EAGER: A Transformative Technology for Producing Transgenic Maize: Pollen Magnetofection

转化，即产生稳定的转基因品系，是基础和应用植物生物学的基本工具，用于从测试基因功能到提高作物产量和对逆境的适应能力。在一些植物物种中，转化是一个常规程序；然而，包括玉米(玉米)在内的几种关键作物很难转化，而且仅限于少数几个品种，这严重限制了对基因功能的研究和最尖端的基因组编辑技术的使用。为了推进研究和加快作物改良，需要新的、可获得的、廉价的植物转化方法。该项目利用简单的磁铁和携带DNA的颗粒将工程DNA导入玉米花粉中，这种花粉很容易收集，然后使用转化后的花粉将导入的DNA传递给下一代后代种子。这种方法的成功将给玉米研究带来革命性的变化，允许大多数实验室快速且廉价地修改任何基因储备，为一步精确的基因组编辑打开大门。将这种方法推广到其他物种，将加快从总体上理解植物基因组功能的进展，并将这种理解转化为改进的商业育种品系。该项目还将帮助培训玉米遗传学、基因组学和细胞生物学方面的研究生和本科生。这个迫切的项目的目标是发展玉米花粉的磁感应作为一种简单快速的转化方法。磁感应利用磁力将携带DNA的纳米颗粒推进细胞内，这是一项相对较新(且价格低廉)的技术，最近已被证明对几种植物的花粉有效，最明显的是棉花。然而，玉米花粉的结构、发育和生理似乎可能为实施磁感应带来重大障碍。该项目将利用两个玉米研究小组的相关专业知识来克服这些障碍，并在全基因组范围内表征通过花粉磁感应转化的结果。该实验计划包括三个目标，这些目标经过校准，以解决在玉米花粉中确定的成功的具体障碍。目的1平行优化玉米花粉体外瞬时磁感应，并确定这种处理对花粉繁殖力(即结籽能力)的影响。目标2将使用目标1的结果来指导稳定转换方案的实施。AIM 3将结合下一代测序使用标准遗传方法来表征稳定转化体的转基因遗传力和全基因组结构。任何成功的转化协议都将在两个实验室进行验证，然后通过预印服务器迅速发布到玉米社区。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

Transformation, the generation of stable, transgenic lines, is a fundamental tool in both basic and applied plant biology used for everything from testing gene function to increasing crop yields and resilience to stress. In some plant species, transformation is a routine procedure; however, several key crop plants, including corn (maize), are very difficult to transform, and success is limited to only a few varieties, severely limiting the study of gene function and use of the most cutting-edge, genome editing technologies. New, accessible, inexpensive methods to transform plants are needed to advance research and accelerate crop improvement. This project utilizes simple magnets and DNA-carrying particles to introduce engineered DNA into maize pollen, which is easily collected, and then uses the transformed pollen to transmit introduced DNA to the next generation progeny, the seed. Success of this method would revolutionize maize research, allowing rapid and inexpensive modification of any genetic stock by most labs, opening the door to one-step, precise genome editing. Expanding this method to other species would speed up progress in understanding plant genome functions in general and translation of that understanding into improved commercial breeding lines. The project will also help train graduate and undergraduate students in maize genetics, genomics and cell biology. The goal of this EAGER project is to develop magnetofection of maize pollen as a simple and rapid method for transformation. Magnetofection, which uses magnetic force to propel DNA-carrying nanoparticles into a cell, is a relatively new (and inexpensive) technology that has recently been proved effective in the pollen of several plant species, most notably cotton. However, the structure, development and physiology of maize pollen appears likely to introduce significant hurdles for implementing magnetofection. The project will utilize the relevant expertise of two maize research groups to overcome these hurdles, and to characterize the outcome of transformation via pollen magnetofection on a genome-wide scale. The experimental plan incorporates three objectives that are calibrated to address the specific barriers to success identified in maize pollen. Aim 1 will, in parallel, optimize transient magnetofection of maize pollen in vitro, and determine the effects of such treatments on pollen fecundity (i.e., ability to produce seeds). Aim 2 will use the results from Aim 1 to guide implementation of a stable transformation protocol. Aim 3 will use standard genetic methods alongside next-gen sequencing to characterize transgene heritability and genome-wide structure in stable transformants. Any successful transformation protocol would be validated in both labs, and thereafter rapidly released to the maize community via preprint server.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.