Gene Expression and Genome Editing Systems by Direct Delivery of Macromolecules Into Rice Egg Cells and Zygotes

Toda, Erika; Okamoto, Takashi

doi:10.21769/bioprotoc.3681

Cited by 2 publications

(1 citation statement)

References 26 publications

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“…This allowed detailed analysis of all stages of embryonic development, from the first zygotic division to the morphology of the mature embryo (Figure 1). Furthermore, transcriptomic and proteomic analyses on isolated egg cells, zygotes, and proembryos developing in vitro were conducted in wheat [415], maize [412,[416][417][418], tobacco [419], and rice [412,420,421] to reveal genes and proteins that play crucial roles from the earliest stages of embryogenesis to full embryo development.…”

Section: Analysis Of Embryogenesis With Embryo Rescue Technical Holdermentioning

confidence: 99%

Embryo Rescue in Plant Breeding

Rogo,

Fambrini,

Pugliesi

2023

Plants

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Embryo rescue (ER) techniques are among the oldest and most successful in vitro tissue culture protocols used with plant species. ER refers to a series of methods that promote the development of an immature or lethal embryo into a viable plant. Intraspecific, interspecific, or intergeneric crosses allow the introgression of important alleles of agricultural interest from wild species, such as resistance or tolerance to abiotic and biotic stresses or morphological traits in crops. However, pre-zygotic and post-zygotic reproductive barriers often present challenges in achieving successful hybridization. Pre-zygotic barriers manifest as incompatibility reactions that hinder pollen germination, pollen tube growth, or penetration into the ovule occurring in various tissues, such as the stigma, style, or ovary. To overcome these barriers, several strategies are employed, including cut-style or graft-on-style techniques, the utilization of mixed pollen from distinct species, placenta pollination, and in vitro ovule pollination. On the other hand, post-zygotic barriers act at different tissues and stages ranging from early embryo development to the subsequent growth and reproduction of the offspring. Many crosses among different genera result in embryo abortion due to the failure of endosperm development. In such cases, ER techniques are needed to rescue these hybrids. ER holds great promise for not only facilitating successful crosses but also for obtaining haploids, doubled haploids, and manipulating the ploidy levels for chromosome engineering by monosomic and disomic addition as well substitution lines. Furthermore, ER can be used to shorten the reproductive cycle and for the propagation of rare plants. Additionally, it has been repeatedly used to study the stages of embryonic development, especially in embryo-lethal mutants. The most widely used ER procedure is the culture of immature embryos taken and placed directly on culture media. In certain cases, the in vitro culture of ovule, ovaries or placentas enables the successful development of young embryos from the zygote stage to maturity.

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Section: Analysis Of Embryogenesis With Embryo Rescue Technical Holdermentioning

confidence: 99%

Embryo Rescue in Plant Breeding

Rogo,

Fambrini,

Pugliesi

2023

Plants

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DNA- and Selectable-Marker-Free Genome-Editing System Using Zygotes from Recalcitrant Maize Inbred B73

Yamada,

Kato,

Ichikawa

et al. 2024

Plant and Cell Physiology

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Genome-editing tools such as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system have become essential tools for increasing the efficiency and accuracy of plant breeding. Using such genome-editing tools on maize, one of the most important cereal crops of the world, will greatly benefit the agriculture and the mankind. Conventional genome-editing methods typically used for maize involves insertion of a Cas9-guide RNA expression cassette and a selectable marker in the genome DNA; however, with such methods, it is essential to eliminate the inserted DNA cassettes to avoid legislative concerns on gene-modified organisms. Another major hurdle for establishing an efficient and broadly applicable DNA-free genome-editing system for maize is presented by recalcitrant genotypes/cultivars, since cell/tissue culture and its subsequent regeneration into plantlets are crucial for producing transgenic and/or genome-edited maize. In this study, to establish a DNA-free genome-editing system for recalcitrant maize genotypes/cultivars, Cas9-gRNA ribonucleoproteins (RNPs) were directly delivered into zygotes isolated from the pollinated flowers of the maize-B73 cultivar. The zygotes were successfully developed and regenerated into genome-edited plantlets by co-culture with phytosulfokine, a peptide phytohormone. The method developed herein made it possible to obtain DNA- and selectable-marker-free genome-edited recalcitrant maize genotypes/cultivars with high efficiency. This method can advance the molecular breeding of maize and other important cereals, regardless of their recalcitrant characteristics.

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Gene Expression and Genome Editing Systems by Direct Delivery of Macromolecules Into Rice Egg Cells and Zygotes

Cited by 2 publications

References 26 publications

Embryo Rescue in Plant Breeding

Embryo Rescue in Plant Breeding

DNA- and Selectable-Marker-Free Genome-Editing System Using Zygotes from Recalcitrant Maize Inbred B73

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