SBIR PHASE I: Genetic Engineering of Regulated Seedlessness in Seed Propagated Crops

该小企业创新研究第一阶段项目旨在开发一种从杂交品种生产无籽水果的方法，同时保留亲本近交系的有籽水果生产。 该策略是允许受精，但随后通过细胞致死功能核糖核酸酶芽孢杆菌RNA酶的种皮特异性表达来阻止种子发育。 芽孢杆菌RNA酶的表达已被证明可以阻止特定植物组织中的细胞生长和发育。 芽孢杆菌RNA酶是一种通常为单体的酶，将被合成为由单独的合成基因编码的两个部分肽，每个部分肽将存在于一个亲本近交系中。 这样，商业水果产品是无籽的，但种子繁殖作物的商业种子生产不会受到损害。 为了初始测试的目的，将用每个嵌合基因分别转化番茄植物，对每个基因使用不同的选择标记。 为了确定当两个嵌合基因一起表达时细胞活力是否被破坏，将通过生物射弹将一个嵌合基因引入已经用另一嵌合基因转化的叶组织中。 随后，将各自表达一个部分芽孢杆菌RNA酶基因的一对品系彼此杂交，以基于对连锁标记的抗性来确认没有获得表达这两种基因的后代。 项目的总体目标是将这种方法应用于西瓜，创造出生产无籽水果的二倍体杂交品种。 为了确保这一目标能够实现，将在第一阶段研究期间根据已发表的方法评估转化西瓜的能力。 使用可育但无籽的二倍体应该会降低生产成本，因为三倍体无籽西瓜的生产需要由于三倍体品种的花粉缺乏而牺牲一部分产量来换取花粉源。 此外，通过消除生产四倍体亲本的需要，将减少开发新的无籽西瓜品种的时间和成本。 最后，这种创建无籽品种的方法对于樱桃和木瓜等其他种子繁殖作物也具有价值，并且可以扩展到葡萄等无性繁殖作物。

This Small Business Innovation Research Phase I project is directed toward the development of a method for producing seedless fruit from a hybrid variety while retaining seeded fruit production from the parental inbreds. The strategy is to allow fertilization but to block seed development soon thereafter through the seed coat-specific expression of a cell lethal function, the ribonuclease barnase. Barnase expression has been shown to block cell growth and development in specific plant tissues. A normally monomeric enzyme, barnase will be synthesized as two partial peptides encoded by separate synthetic genes, each of which will be present in one parental inbred. In this way, the commercial fruit product is seedless, yet commercial seed production of a seed-propagated crop would not be impaired. For the purpose of the initial test, tomato plants will be transformed separately with each chimeric gene, using different selectable markers for each gene. To determine whether cell viability is disrupted when the two chimeric genes are expressed together, one chimeric gene will be introduced through biollistics into leaf tissue already transformed with the other chimeric gene. Later, a pair of lines, each expressing one partial barnase gene will be crossed to each other to confirm that no progeny are obtained in which both genes are expressed, based on resistance to the linked markers. The overall project goal is to apply this method to watermelon to create diploid hybrid varieties producing seedless fruits. To ensure that this can be achieved, the ability to transform watermelon, based on published methods, will be evaluated during Phase I research. The use of fertile but seedless diploids should reduce costs of production since triploid seedless watermelon production requires that a portion of yield be sacrificed to a pollen source due to the pollen-deficiency of triploid varieties. In addition, the time and cost to develop new seedless watermelon varieties would be reduced by eliminating the need to produce a tetraploid parent. Finally, this method for creating seedless varieties will have value in other seed-propagated crops, such as cherry and papaya, and it may be extended to vegetatively-propagated crops such as grape.