MULTIPLE TRANSGENE ENGINEERING FOR MAIZE (Zea mays L.) DROUGHT AND SALT TOLERANCEByThang Xuan Nguyen Maize (Zea mays L.) is an important basic food and feed grain, and its stover is used for animal feed and biofuel. Increased soil-salinity and water-deficiency are the two major factors limiting the maize plant growth and development and subsequently affecting its grain and biomass yields, and the yield. The research in this dissertation is focused on multiple transgene engineering (transgene pyramiding) for drought and salt tolerance in maize. In the research presented in this dissertation, genetic transformation of maize was performed via the gene gun bombardment of embryogenic immature embryos of maize using single and multiple constructs, pBY520 containing the barley (Hordeum vulgare) HVA1 and the JS101 containing the bacterial mannitol 1 phosphate dehydrogenase (mtlD), both genes regulated by rice actin promoter and potato protease inhibitor II terminator. There were two gene linked cassettes in each of these two constructs, one cassette containing the abiotic stress tolerance gene of interest (HVA1 or mtlD) and the other containing the bar herbicide resistance gene regulated by cauliflower mosaic virus 35S promoter and nos terminator. The pBY520 and JS101 constructs were co-bombarded in 1:1 ratio into maize genome for transgene pyramiding. The confirmation of transgene integration and expression were made via molecular techniques, including polymerase chain reaction (PCR) for transgene integration, and reverse transcriptase (RT) PCR and Northern blotting for transgene transcription. Southern blotting was performed to find the number of copies of each transgene in transgenic plants. Results showed stable integration and expression of the HVA1, mtlD and HVA1-mtlD in transgenic maize plants. Up to 4th generation transgenic (T3) plants were produced, with all progenies showing the co-integration of abiotic stress tolerance genes of interests and the bar gene with a frequency of 100%. The single HVA1 or mtlD transgenic plants showed higher leaf relative water content (RWC) and higher percent of plant survival as compared to their wild-type non-transgenic control plant counterparts under water withholding condition for 15 days followed by 7 days of re-watering. When exposed to different salt concentrations (0, 100, 200 and 300 mM NaCl) for 10 days, the HVA1 and mtlD transgenic plants showed higher fresh and dry shoot and dry root biomass matter as compared to their wild-type non-transgenic control plants. The research also demonstrated that the mtlD transgenic plants that were salt tolerant, also accumulated mannitol in their cells. More research is needed on mannitol accumulation in transgenic plants to see whether the mannitol level measured by gas chromatography (GC) was not partially or totally representing sorbitol accumulation. Considering that mannitol and sorbitol are both osmoprotectants, the salt tolerance of the mtlD transgenic plants might be due to the accumulation of one or both osmoprotectants in mtlD transgenic plants. Co-transformation of two transgenes (HVA1+mtlD) in maize plants also demonstrated the co-integration and co-expression of these two stacked genes in up to T3 plants, resulting in improved plant survival rate under 15 and 20 days of water withholding and enhanced shoot and root biomass weight at 100mM NaCl as compared to single transgene (HVA1 or mtlD) transgenic plants and as compared to the wild-type control plants. This dissertation also covers genetic transformation of maize plants with a construct containing the sorghum dehydration responsive element binding 2 (DREB2) transcription factor regulated by the Arabidopsis rd29 drought inducible promoter and nos terminator. The preliminary results showed the integration of DREB2 into maize plants via PCR analysis. The DREB2 transgenic maize research will be continued in Sticklen's laboratory by another researcher in the near future. Although salt stress is predictable, drought is not predictable in most counties on earth. The dissertation research presented here is a step towards production of maize plants that can tolerate the harsh abiotic stress conditions of drought and salt.
Read
