Preliminary investigations of Agrobacterium-mediated transformation in indica rice MR219 embryogenic callus using gusA gene

Abstract

Preliminary steps in the genetic transformation of indica rice MR219 was investigated in the plant-Agrobacterium tumefaciens interaction. Agrobacterium tumefaciens strain LBA 4404 carrying a binary vector pCAMBIA 1305.2 harboring the modified GUS gene driven by the CaMV 35S promoter was used. Various transformation parameters influences were optimized using embryogenic calli via βglucuronidase (GUS) as a reporter marker. Various transformation parameters were optimized including bacterial concentration, age of embryogenic callus, pre-culture period, wounding technique, cocultivation period, immersion time and dry time before co-cultivation, acetosyringone (AS) concentration, pH of co-cultivation media and temperature of the co-cultivation period. The expression of the transient gusA gene in the plant genome was preliminary confirmed by histochemical GUS assay activity (as blue spots). The results from transient gusA gene expression of calli suggested that the Agrobacterium-mediated transfer system of T-DNA in indica rice MR219 was highly efficient. Therefore, the investigation of factors that influence T-DNA delivery is an important first step in the utilization of Agrobacterium in the transformation of indica rice MR219 calli.

Development of partial abiotic stress tolerant Citrus reticulata Blanco and Citrus sinensis (L.) Osbeck through Agrobacterium-mediated transformation method

“…Zuraida et al 2011 [25] showed that pre-culture period for more than 3 days improved transformation frequency. Four-day pre-cultured calli were used for Agrobacterium infection.…”

“…The infected calli were dried for 30 mins on sterilized filter paper. Eighty-five transformation rate was recorded when calli were infected for 90 mins and dried for 30 mins [25]. In this study, infected calli were co-cultured for 3 days [25].…”

Optimization of various factors affecting Agrobacterium-mediated transformation and regeneration of putative transgenic Malaysian MR219 rice with Bacillus SP 289 endo-β-1,3-1,4-glucanase gene

“…This result was contrast to Sreeramanan et al (2009), who reported that low transformation efficiency with Agrobacterium at a cell density of 0.2 at OD 600nm was attributed due to lack of sufficient Agrobacterium cells to transfer T-DNA into orchid cells. According to Zuraida et al (2011), delivery of gusA gene was most efficient in rice cultivar MR232 by using Agrobacterium cell density of OD 600nm 0.6. Yong et al (2006) reported that the highest rate of genetic transformation of members of the Melastomataceae family with Agrobacterium LBA4404 was obtained at a cell density of 0.8 OD 600nm (1x10 7 cfu ml -1 ).…”

“…The transformed whitish embryos were transferred to MS regeneration media (Zuraida et al 2011) supplemented with 30 mg/L hygromycin B, 100 mg/L carbenicillin and 100 mg/L cefotaxine for further regeneration. The cultures were then kept at 26 ± 2 ºC under 16-h photoperiod.…”

Efficient regeneration and Agrobacterium-mediated transformation protocol for recalcitrant indica rice (Oryza sativa L.).

“…However, Agrobacterium transformation of Malaysian rice variety remained largely untapped probably due to its recalcitrance towards Agrobacterium. The optimization study on the Agrobacterium method using mature-seed derived embryogenic callus MR219 was only carried out recently (Zuraida et al, 2011). It was reported that a 2.4% of transformation efficiency was recorded by using somatic embryos and embryogenic calli MR219 as the target tissues (Zuraida et al, 2013).…”