STTR Phase I: Enhancing plant genetic transformation by Agrobacterium

这一小型企业技术转让项目的更广泛的影响/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年转基因植物的研究和商业化导致了农艺性状得到改善的作物品种，如抗除草剂和抗虫害，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的，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，对于科学家来说，在不同时引入外来DNA的情况下将理想的基因引入植物一直是一项挑战，导致了技术和监管方面的复杂问题。这些技术问题限制了转型顽固工厂包括橡胶在内的重要产品的生产。这项提案中开发的技术将通过减少产品开发成本和监管障碍，使更多的小型和工业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，包括在非传统作物上可持续的国内天然橡胶生产，以及能够创造更高效的粮食作物。这个STTRI期项目建议开发一个基于农杆菌的新型植物转化平台，以提高顽固作物转化事件的效率和质量。这项技术结合了植物组蛋白基因或蛋白质的使用，这提高了转化效率，并从细菌染色体上“启动”了农杆菌T-DNA，这减少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。专门设计的农杆菌菌株将接受测试，这些菌株可以与含有选择基因和报告基因的T-DNA同时转移组蛋白H_2A基因或蛋白质。将对拟南芥(因为很容易获得大量的转化子进行分析)和番茄(一种顽固的但在农学上很重要的作物)进行转化。目标是确定这些新的农杆菌菌株是否能够将转化频率提高一倍。此外，该计划将使用qPCR和DNA印迹策略分析数百个转基因事件的基因组，以确定这些细菌菌株的转化是否导致高比例的转基因植物包含引入的转基因的单一副本，而T-DNA(载体主干或染色体DNA序列)两侧没有外来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.