Studies on Transformation of Tomato(Solanum lycopersicum L.) and Arabidopsis thaliana using Chimerical constructs of varying Tospoviral Origin
Pathogen derived resistance (PDR) is a recent breakthrough where plant hosts can be made to be resistant to viral infections through transformation with conserved viral genes. Given the severity of Tospovirus diseases worldwide (particularly in tomato), PDR has the potential to garner large yield returns where pathogen populations have overcome the established resistance. Tomato breeding lines FLA7804, FLA8044, and the research line MP1 were used in transformation experiments with potions of the Tomato spotted wilt virus (TSWV) N-gene, and two other chimerical viral nucleocapsid gene constructs from, Impatiens necrotic spot virus (INSV), and Groundnut ringspot virus (GRSV). We conducted 19 independent transformations consisting of 300 to 700 14-day old whole cotyledons each for a total number of approximately 9,000 potentially transformed explants. Of those, approximately 6,300 explants failed to produce regenerants, 2,419 explants underwent abnormal development on elongation media, 187 failed to root, and 215 plants to be characterized genetically. Of the 215 plants, 9 were from FLA 7804, 96 from FLA 8044, and 110 from MP1. Both PCR and Southern blot hybridization analysis later confirmed that none of the 215 plants were transgenic. Opposite to tomato, we were able to transform Arabidopsis thaliana ecotype wassilewskija (Ws) via floral dip with the above listed constructs demonstrating that constructs were not deleterious within a plant once fully introgressed. Sixteen independent transformants in the T0 generation resulted from 19,000 germinated seed from three dipped plants resulting in a total transformation rate of 0.08%. Of the 1,000 T1 seed germinated on kanamycin media from each of the 16 putative Arabidopsis plants transformed with the construct containing elements of the N-gene from all three of the aforementioned tospoviruses, four populations exhibited simple Mendelian inheritance of the transgene. DNA walking analysis yielded amplification of the unknown region outside the nptII region of the insert for three of the four remaining transformants, which was subsequently sequenced and mapped to chromosomes 1, 3, and 4. There were 25 T1 individuals selected from each population and transferred to soil for DNA extraction and zygosity determination. Homozygous T2 seed was collected for future resistance studies.
College and Department
Life Sciences; Plant and Wildlife Sciences
BYU ScholarsArchive Citation
Cobb, Joshua Nathaniel, "Studies on Transformation of Tomato(Solanum lycopersicum L.) and Arabidopsis thaliana using Chimerical constructs of varying Tospoviral Origin" (2008). Theses and Dissertations. 1478.
tomato, TSWV, Tospovirus, pathogen derived resistance, PDR, plant transformation, Solanum lycopersicum, Arabidopsis thaliana