Abstract:

The effects of preculture time, bacteria concentration, infection time, time and temperature of co-culture on Agrobacterium-mediated transformation were studied through introducing expression vector PBI121-4CL-RNAi, that decreases lignin content, into callus from a mature embryo of Dendrocalamus farinosus. Our purpose is to establish an Agrobacterium-mediated transformation method of Dendrocalamus farinosus and to obtain transgenic plants. This study could lay a foundation for transformation of Dendrocalamus farinosus. The results showed that the loose and fragile callus of Dendrocalamus farinosus with pale yellow color and granular shape was a good donor to be used for Agrobacterium-mediated transformation. The callus was precultured in the inducing medium for 8 days, before it was immersed for 20 min in Agrobacterium suspension (EHA105, Agrobacterium concentration is OD₆₀₀ = 0.05), and then it were co-cultured in the co-cultivation medium with single filter paper at 25 ℃ for 2 days in the dark. The co-cultured callus were transferred into Kana-resistant medium containing Kana (55 mg·L^-1) for 30 days. The Kana-resistant callus was accounted for 90% of the total callus. PCR identification of the Kana-resistant callus showed that the 4CL gene from Neosinocalamus affinis was introduced into Dendrocalamus farinosus callus. The Kana-resistant calli were transferred to the shooting medium and cultured for 30 days to obtain clustered shoots. After growing to 3—5 cm, the shoots were transferred into the rooting medium for 20—30 days. The regeneration plants with 1—8 roots were obtained. PCR identification of resistant plants showed that the 4CL gene from Neosinocalamus affinis was introduced into the resistant plants. The transgenic plant was obtained. The transformation efficiency with the system was 9%. The results showed that the endogenous 4CL gene expression in the transgenic callus and plants was effectively suppressed. The expression in the transcription level is significantly lower than the control by RT-PCR detection.

Key words: Dendrocalamus farinosus, callus, Agrobacterium-mediated method, genetic transformation