Transmission of a Sugarcane yellow leaf virus resistance transgene to sexual progeny and screening by kanamycin inoculation

Sugarcane (a complex Saccharum hybrid) yellow leaf is caused by the Sugarcane yellow leaf virus (SCYLV) and is endemic in many growing regions. Inadequate sources of SCYLV resistance limit conventional breeding for resistance. Although transgenics offer the potential for improving SCYLV resistance, previous studies have shown that agronomic performance may be negatively impacted in primary (T0) transgenic lines due to somaclonal variation or transgene position effects. These problems could be overcome or reduced by transgene transfer to sexual progeny. The objectives of this study were to evaluate the utility as parents of two sugarcane genotypes (6-1 and 6-2) previously transformed with the nptII selectable marker and U-SCYLV-CP inserts and to examine the potential of kanamycin sensitivity as a selectable marker in progeny derived from these crosses. 6-1 and 6-2 were used as parents in 31 crosses in four crossing seasons. Flowering characteristics and germination rates of the resulting true seed were comparable with those for the untransformed parent, CP 92-1666, and with other crosses made in the same seasons. Inheritance and function of the nptII insert were tested in 215 seedlings from three crosses through the application of a solution containing 3.0 g L−1 kanamycin. Diagnostic PCR confirmed the presence of the nptII insert transgene in 158 (98.1%) of the 161 kanamycin-resistant progeny. Segregation analysis of the data suggests that the nptII insert is independently integrated at two linked loci in 6-1 one of which carries a functional copy of the nptII insert and the U-SCYLV-CP insert. Whereas for 6-2, nptII is inserted at a single, functional loci carrying the U-SCYLV-CP insert. ΔΔCt values were determined from real-time PCR data of each insert among progeny testing positive for both inserts using diagnostic PCR. This novel application of the ΔΔCt method allowed the determination of copy number relative to the T0 clone without the use of an exogenous control and showed that all copies of the U-SCYLV-CP insert were inherited among almost all progeny of either 6-1 or 6-2. These results demonstrate the utility of T0 transformed sugarcane genotypes for transmission of transgenes to additional sugarcane genotypes and the effectiveness of kanamycin application for screening sexual progeny and will be useful in guiding breeding strategies to improve resistance to the SCYLV through transgenics.