Early infection of scutellum tissue with Agrobacterium allows high‐speed transformation of rice

Abstract

SummarySeveral approaches have recently been adopted to improve Agrobacterium-mediated transformation of rice, both to generate the large number of T-DNA insertion plants needed for functional analysis of the rice genome, and for production of rice with additional agronomical value. However, about 3 months of in vitro culture is still required for isolation of transgenic rice plants. Here, we report the competency of scutellum tissue from 1-day pre-cultured seeds for Agrobacterium-mediated transformation. Furthermore, early infection of rice seeds with Agrobacterium enhanced efficient selection of transformed calli. Using our system, we successfully regenerated transgenic rice plantlets within a month of the start of the aseptic culture of mature seeds. Our new system should reduce the somaclonal variation accompanying prolonged culture of rice cells in the dedifferentiated state and facilitate the molecular breeding of rice.

Potential Candidate Genes for Improving Rice Disease Resistance

“…Rice stable transformation via Agrobacterium tumefaciens has become a routine technique in laboratories (Toki et al 2006). Nowadays, even if japonica is frequently used because it is highly amenable to transformation, indica transformation efficiency has sufficiently improved to allow its use in functional validation experiments and field trials in the appropriate environments.…”

Visual selection allows immediate identification of transgenic rice calli efficiently accumulating transgene products

“…1a) used in this study (Toki 1997;Toki et al 2006) was transferred into Agrobacterium tumefaciens strain EHA105 by electroporation (Hood et al 1993). …”

“…Selection against hygromycin (Hyg) and regeneration of transgenic plants were performed following the method of Toki et al (2006). Visual selection of GFP was performed as follows: after 3-days of co-cultivation with Agrobacterium at 25°C under constant dark, the calli were washed and cultured on N6D medium containing 400 mg/L carbenicillin (Nakalai tesque, Kyoto, Japan) for 4-8 weeks at 33°C (light for 10 h)/30°C (dark for 14 h).…”

“…Strong green fluorescence emitted from rice callus was considered to be derived from the rice cells because only a faint GFP signal was shown to be detected from Agrobacterium harboring pCAMBIAsGFP . In parallel, antibiotic selection using Hyg was performed following the method of Toki (1997;Toki et al 2006); 2 weeks after Agrobacterium infection, sectors growing vigorously in the presence of Hyg were isolated under white light and sub-cultured (Fig. 1c).…”

“…Until now, routine and efficient Agrobacterium-mediated transformation systems have been developed for japonica, javanica and indica cultivars of Asian rice (Oryza sativa L.) and for the new rice for Africa (NERICA, O. sativa 9 O. glaberrima) (Dong et al 1996;Hiei and Komari 2006;Hiei et al 1994;Ishizaki and Kumashiro 2008;Rashid et al 1996;Toki 1997;Toki et al 2006). In Nipponbare, a model variety of japonica rice, the generation of transgenic plantlets at high frequency was successfully achieved within a month of inoculation of mature seeds .…”