McCaw Morgan E, Lee Keunsub, Kang Minjeong, Zobrist Jacob D, Azanu Mercy K, Birchler James A, Wang Kan
Department of Agronomy, Iowa State University, Ames, IA, United States.
Crop Bioengineering Center, Iowa State University, Ames, IA, United States.
Front Genome Ed. 2021 Jan 11;2:622227. doi: 10.3389/fgeed.2020.622227. eCollection 2020.
Maize ( ssp. ) is a popular genetic model due to its ease of crossing, well-established toolkits, and its status as a major global food crop. Recent technology developments for precise manipulation of the genome are further impacting both basic biological research and biotechnological application in agriculture. Crop gene editing often requires a process of genetic transformation in which the editing reagents are introduced into plant cells. In maize, this procedure is well-established for a limited number of public lines that are amenable for genetic transformation. Fast-Flowering Mini-Maize (FFMM) lines A and B were recently developed as an open-source tool for maize research by reducing the space requirements and the generation time. Neither line of FFMM were competent for genetic transformation using traditional protocols, a necessity to its status as a complete toolkit for public maize genetic research. Here we report the development of new lines of FFMM that have been bred for amenability to genetic transformation. By hybridizing a transformable maize genotype high Type-II callus parent A (Hi-II A) with line A of FFMM, we introgressed the ability to form embryogenic callus from Hi-II A into the FFMM-A genetic background. Through multiple generations of iterative self-hybridization or doubled-haploid method, we established maize lines that have a strong ability to produce embryogenic callus from immature embryos and maintain resemblance to FFMM-A in flowering time and stature. Using an -mediated standard transformation method, we successfully introduced the CRISPR-Cas9 reagents into immature embryos and generated transgenic and mutant lines displaying the expected mutant phenotypes and genotypes. The transformation frequencies of the tested genotypes, defined as the numbers of transgenic event producing T1 seeds per 100 infected embryos, ranged from 0 to 17.1%. Approximately 80% of transgenic plants analyzed in this study showed various mutation patterns at the target site. The transformable FFMM line, FFMM-AT, can serve as a useful genetic and genomic resource for the maize community.
玉米（亚种）因其易于杂交、成熟的工具包以及作为全球主要粮食作物的地位，是一种广受欢迎的遗传模型。基因组精确操纵的最新技术发展进一步影响了基础生物学研究和农业生物技术应用。作物基因编辑通常需要一个遗传转化过程，即将编辑试剂引入植物细胞。在玉米中，对于有限数量适合遗传转化的公共品系，该程序已经成熟。快速开花迷你玉米（FFMM）品系A和B最近被开发为一种开源玉米研究工具，可减少空间需求和世代时间。FFMM的两个品系都不能使用传统方案进行遗传转化，而这是其作为公共玉米遗传研究完整工具包的必要条件。在此，我们报告了已培育出适合遗传转化的新型FFMM品系。通过将可转化的玉米基因型高II型愈伤组织亲本A（Hi-II A）与FFMM的A品系杂交，我们将Hi-II A形成胚性愈伤组织的能力导入到FFMM-A遗传背景中。通过多代迭代自交或双单倍体方法，我们建立了从幼胚中产生胚性愈伤组织能力强且在开花时间和株高上与FFMM-A相似的玉米品系。使用农杆菌介导的标准转化方法，我们成功地将CRISPR-Cas9试剂导入幼胚，并产生了显示预期突变表型和基因型的转基因和突变品系。测试基因型的转化频率定义为每100个感染胚产生T1种子的转基因事件数量，范围为0至17.1% 。本研究中分析的约80%转基因植物在靶位点显示出各种突变模式。可转化的FFMM品系FFMM-AT可作为玉米群体有用的遗传和基因组资源。
