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Abstract The production of embryogenic callus and somatic embryos is integral to the genetic improvement of crops via genetic transformation and gene editing. Regenerable embryogenic cultures also form the backbone of many micro‐propagation processes for crop species. In many species, including maize, the ability to produce embryogenic cultures is highly genotype dependent. While some modern transformation and genome editing methods reduce genotype dependence, these efforts ultimately fall short of producing truly genotype‐independent tissue culture methods. Recalcitrant genotypes are still identified in these genotype‐flexible processes, and their presence is magnified by the stark contrast with more amenable lines, which may respond more efficiently by orders of magnitude. This review aims to describe the history of research into somatic embryogenesis, embryogenic tissue cultures, and plant transformation, with particular attention paid to maize. Contemporary research into genotype‐flexible morphogenic gene‐based transformation and genome engineering is also covered in this review. The rapid evolution of plant biotechnology from nascent technologies in the latter half of the 20th century to well‐established, work‐horse production processes has, and will continue to, fundamentally changed agriculture and plant genetics research.
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Development of a Transformable Fast-Flowering Mini-Maize as a Tool for Maize Gene Editing
Maize ( Zea mays ssp. mays ) 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 Agrobacterium -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.
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- Award ID(s):
- 1917138
- PAR ID:
- 10291957
- Date Published:
- Journal Name:
- Frontiers in Genome Editing
- Volume:
- 2
- ISSN:
- 2673-3439
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3389/fgeed.2020.622227
