Date of Award
Doctor of Philosophy
David J. Hannapel
Loren C. Stephens
Two major research approaches have been used to study the tuber-related gene expressions in different Solanaceous species. The first part of the research is using Agrobacterium-mediated gene transformation to study potato proteinase inhibitor II (pinII) gene which highly expressed in developing tuber in a wild nontuberizing specie Solanum brevidens. To establish the transformation system, leaf pieces of in-vitro cultured S. brevidens were cocultivated with Agrobacterium tumefaciens that contained nptII and GUS genes on the disarmed plasmid pBI121. Independent transgenic shoots were regenerated from both solidified and liquid medium with kanamycin. Agrobacterium strains GV2260 resulted in a higher transformation frequency than LBA4404. All kanamycin-resistant, putatively transformed plantlets were confirmed positive by GUS assays. Southern analysis of randomly selected transgenic plants showed that each transgenic plant contained at least one copy of the GUS gene. S. brevidens were then cocultivated with Agrobacterium tumefaciens strain GV2260 with pinII-GUS fusion gene on the disarmed plasmid pRT210. Analysis of polymerase chain reaction (PCR) products followed by Southern blot hybridization demonstrated the presence of GUS genes in three of the transforments. Transgenic plants were tested for methyl jasmonate, sucrose, and wounding induction of the pinII-GUS gene at the RNA and protein level using leaf-petiole cuttings and whole plants. Northern blot analysis showed the pinII-GUS transgene in the transgenic plant can be induced by methyl jasmonate and sucrose. Wounding of transgenic S. brevidens leaves on whole plants resulted in high expression of GUS activity both locally and systemically;The second approach is to study the expression of cathepsin D inhibitor genes which is also expressed in developing tuber in both tuberizing and nontuberizing species using blot hybridization. Three Solanaceous species, tomato (Lycopersicon esculentum), potato (S. tuberosum cv. Superior) and S. brevidens, were tested for MJ induction at the RNA level using a petiole/leaf system. The results of this study showed that the cathepsin D inhibitor is induced by MJ. A dose response test was performed for MJ induction of CDI in the leaves of petiole/leaf cuttings. For Superior and tomato, both had highest level of expression at 50 [mu]M. The maximum expression level for S. brevidens was at 500 [mu]M MJ. The induction of MJ was also dependent on the duration of exposure. Twelve hours of incubation were enough to give relatively abundant accumulation for all three species. There is no light/dark effect on MJ induction from 0 to 48 hours for either Superior or S. brevidens. But it has a slight decrease of expression using tomato leaf samples in the same treatment. Leaf samples of Superior and S. brevidens were also treated with 50 [mu]M of GA either before or after MJ treatment to observe the interaction of these two hormones. GA had no effect on the MJ induction of CDI expression in either Superior or S. brevidens. Immunoblot analysis showed CDI proteins were induced in 'Superior' leaves after MJ treatment.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Tsu-Hwie Annie Liu
Liu, Tsu-Hwie Annie, "Induction of tuber gene expression in solanaceous species by methyl jasmonate and wounding " (1995). Retrospective Theses and Dissertations. 11012.