5.3.21
TRANSFER OF PLUM POX VIRUS RESISTANCE FROM A TRANGENIC CLONE OF PRUNUS DOMESTICA THROUGH HYBRIDIZATION

L LEVY1, R SCORZA2, V DAMSTEEGT3, A CALLAHAN2 and M RAVELONANDRO4

1USDA-APHIS, PPQ, Beltsville, Maryland 20705, USA; 2USDA-ARS, AFRS, Kearneysville, West Virginia 24530, USA; 3USDA-ARS, FDWSRU, Frederick, Maryland 21702, USA; 4INRA, Centre de Recherches de Bordeaux, BP 81 33883, France

Background and objectives
Plum pox virus (PPV), a member of the Potyviridae, is the causal agent of Sharka disease of stone fruits.There is little resistance to PPV in most Prunus species, thus genetic engineering represents a potentially useful approach to obtain resistant germplasm. In earlier experiments, the heterologous coat protein (CP) from papaya ringspot virus (PRV) was transferred to plum, and transgenic plum clones remained resistant to PPV infection for 19 months [1]. The PPV-CP has been transferred to plum and we have shown one transgenic plum line, C5, is resistant to PPV infection for over 3 years [2]. It is of interest to transfer the PPV resistance observed in plum clone C5 to the progeny of a C5 cross so that resistant breeding lines can be developed and evaluated for fruit quality and other important agronomic qualities, specifically resistance to PPV infection.

Materials and methods
Transgenic clone C5 was used as the pollen parent for hybridization. Pollen from C5 plants was collected at 27 months of greenhouse growth which included two artificially induced dormancy cycles. Transgenic plum clones carrying the PRV-CP gene were maintained in the field under USDA-APHIS field test permit. At flowering, trees were maintained in a box container to prevent natural cross-pollination. Flowers on the PRV-CP clones were hand-emasculated and pollinated with C5 pollen. Fruits were harvested at a green-ripe stage, and seeds extracted and stratified. Following stratification, seeds were planted in a quarantine greenhouse, and progeny buds were propagated on Myrobalan plum rootstocks and inoculated with chip-buds from PPV-infected Prunus tomentosa. Prior to inoculation, inheritance of the PPV-CP and PRV-CP transgenes was confirmed by PCR [1, 2]. PPV infection in C5 progeny was evaluated by ELISA and observation of symptom expression. We have previously shown that C5 does not produce measurable levels of CP using immunoblotting and in the ELISA system used [2].

Results and conclusions
C5 progeny were obtained containing only PPV-CP, only PRV-CP, or both PPV-CP and PRV-CP transgenes. After 11 months post-inoculation, seedlings containing only the PPV-CP gene from C5 were symptomless and ELISA-negative. Seedlings containing only the PRV-CP gene or non-transformed controls showed PPV symptoms and were ELISA-positive. The results indicate that the PPV-CP transgenes can be transferred to progeny through hybridization, and that the genes impart resistance to the PPV transgenic seedlings. The inheritance of the multi-copy inserts of the PPV-CP and PRV-CP transgenes is currently under study.

References
1. Scorza R, Levy L, Damsteegt V et al., 1995. Journal of the American Society for Horticultural Science 120, 943-952.
2. Ravelonandro M, Scorza R, Bachelier J et al., 1997. Plant Disease 81, 1231-1235.