Genome editing approaches using reproductive cells/tissues in flowering plants

Abstract

Targeted mutagenesis via programmable nucleases including the clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) system has been broadly utilized to generate genome-edited organisms including flowering plants. To date, specific expression of Cas9 protein and guide RNA (gRNA) in reproductive cells or tissues is considered one of the most effective genome-editing approaches for heritable targeted mutagenesis. In this report, we review recent advances in genome editing methods for reproductive cells or tissues, which have roles in transmitting genetic material to the next-generation, such as egg cells, pollen grains, zygotes, immature zygotic embryos, and shoot apical meristems (SAMs). Specific expression of Cas9 proteins in initiating cells efficiently induces targeted mutagenesis viaAgrobacterium-mediated in planta transformation. In addition, genome editing by direct delivery of CRISPR/Cas9 components into pollen grains, zygotes, cells of embryos and SAMs has been successfully established to generate genome-edited plant lines. Notably, DNA-free genome editing by the delivery of Cas9-gRNA ribonucleoproteins (RNPs) is not associated with any legislative concerns about genetically modified organisms. In summary, the genome editing methods for reproductive cells or tissues have enormous potential for not only basic studies for plant reproduction but also applied sciences toward molecular plant breeding.

New Frontiers in Potato Breeding: Tinkering with Reproductive Genes and Apomixis

“…The strategies used so far focus on annulling key meiotic steps to produce gametes with a somatic number of non-recombinant chromosomes (mimicking apomeiosis), and then to develop the unreduced egg cell without male fertilization (mimicking parthenogenesis). Knocking out multiple genes simultaneously using CRISPR−Cas9 has produced site-specific mutant phenotypes in different plant species without side effects, both by exploiting reproductive-cell- or tissue-specific features [ 125 , 126 ], and by generating specific reproductive plant phenotypes such as a male-sterile Eucalyptus [ 127 ] or a hybrid rice producing clonal seeds [ 128 ]. In potato, the prospect for using similar approaches to create specific reproductive phenotypes is promising.…”

Preface to the special issue “Current Status and Future Prospects for the Development of Crop Varieties and Breeding Materials Using Genome Editing Technology”

“…Various strategies for null segregant production have been proposed (reviewed in detail by Bhattacharjee et al 2023), including crossing or direct transformation of primary transformants. Additionally, methods involving Ribonucleoproteins (RNPs), protoplasts, pollen, or reproductive techniques have been explored (Toda et al 2022). Recently, a graftingmediated genome editing system was also reported (Yang et al 2023), adding to the array of available techniques.…”