Improved protoplast fusion technologies using metabolic inhibitors and microfluidics

植物育种极大地提高了农业生产率，并将在适应气候变化和养活日益增长的人口方面发挥重要作用。在许多情况下，所需的性状，无论是抗病/抗虫，还是对冷、热或干旱胁迫的适应，都不存在于作物中，但可以通过与近缘野生植物杂交来整合。然而，这需要物种之间的性别相容，但情况往往并非如此。*原生质体融合技术，其中来自两个物种的细胞可以融合在一起，可以克服性别障碍，使作物能够与更远缘物种杂交。这项技术对树木育种特别有吸引力，因为传统的方法可能需要几十年的时间，而且不可行。然而，树木带来了一些尚未完全解决的技术挑战，限制了这项技术的应用。这方面的一个例子是美洲榆树，当地人口几乎被入侵的荷兰榆树病(DED)灭绝。早期的研究人员发现，美国榆树种群中不包含抗DED的基因，尽管传统育种已有50多年的历史，但仍然需要真正的抗DED树。虽然一些亚洲榆树是抗性的，但由于性别不相容，早期的杂交尝试失败了，早在1980年就提出了体细胞杂交来克服这一点。然而，与原生质体分离和再生相关的一些问题阻碍了成功。*在最近的工作中，我们已经确定了问题的主要来源，并开发了一种新的方法来解决其中的一些问题。这导致了美国榆树第一个原生质体植株再生系统的建立，并为原生质体融合提供了一个框架。促成这一过程的关键突破是通过操纵植物的一条新陈代谢途径来改变细胞壁的化学成分，使其更容易适应这一过程。然而，目前还没有对这一系统进行深入的研究，一些问题需要进一步研究，以了解美国榆树的潜在机制，评估这种方法是否适用于其他树种，并开发新的方法来提高融合效率。这项提案将建立一个研究计划，以完善这项技术，并利用微流控方法开发更受控制的原生质体融合技术，用于经济和生态上重要的植物，重点是木本植物。这些技术可以在植物遗传改良方面发挥重要作用，以适应新的气候条件，并帮助养活不断增长的人口。********

Plant breeding has dramatically increased agricultural productivity and will play an important role in adapting to a changing climate and feeding the growing population. In many cases the desired trait, be it disease/insect resistance, or adaptation to cold, heat, or drought stress, is not present in the crop but can be incorporated by hybridization with closely related wild relatives. However, this requires that the species are sexually compatible with each other, which is often not the case.***Protoplast fusion technologies, in which cells from two species can be fused together, can overcome sexual barriers and allow crops to be crossed with more distantly related species. This technology is particularly attractive for tree breeding, where traditional approaches can take decades and are not feasible. However, trees present a number of technical challenges that have not been fully addressed and limit the application of this technology. One example of this is American elm, where the native population was nearly eradicated by the invasive Dutch elm disease (DED). Early researchers found that the American elm population does not contain genes for DED resistance and despite over 50 years of traditional breeding there remains a need for true DED resistant trees. While some Asiatic elms are resistant, early attempts at hybridization failed due to sexual incompatibility and somatic hybridization was proposed as early as 1980 to overcome this. However, a number of problems related to isolation and regeneration of protoplasts have prevented success.***In recent work, we have identified the primary source of the problem and developed a novel approach that resolved some of these issues. This has led to the first protoplast to plant regeneration system for American elm and provides a framework for protoplast fusion. The key breakthrough that facilitated this was through manipulating one of the plant's metabolic pathways to alter the chemical composition of the cell wall, making it more amenable to the process. However, an in depth investigation of this system has not been conducted and a number of issues require further study to understand the underlying mechanism in American elm, evaluate if this approach will work in other tree species, and develop new approaches to improve fusion efficiency. This proposal will establish a research program to refine this technique and develop more controlled protoplast fusion technologies using a microfluidic approach for economically and ecological important plants, with a focus on woody species. These technologies could play an important role in the genetic improvement of plants to adapt to new climatic conditions and help feed the growing population. ********