RESUMEN
Large number of primary transgenic events were generated in groundnut by an Agrobacterium mediated, in planta transformation method to assess the efficacy of cry1AcF against the Spodoptera litura. The amplification of required size fragment of 750 bp with npt II primers and 901 bp with cry1AcF gene primers confirmed the integration of the gene. The expression of the cry gene was ascertained by ELISA in T2 generation, and the maximum concentration of cry protein in transgenic plants reached approximately 0.82 µg/g FW. Further, Southern blot analysis of ten T2 transgenic plants proved that transgene had been integrated in the genome of all the plants and Northern analysis of the same plants demonstrated the active expression of cry1AcF gene. The highest mean % larval mortalities 80.0 and 85.0 with an average mean % larval mortalities 16.25 (n = 369) and 26.0 (n = 80) were recorded in T1 and T2 generations, respectively. Segregation analysis of the selected lines in the T3 generation demonstrated homozygous nature. This clearly proved that though there is considerable improvement in average mean % larval mortality in T2 generation, the cry1AcF gene was effective against S. litura only to some extent.
RESUMEN
A rapid and efficient sap inoculation method for tobacco streak virus (TSV) was developed in sunflower. Sap from TSV-infected sunflower plants was freshly extracted in phosphate buffer and diluted serially from 10(-1) to 10(-8). Two-day old seedlings of sunflower were injured at the meristem and immersed in the sap for 10 min, maintained at 20 °C for 2-3 days and shifted to greenhouse. The surviving seedlings in the respective sap dilution were scored for symptoms of sunflower necrosis disease (SND). SND symptoms were seen in 80 % of the seedlings inoculated with a sap dilution of 10(-5). ELISA and RT-PCR analysis of coat protein and movement protein of TSV confirmed SND symptoms. The methodology was also found to be reproducible when the sap from the infected plants was inoculated onto healthy plants. The main aim of the study was to develop a primary screening strategy for the selection of transgenics developed for SND resistance. This methodology can also be extended for the analysis of resistance against other viruses.