An improved rice transformation system using the biolistic method

Abstract

Abstract. Immature embryos and embryogenic calli of rice, both japonica and indica subspecies, were bombarded with tungsten particles coated with plasmid DNA that contained a gene encoding hygromycin phosphotransferase (HPH, conferring hygromycin resistance) driven by the CaMV 35s promoter or Agrobacterium tumefacieiis NOS promoter. Putatively transformed cell clusters were identified from the bombarded tissues 2 weeks after selection on hygromycin B. By separating these cell clusms from each other, and by stringent selection not only at the callus growth stage but also during regeneration and plantlet growth, the overall transformation and selection efficiencies were substantially improved over those previously reported. From the most responsive cultivar used in these studies, an average of one transgenic plant was produced from 1.3 immature embryos or from 5 pieces of embryogenic calli bombarded. Integration of the introduced gene into the plant genome, and inheritance to the offspring were demonstrated. By using this procedure, we have produced several hundred transgenic plants. The procedure described here provides a simple method for improving transformation and selection efficiencies in rice and may be applicable to other monocots.

Transient expression of - glucuronidase gene in indica and japonica rice (Oryza sativa L.) callus cultures after different stages of co-bombardment

“…In most of the samples gus gene expression exhibiting blue stains were unevenly distributed on the surfaces. Similar observations have been previously reported for embryogenic calli 48 h after bombardment (Li et al, 1993 andSivamani et al, 1996).…”

“…In some of the clumps intensive blue staining could be detected ( Figure 3C) and this could be due to the diffusion of gus reaction product to neighboring region. Similar expression patterns were reported earlier following particle bombardment in rice (Li et al, 1993), turf type Bermuda grass (Li and Qu, 2004), cowpea (Kononowicz et al, 1995), and Sweet potato (Prakash and Vardarajan, 1992).…”

“…For callus induction, indica seeds were plated on Petri dishes (6 cm diameter) containing LS (Linsmaier and Skoog, 1965) supplemented with 11.3 M 2,4-D (2,4-dichlorophenoxyacetic acid), 30 g/L sucrose and 2.5 g/L phytagel (pH 5.8). Mature seeds of Taipei 309 were cultured on NB medium (Li et al, 1993) containing 9.0 M 2,4-D. After 4 weeks of culturing at 26°C, friable loosely arranged embryogenic calli were separated and sub cultured onto fresh medium for proliferation.…”

“…E-Mail: ramesh_biotek@yahoo.co.in japonica cultivars (Christou et al, 1991;Cao et al,1992;Li et al,1993;Duan et al,1996;), indica cultivars (Li et al,1993;Sivamani et al, 1996;Zhang et al,1996;Jain et al, 1996;Ghosh Biswas et al, 1998), elite cultivars belong to Central America and West Africa (Valdez et al,1998), U.S. rice lines (Jiang et al ., 2000) and Australian rice cultivars (Abedinia et al, 2004) have been successfully used as target tissue to develop transgenic plants expressing various traits. Earlier, optimization of various parameters for the introduction of -glucuronidase gene into embryogenic callus cultures of indica rice variety Basmati 370 (Minhas et al, 1996) and suspension cells of Pusa Basmati 1 (Jain et al, 1996) have been reported.…”

Selection of large quantities of embryogenic calli from indica rice seeds for production of fertile transgenic plants using the biolistic method

“…In the past decade, extensive research efforts have been focused on rice (Oryza sativa L) with the aim of establishing an efficient and reliable rice transformation system. To date, two transformation techniques, protoplast transformation mediated by polyethylene glycol or by electroporation, and microprojectile bombardment of immature embryos or embryogenic calli, have been used routinely to transform thejaponka subspecies of rice (Toriyama et aL, 1988; B a n g et al., 1988;Zhang and Wu, 1988;Shimamoto et al, 1989;Datta et al, 1990;Peng et al, 1992;Li et al, 1993). While fertile transgenic plants can be obtained using either method, the biolistic method is becoming the method of choice because it alleviates the need of preparing protoplasts, reduces the time needed to regenerate transgenic plants, and results in transgenic plants with higher fertility.…”