Field Evaluation of Transgenic Squash Containing Single or Multiple Virus Coat Protein Gene Constructs for Resistance to Cucumber Mosaic Virus, Watermelon Mosaic Virus 2 and Zucchini Yellow Mosaic Virus

Abstract

Transgenic inbred squash lines containing various combinations of the cucumber mosaic virus (CMV), watermelon mosaic virus 2 (WMV 2) or zucchini yellow mosaic virus (ZYMV) coat protein (CP) genes were produced using Agrobacterium-mediated transformation. Progeny from lines transformed with single or multiple CP gene constructs were tested for virus resistance under field conditions, and exhibited varying levels of resistance to infection by CMV, WMV 2 or ZYMV. Most transgenic lines remained nonsymptomatic throughout the growing seasons and produced marketable fruits, while other lines showed a delay in the onset of symptoms and/or a reduction in symptom severity. A few lines failed to display any level of resistance. Depending on the CP gene used, 40 to 95% of the transgenic lines containing single CP constructs of either CMV, WMV 2 or ZYMV were resistant to the virus from which the CP gene was derived. Transgenic lines transformed with a double CP construct containing the CP genes from CMV and WMV 2, designated CW, displayed high level of resistance to CMV and WMV 2. A transgenic line, designated ZW-20, which contained the CP genes from ZYMV and WMV 2 displayed excellent resistance to ZYMV and WMV 2 in that most of the plants showed complete resistance. A few plants developed localized chlorotic dots or blotches, yet fruits remained asymptomatic. Southern blot analysis revealed that the CP inserts of some resistant plants of line ZW-20 were no longer linked to the neomycin phosphotransferase II (NPT II) gene. This loss of linkage allowed the marker gene to be separated from the virus resistance trait by Mendelian segregation. Further analysis of these plants showed that they contained multiple WMV 2 inserts which were designated B and H, the latter consisting of two hybridization signals. Analysis of inoculated plants showed that plants with the H inserts were symptomless or developed only chlorotic dots, while those without the H insert developed more prominent chlorotic blotches. In addition to lines with resistance to two viruses, a line with resistance to three viruses was also identified. Transgenic line CZW-3, transformed with the triple CP construct containing the CMV, WMV 2 and ZYMV CP genes, exhibited resistance to all three viruses. These two transgenic inbred lines, ZW-20 and CZW-3, have allowed for the development of commercial squash hybrids with multiple virus resistance.