Abstract：Agrobacterium-mediated transformation technology has been widely used in japonica rice (Oryza sativa ssp. japonica) varieties, but so far inaccessible to indica rice(O. sativa ssp. indica) varieties, mainly attributed to calli browning in following regeneration, which has severely hindered the application of transformation in indica rice. In this study, cv. 93-11, a typical indica variety, was used and the mathematical model was established by central composite design combined with response surface methodology and the media component for callus subculture were optimized. Results showed that MS media was more suitable for primary callus induction than N6, B5, N6B and NMB, which callus browning was significant inhibited through observation and comparison. Based on MS media, the effects of single factors such as hormone, carbon source were analyzed through single factor experiment. It was found that the best 2,4-dichlorophenoxyacetic acid (2,4-D) was 3 mg/L, kinetin (KT) was 0.5 mg/L, sucrose was 15 g/L and maltose was 15 g/L. And then Box-Behnken design was used to obtain the best value. The determination coefficient R2 of the model was 0.9 476 and the optimal combination of media component for inducing yellow, compact and organized calli was 2,4-D 2.92 mg/L, KT 0.59 mg/L, sucrose 16.37 g/L and maltose 13.63 g/L. Histological and anatomical analysis revealed the remarkable morphological differences between normal calli and brown calli: The surface of normal calli was quite irregular and was full with distinctive globular nodules and the globular nodules were full with tightly packed cells. The somatic embryogenesis consisted of white, sheet-shaped non-embryogenesis structures could be descried from the yellowish opaque sectors of a calli. In contrast, the smooth surface of brown calli was due to the fact that nearly all cells distorted from round shape to flat shape and the inactivation cells were unlikely to occur globular nodules. qRT-PCR revealed that the transcript levels of artificial wound related gene auxin 1 gene (AUX1) and sucrose non-fermenting 1-related nrotein kinase 2 gene (SnRK2) had obviously difference between normal calli and brown calli, the mRNA accumulation of the AUX1 in normal calli with roughly a 5-fold increase compared to brown calli and SnRK2 transcription in brown calli was about 2.5-fold higher than in normal calli, while phenolic metabolism related gene phenylalanine ammonia-lyase gene (PAL) and polyphenol oxidase gene (PPO) could neither be detected in normal calli nor in brown calli, these expression patterns indicated that artificial wound was the main reason for calli brown. The results would be helpful to understand the mechanism of browning occurrence in plant tissue culture.
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