Agrobacterium-Mediated Genetic Transformation of Wild Oryza Species Using Immature Embryos

Abstract

Genetic transformation is one of the most important technologies for revealing or modulating gene function. It is used widely in both functional genomics and molecular breeding of rice. Demands on its use in wild Oryza species is increasing because of their high genetic diversity. Given the difficulties in genetic crosses between distantly related species, genetic transformation offers a way to alter or transfer genetic traits in wild rice accessions. However, transformation of wild Oryza accessions by conventional methods using calli induced from scutellum tissue of embryos in mature seeds often fails. Here, we report methods using immature embryos for the genetic transformation of a broad range of Oryza species. First, we investigated the ability of callus induction and regeneration from immature embryos of 192 accessions in 20 species under several culture conditions. We regenerated plants from immature embryos of 90 accessions in 16 species. Next, we optimized the conditions of Agrobacterium infection using a vector carrying the GFP gene driven by the maize ubiquitin promoter. GFP signals were observed in 51 accessions in 11 species. We analyzed the growth and seed set of transgenic plants of O. barthii, O. glumaepatula, O. rufipogon, and O. brachyantha. The plants grew to maturity and set seeds normally. Southern blot analyses using DNA from T 0 plants showed that all GFP plants were derived from independent transformation events. We confirmed that the T-DNAs were transmitted to the next generation through the segregation of GFP signals in the T 1 generation. These results show that many Oryza species can be transformed by using modified immatureembryo methods. This will accelerate the use of wild Oryza accessions in molecular genetic analyses and molecular breeding.

An Efficient Genetic Transformation and CRISPR/Cas9-Based Genome Editing System for Moso Bamboo (Phyllostachys edulis)

“…Immature embryos are easier to sterilize, and calli can be induced from the scutellum with parenchyma cells. Regeneration systems from immature embryos have been widely established in rice, wheat, corn, and other gramineous crops (Özgen et al, 1998;Frame et al, 2011;Shimizu-Sato et al, 2020). In this study, although bamboo rarely blooms, immature seeds were collected from experienced bamboo farmers every year.…”

Agrobacterium-Mediated High-Efficiency Genetic Transformation and Genome Editing of Chaling Common Wild Rice (Oryza rufipogon Griff.) Using Scutellum Tissue of Embryos in Mature Seeds

“…The genetic transformation of several wild Oryza accessions had been reported, however, its regeneration and transformation efficiency were low ( Zhang et al, 2019 ; Shimizu-Sato et al, 2020 ; Yu et al, 2021 ). An efficient genetic transformation system for wild rice is needed to be well established.…”

“…Variant concentrations of 2,4-D are used for callus induction of different genotypic Oryza accessions. The concentration of 2,4-D used for callus induction of cultivated rice Nip ( japonica ), Kasalath ( indica ), and common wild rice is 2 mg/L, meanwhile 2.5 mg/L for IR64 ( indica ) ( Hiei et al, 1994 ; Saika and Toki, 2010 ; Sahoo and Tuteja, 2012 ; Shimizu-Sato et al, 2020 ). In this study, 2.5 mg/L 2,4-D was used for CLCWR and shows the best induction efficiency ( Figure 1 ).…”

“…Compared with the genetic transformation system of PPR1, it took about 120 days to obtain T 0 transgenic plants, however, which took about 100 days for CLCWR, which markedly reduce the time of tissue culture. Compared with callus-induced material as used in the immature embryos ( Shimizu-Sato et al, 2020 ), we used mature seeds to induce callus as the transformation receptor, which makes the experimental operation fast and convenient. Three hormones (2,4-D, NAA, and 6-BA) were used to induce callus when immature embryos were used for transformation receptors ( Shimizu-Sato et al, 2020 ), however, only one hormone (2,4-D) was used and got the same effect in our study.…”

“…However, its infection efficiency was only 12.5–37.5%, which is lower than that of Nip. In addition, compared with scutellum tissue of embryos in mature seeds which can be conveniently collected and stocked enough mature seeds to run experiments, the operation of callus induction using immature embryosis is more cumbersome ( Shrawat and Good, 2011 ; Shimizu-Sato et al, 2020 ). Therefore, it is necessary to establish an Agrobacterium -mediated high-efficiency genetic transformation system of CWR by using scutellum tissue of embryos in mature seeds.…”