Improved protoplast fusion technologies using metabolic inhibitors and microfluidics

植物育种极大地提高了农业生产力，并将在适应气候变化和养活不断增长的人口方面发挥重要作用。在许多情况下，期望的性状，无论是抗病/抗虫，还是对寒冷、炎热或干旱胁迫的适应，都不存在于作物中，但可以通过与密切相关的野生近缘种杂交而纳入。然而，这要求物种之间有性相容，而事实往往并非如此。原生质体融合技术可以将两个物种的细胞融合在一起，这种技术可以克服性障碍，使作物能够与更远的物种杂交。这项技术对树木育种特别有吸引力，传统方法可能需要几十年的时间，而且不可行。然而，采油树面临着许多技术挑战，这些挑战尚未得到充分解决，并限制了该技术的应用。美洲榆树就是一个例子，当地的榆树几乎被入侵性荷兰榆树病（DED）消灭了。早期的研究人员发现，美国榆树种群不含有抗DED的基因，尽管经过了50多年的传统育种，仍然需要真正抗DED的树木。虽然一些亚洲榆树具有抗性，但由于性不相容，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问题。然而，与原生质体的分离和再生有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，并开发了一种解决其中一些问题的新方法。这导致了美洲榆树原生质体到植株的第一个再生系统，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代谢途径之一来改变细胞壁的化学成分，使其更容易适应这一过程。然而，对这一系统的深入研究尚未进行，许多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。本提案将建立一个研究项目，以改进该技术，并利用微流体方法开发更具控制的原生质体融合技术，用于经济和生态重要的植物，重点是木本物种。这些技术可以在植物的遗传改良中发挥重要作用，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.