to aphids and farmers use a large quantity of aphidicidal drugs to keep aphids out of their lettuce crops. Aphids not only destroy crops through cell sap sucking, but also they serve as the most important vectors for many viral diseases. The present study was carried out with a view to develop transgenic aphid resistance lettuce (cv. "Evola") with Penellia ternate agglutinin an aphidicidal gene from Penellia ternate, a Chinese traditional medicinal plant. PTA protein has been demonstrated to possess aphidicidal properties. In order to develop transgenic plants true leaves (1st and 2nd leaves) from aseptically grown seedlings were transformed with Agrobacterium tumefaciens bearing Ti based pBIPTA plasmid containing selectable marker neomycin phosphotransferase (nptll) and pta gene. By using optimal parameters of 50 mg/I kanamycin, 1.5 OD (at 600 nm) of bacterial suspension, 3 min incubation period, 24 h co-cultivation period successful transformation experiment was carried out. Putative transgenic plants were regenerated from the infected leaves cultured onto regeneration medium containing 0.1 mg/I NAA + 0.01 mg/I BA and 50 mg/I kanamycin. Fifty putative kanamycin resistant plants were analysed with PCR and RT-PCR for the detection of pta gene and its transcript. Out of 50 putative trangenetic plants 22 plants were found PCR and RT-PCR positive, which reveal that pta gene were integrated into the plant genome. Among 22 PCR and RT-PCR positive T0 plant, 10 plants were grown to maturity and TI seeds were collected. Homozygous pta plants were selected by germinating T1 seeds on to 200 mg/I Kanamycin supplemented MS0 medium. Kanamycin resistant TO plants were segregated npt II gene into 3: 1 Mendilian ratio. Fifty transgenic plants were grown in a field, individually covered with aphid protecting nylon net. The plants were individually challenged with Turnip aphids (Lipaphis erysimi Kaltenbach). In transgenic plants aphid population was significantly decreased than those of control plants. Insect bioassays showed that transgenic expressing PTA were significantly inhibit the growth of Turnip aphids. Present study reveals that the pta gene can be used as a supplement in the control of aphids in lettuce crop. This is the first time report on lettuce (cv. 'Evola') that transgenic plants expressingpta gene confer enhanced resistance to aphids.