In Vitro Regeneration of Indica Rice (Oryza sativa L.) cultivar Sangyod and Ist Transformation by Agrobacterium tumefaciens

Abstract

Indica rice cultivar Sangyod is one of the most important commercial rice cultivars in Thailand. However, a review of the literature was unable to trace any published research considering the improvement by gene transformation of Sangyod using Agrobacterium tumefaciens. The present study investiagted to develop a tissue culture and plantlet regeneration systems, established Agrobacterium-mediated transformation systems and molecular analaysis to confirm introduction of foreign gene into putative rice transformants. Mature embryos from seed were used as explant for callus induction and plantlet regeneration system. The result of experiments showed that the highest frequency of callus induction (73.08 ± 2.65%) and mean callus fresh weight (67.5 ± 7.4 mg) were obtained on MS (Murashige and Skoog, 1962) medium supplemented with 2 mg/L 2, 4-dichlorophenoxyacetic acid (2,4-D), 750 mg/L casein hydrolysate (CH) and 200 mg/L L-proline. The combination of 0.5 mg/L a-naphthaleneacetic acid (NAA), 1 mg/L 6- benzyladenine (BA), 0.5 mg/L Kinetin (Kn) and containing solidified MS medium gave the maximum mean fresh weight of callus (938.9 ± 44 mg), the highest percentage of green spot formation (64.17 ± 7.08%), maximum shoot induction frequency (66.25 ± 6.80%) and mean number of shoots/callus (6.12 ± 0.36 shoots). Furthermore, the greatest mean number of shoots/explant (14.93 ± 0.97 shoots) and root formation (82.71 ± 3.03%) was observed in liquidified MS medium supplemented with 0.5 mg/L NAA and 1 mg/L BA. The culture medium and plant growth regulators (PGRS) play a significant role in callus induction, fresh weight of callus, the callus growth index and the plantlet regeneration protocol of mature rice seeds. The highest callus induction (75.63 ± 5.28%) and fresh weight of callus (68.05 ± 20.04 mg) were achieved on agricultural research development agency (ARDA) medium supplemented with 2 mg/L 2, 4-D, 750 mg/L CH and 200 mg/L L-proline after 4 weeks of culture. The optimum mean fresh weight of callus (1272.83 ± 48.63 mg) and the highest callus growth index (11.73-fold) were obtained on day 35 of sub-culture. The combination of 0.5 mg/L NAA, 0.5 mg/L 6-benzylaminopurine (BAP), 0.5 mg/L Thidiazuron (TDZ) and 1.0 mg/L Kn gave the maximum percentage of green spot formation (72.34 ± 8.75%), plantlet regeneration frequency (67.25 ± 6.14%) and the mean number of plantlets/ callus (6.63±0.47 plantlets). Finally, an efficient transformation system for the indica rice cultivar Sangyod was carried out by optimizing some key factors including inoculation time and optical density (OD) of Agrobacterium suspension, various concentrations of acetosyringone (AS) and cefotaxime. These results showed 6-week-old Sangyod calluses derived from mature seeds were inoculated at an OD600 at 0.6 for 30 minutes, Then the calluses were cultured on co-cultivation medium containing ARDA medium supplemented with 100 mg/L myo-inositol and 200 μM AS for 2 days. After co- cultivation, the explants were washed with liquid ARDA medium containing 300 mg/L cefotaxime for 20 minutes. Finally, the inoculated calluses were placed on selection medium (ARDA medium containing, 300 mg/L cefotaxime, 100 mg/L myo- inositol and 0.6 mM glyphosate). The highest transformation efficiency detected by measuring transient GUS expression (70.43%) and callus survival rate (43.5%) was achieved with the foregoing conditions, the stable transfer of the GUS gene were confirmed by PCR analysis.