College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, China/Zhangjiakou Academy of Agricultural Sciences, Zhangjiakou 075000, China
Abstract：Maintaining good callus status and improving plantlet regeneration frequency (PRF) are the key points of successful rice (Oryza sativa L.) genetic transformation. In this paper, we studied the effect of sorbitol on callus status and regeneration of a japonica-type upland rice cultivar HD297 and an indica-type paddy rice cultivar 9311. We supplied different concentrations of sorbitol in callus induction, subculture and regeneration media respectively and found the 10-20-40 treatment (adding 10, 20 and 40 g/L sorbitol, respectively) got the highest PRF, up to 88.9% and 86.1% for the two cultivars with 45.1% and 32.6% increase compared to the control (0-0-0). This result was also verified in activator(Ac) gene transformation for HD297. On the basis of adding different concentrations of sorbitol in induction and subculture media, we supplied sufficient sorbitol and reduced sucrose concentration from 30 g/L to 5.5 g/L and 0 g/L respectively in regeneration medium, and amazingly, we found the combination of sucrose concentration at 5.5 g/L and sorbitol concentration at 40 g/L improved PRF greatly, up to from 82.7% to 95.2% for both cultivars no matter whether we added sorbitol in induction or subculture media. Calli were compact and fresh yellow with much more green shoots and callus browning of 9311 was suppressed significantly. These results showed that sorbitol can keep callus in good state, inhibit callus browning of indica rice and improve regeneration rate and transgenic efficiency. It is of direct application and reference value on plants tissue and organ culture such as rice and other crops.
