Genetic transformation of soybean and tobacco with β-1,3-glucanase genes [thesis]

Liying Fan
f3-l,3-glucanases (also known as callases) are enzymes that hydrolyze f3-l ,3-linked glucans (calloses). They are involved in various important plant physiological and developmental processes, such as seed germination, cell growth, plant defense against pathogens, environmental stresses, and flowering . In this research project I was particularly interested in their roles during pollen development and maturation. Premature or delayed f3l ,3-glucanase function in tapetal tissue is the apparent
more » ... use of male sterility in some plants. Genetic engineering has been used successfully to manipulate callase expression in tobacco and lettuce and resulted in partial to complete male sterility in the transgenic plants, while leaving female function unimpaired. The objective of this research was to alter callase expression by genetic transformation of soybean and tobacco with f3-l ,3-glucanase genes. To familiarize myself with the method of Agrobacterium-mediated transformation of soybean cotyledonary nodes, several months' training was carried out. From the training program, a few putative transgenic plants were obtained from soybean cultivars ' Jack' and 'Thorne', based on GUS assay results and/or resistance to herbicide 'Liberty' . Down-regulation of callase expression was carried out in soybean by introducing an anti-sense version of a soybean flower bud specific partial f3-l ,3-glucanase gene (SGlu 7) driven by a tapetum-specific promoter T A-29. Due to insufficient funding, high cost and low efficiency of soybean transformation, this work was terminated one year later. No transgenic plants were recovered. Binary vector pL Y200. l generated in this research could be used as a generic mother vector for expressing genes in tapetal tissue. Blast search of SGlu7 in EST database of GenBank located two soybean f3-l,3glucanase genes. Sequencing results showed that one of them, A W-Glu, contained the complete coding sequence of the callase gene. Constructs pL Y500 and pL Y 450 were made by inserting AW-Glu after TA-29 in pLY200.l , with pLY500 having AW-Glu in sense orientation and pL Y 450 having A W-Glu in anti-sense orientation. pL Y 450 could be used to down-regulate callase expression in soybean. pL Y500 was subsequently introduced into tobacco by a leaf disk transformation method, in order to alter the timing and/or to increase V the amount of callase expression, which may cause male sterility. While most of the transgenic plants did not show the expected male sterility, one plant L YS00-9 displayed two distinct kinds of branches, L Y500-9AB and LY500-9N. Flowers from branches of LY500-9AB had stigmas higher than the anthers, while flowers from branches of L Y500-9N had stigmas about the same height as the anthers. Compared with LY500-9AB or L Y500-9N, non-trangenic plants had anthers significantly taller than the stigmas. The unique floral characteristics made L Y500-9AB functionally male sterile. Further research is needed to investigate the cause of the functional male sterility in L Y500-9AB and its relation with the transgene A W-Glu expression.
doi:10.31274/rtd-20200803-66 fatcat:oph3kl5bjzfoxoao543qwqbala