Abstract
Key messageBiolistic delivery into pollen.In recent years, genome editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells. However, this method has general limitations, such as the limited host range of Agrobacterium and difficulties in tissue culture, including callus induction and regeneration. To avoid these issues, we developed a method to genetically modify germ cells without the need for Agrobacterium-mediated transfection and tissue culture using tobacco as a model. In this study, plasmid DNA containing sequences of Cas9, guide RNA, and fluorescent reporter was introduced into pollen using a biolistic delivery system. Based on the transient expression of fluorescent reporters, the Arabidopsis UBQ10 promoter was found to be the most suitable promoter for driving the expression of the delivered gene in pollen tubes. We also evaluated the delivery efficiency in male germ cells in the pollen by expression of the introduced fluorescent marker. Mutations were detected in the target gene in the genomic DNA extracted from CRISPR/Cas9-introduced pollen tubes, but were not detected in the negative control. Bombarded pollen germinated pollen tubes and delivered their contents into the ovules in vivo. Although it is necessary to improve biolistic delivery efficiency and establish a method for the screening of genome-modified seeds, our findings provide important insights for the detection and production of genome-modified seeds by pollen biolistic delivery.
Highlights
Rapid progress has been made in the development of genome engineering techniques, which have made it possible to perform specific modifications of selected target genes
Fluorescent proteins are driven under the control of cauliflower mosaic virus (CaMV) 35S, A. thaliana ribosomal protein S5A (AtRPS5A), and A. thaliana UBIQUITIN10 (AtUBQ10) promoters, which have previously been used as constitutive promoters (Liang et al 2018; Nekrasov et al 2013; Tsutsui and Higashiyama 2017), as well as the pollen vegetative cellspecific LAT52 promoter derived from S. lycopersicum (Eady et al 1995)
The other 70% of angiosperms, including N. benthamiana and N. tabacum, produce bicellular pollen grains that contain a generative cell as a precursor of sperm cells
Summary
Rapid progress has been made in the development of genome engineering techniques, which have made it possible to perform specific modifications of selected target genes. One of the most widely used methods is targeted genome editing using clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 nuclease (Cas9) (Cong et al 2013). This method is widely used in various organisms, including plants, because of its suitability for genetic engineering (Li et al 2013; Nekrasov et al 2013; Osakabe and Osakabe 2015; Osakabe et al 2016). Flowers contain male and female gametophytes that produce gametes. Pollen tubes serve as vector cells that deliver copies of the male genome to the female gametophyte to produce seeds that give rise to the generation. Pollen is easier to access and handle than egg cells or zygotes, and such features are common to a wide range of angiosperms
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