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

转化，产生稳定的转基因株系，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增加作物产量和对胁迫的适应性的一切。 在某些植物物种中，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植物很难转化，成功仅限于少数品种，严重限制了基因功能的研究和最前沿的基因组编辑技术的使用。需要新的、可获得的、廉价的植物转化方法来推进研究和加速作物改良。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，这很容易收集，然后使用转化的花粉将导入的DNA传递给下一代后代，种子。这种方法的成功将彻底改变玉米研究，使大多数实验室能够快速、廉价地修改任何基因库，为一步精确的基因组编辑打开大门。将这种方法扩展到其他物种将加快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系。该项目还将帮助培养玉米遗传学、基因组学和细胞生物学方面的研究生和本科生。本EAGER项目的目标是开发玉米花粉的磁转染作为一种简单快速的转化方法。 磁转染，利用磁力将携带DNA的纳米颗粒推进细胞，是一种相对较新（且廉价）的技术，最近已被证明在几种植物的花粉中有效，最显着的是棉花。 然而，玉米花粉的结构、发育和生理学似乎可能为实施磁转染引入重大障碍。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描述花粉磁转染转化的结果。 该实验计划包括三个目标，这些目标经过校准，以解决玉米花粉中确定的成功障碍。 目标1将同时优化玉米花粉的体外瞬时磁转染，并确定这种处理对花粉生殖力的影响（即，种子生产能力）。 目标2将使用目标1的结果来指导实现稳定的转换协议。 目标3将使用标准遗传学方法以及下一代测序来表征稳定转化体中的转基因遗传性和全基因组结构。 任何成功的转化方案都将在两个实验室进行验证，然后通过预印本服务器迅速发布给玉米社区。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的知识价值和更广泛的影响审查标准进行评估来支持。

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.