5.3.14
RESISTANCE EVALUATIONS OF TRANSGENIC TOMATOES EXPRESSING THE TOMATO MOTTLE GEMINIVIRUS AC1-REP GENE

JE POLSTON1, WB HUNTER1, AM ABOUZID2 and E HIEBERT2

1Plant Pathology, University of Florida, GCREC Bradenton, FL, USA; 2Plant Pathology, University of Florida, Gainesville, FL, USA

Background and objectives
Whitefly-transmitted geminiviruses are a serious problem for tomato production in Florida and in Latin America [1]. Tomato mottle geminivirus (ToMoV) was discovered in Florida's tomato production in 1989 [2]. Currently ToMoV is managed in Florida through the use of imidacloprid, a systemic insecticide, effective against the whitefly vector. The widespread use of this insecticide in tomato and many other crops in Florida (citrus, turf and many different ornamentals and vegetables) may lead to the development of resistance in the whitefly. The use of insecticides is an expensive method of virus management. A less expensive and most effective method is the use of resistant cultivars. No tolerant or resistant cultivars are available at this time for use against ToMoV or any other geminiviruses native to this hemisphere [1]. We have initiated a pathogen-derived approach to develop tomatoes with resistance to ToMoV. We successfully transformed tomato with the ToMoV replicase-associated protein gene (AC1-REP) utilizing a non-enhanced 35S promoter vector system. Two advanced breeding lines (from the breeding program of JW Scott, GCREC, Bradenton, FL) were transformed with ToMoV AC1-REP in both sense and antisense orientations. Our approach for resistance evaluation was to simulate natural inoculation as much as possible.

Results and conclusions.
28 and nine plants, respectively, were determined to be transformed with AC1-REP in the sense and antisense orientations. T1 progeny of these plants were evaluated for resistance to ToMoV in the greenhouse. Ten T1 plants of each T0 plant were inoculated with ToMoV using an average of 10 whiteflies per plant at the two true-leaf stage. ToMoV resistance was detected only in the plants transformed with the sense orientation AC1-REP. We evaluated 26 T2 lines (40 plants each), 23 T3 lines and 20 T4 lines in the field for horticultural qualities and for resistance to ToMoV (using dot spot hybridization or PCR to detect virus). Tomato lines were inoculated by viruliferous whiteflies at the seedling stage before being transplanted into the field for evaluation under conditions of high whitefly populations during the fall 1996, and spring and fall 1997. The inoculation pressure resulted in 100% infection within 30 days in the untransformed, control plants. Two out of 23 T0 plants were the source of stable and high levels of resistance to ToMoV under field conditions through four generations. Yields of the resistant lines in the T4 generation were found to be as high or higher than the untransformed and uninfected parent during yield trials in the fall 1997. Preliminary Southern analysis has indicated that resistant plants have either one or two copies of the transgene. The AC1-REP gene provides stable ToMoV resistance and has been successfully passed through four generations of tomato. Several lines of transformed tomatoes have been identified which confer very good levels of resistance to ToMoV under the pressure of an inoculation protocol which is more severe than the conditions experienced in a grower's field.

References
1. Polston JE, Anderson PK, 1997. Plant Disease 81, 1358-1369.
2. Abouzid AM, Polston JE, Hiebert E, 1992. Journal of General Virology 72, 3225-3229.