STTR Phase I: Enhancing plant genetic transformation by Agrobacterium

这个小企业技术转让（STTR）项目更广泛的影响/商业潜力是改进植物遗传转化（通过生物技术方法引入新基因）。过去20年来，转基因植物的研究和商业化已经产生了具有改进的农艺性状的作物品种，如除草剂耐受性和抗虫害性，这使农民和消费者都受益。尽管目前美国种植的绝大多数玉米和大豆都是转基因作物，但棉花和包括向日葵在内的许多其他作物的转基因仍然很困难。此外，科学家们一直面临着将所需基因引入植物而不同时引入外源DNA的挑战，这导致了技术和监管方面的复杂性。 这些技术问题限制了包括橡胶在内的重要产品在转化-再生柠檬植物中的生产。本提案中开发的技术将通过降低产品开发成本和监管障碍，使更多的次要和经济作物品种得以广泛转化，从而实现商业化。这些转化技术将应用于工业产品的表达，包括在非传统作物中可持续地生产天然橡胶，以及创造更高效的粮食作物。STTR第一阶段项目提出开发一种新型的基于农杆菌的植物转化平台，以提高柑橘类作物转化事件的效率和质量。该技术将植物组蛋白基因或蛋白质的使用与从细菌染色体“发射”农杆菌T-DNA相结合，所述植物组蛋白基因或蛋白质提高了转化效率，所述农杆菌T-DNA降低了整合的转基因拷贝数并消除了存在调控问题的外来载体序列的整合。 将测试特殊设计的农杆菌菌株，其可以同时转移组蛋白H2 A cDNA或蛋白质与含有选择和报告基因的T-DNA。 转化将在拟南芥（因为容易获得大量转化体以进行分析）和番茄（一种转化不稳定但在农学上重要的作物物种）上进行。 目的是确定这些新的农杆菌菌株是否能够使转化频率加倍。此外，计划使用qPCR和DNA印迹策略分析数百个转基因事件的基因组，以确定通过这些细菌菌株的转化是否导致高百分比的转基因植物含有引入的转基因的单拷贝，而没有T-DNA侧翼的外源DNA序列（载体骨架或染色体DNA序列）。最好的菌株将在第二阶段进行额外的测试。

The broader impact/commercial potential of this Small Business Technology Transfer (STTR) project is the improvement of plant genetic transformation (the introduction of new genes via biotechnology approaches). The past two decades of research and commercialization of genetically modified plants have resulted in crop species with improved agronomic traits, such as herbicide tolerance and pest resistance, which have benefitted both farmers and consumers. Whereas the vast majority of corn and soybeans currently grown in the US is genetically modified, transformation of cotton and many other crops, including sunflower, remains difficult. In addition, it has been challenging for scientists to introduce desirable genes into plants without the concomitant introduction of extraneous DNA, resulting in both technical and regulatory complications. These technical problems have limited production of important products, including rubber, in transformation-recalcitrant plants. Technology developed in this proposal will enable widespread transformation, and thereby commercialization, of a greater number of minor and industrial crop species by reducing product development costs and regulatory barriers. These transformation techniques will be applied toward the expression of industrial products including the sustainable domestic production of natural rubber in non-traditional crops, as well as enabling the creation of more efficient food crops.This STTR Phase I project proposes to develop a novel Agrobacterium-based plant transformation platform to improve the efficiency and quality of transformation events in recalcitrant crops. This technology combines the use of plant histone genes or proteins, which increases transformation efficiency, with "launching" Agrobacterium T-DNA from the bacterial chromosome, which decreases integrated transgene copy number and eliminates integration of extraneous vector sequences that present regulatory problems. Specially designed Agrobacterium strains will be tested that can transfer a histone H2A cDNA or protein simultaneously with T-DNA containing selection and reporter genes. Transformation will be conducted on Arabidopsis (because of the ease of obtaining large numbers of transformants to analyze) and on tomato (a transformation-recalcitrant but agronomically important crop species). The goal is to determine whether these novel Agrobacterium strains are able to double the transformation frequency. In addition, the plan is to analyze the genomes of hundreds of transgenic events, using qPCR and DNA blotting strategies, to determine whether transformation by these bacterial strains results in a high percentage of transgenic plants containing a single copy of the introduced transgenes without extraneous DNA sequences flanking the T-DNA (vector backbone or chromosomal DNA sequences). The best strain will be advanced for additional testing in Phase II.