The Stress Condition Needed For Kranz Structure Forming In Submerged Eleocharis Baldwinii & The Exploration Of Setaria Viridis Genetic Transformation System

Photosynthesis is the world’s most massive energy and material conversion process, and is the material basis for almost all life’s survival and development. It has been awarded as “the most important chemical reaction on earth” by the Nobel foundation. As the growing of population, decreasing of fields, how to increase the crop yield has become the primary problem. Compared with rice, wheat and other C₃ plants, the existence of Kranz structure in C₄ plants allow the enrichment of CO2 between bundle sheath cells, so as to improve photosynthetic efficiency. Previously, scientists dedicated to engineer C₄ core cycle related enzymes into C₃ plants, but haven’t achieved any big breakthrough. In this study, we hope to study the mechanism of C₄ photosynthesis by investigate the formation of Kranz structure.Eleocharis baldwinii is a kind of amphibians freshwater aquatic plants, it performs different modes of photosynthesis when grows in different environments. The mode of photosynthesis is C₃-C₄ intermediate in the submerged forms and C₄ in the terrestrial forms. In this study, submerged forms of E. baldwiniinnii was treated with different concentration of NaCl and PEG-6000, the anatomical section and enzymatic activity were further investigated to understanding the basis of formation of Kranz structure. At the same time, we chose several candidate genes which showed differentially expression when comparing S. viridis and E. baldwinii transcriptome database, RNAi vectors was constructed and introduced into S. viridis using Agrobacterium-mediated genetic transformation. In addition, we optimized the tissue culture and genetic transformation system procedure, which will help to explore the key genes in the process of the development of C₄ photosynthesis.The main results of this study are summarized as follows:1. By combining anatomical observation, determination of chlorophyll content and key enzyme activity of C₄ cycle, we could show that when materials induced by 0.4% NaCl and 3% PEG-6000, the anatomical structure of culms appear Kranz type and present C₄-like phenotype.2. Based on transcriptome date, we selected 18 differentially expressed genes, constructed RNAi vectors for S. viridis endogenous genes and conducted Agrobacterium transformation. A few T0 generation transgenic lines were selected, ds1-1, ds3-1, ds4-1, ds5-1, ds6-1, ds7-1, ds7-2, ds9-1, ds10-1, which will be further characterized in the next step.3. In order to improve callus status, the types of cytokinin and auxin and their relative proportion were adjusted, as well as using different supplements in the culture medium. When 40 g sucrose was used to replace maltose, the callus quality was improved slightly but significantly, which indicated that the addition of sucrose is important for callus induction using mature S. viridis seeds.