Abstract:

The purpose of this study is to explore the genetic stability and DNA methylation changes of test tube plantlets of Atractylodes lancea (Thunb.) DC in long-term subculture, so as to ensure the quality of plantlets produced in factory. We established an efficient in vitro rapid propagation system for long-term subculture with young terminal buds of Atractylodes lancea and analyzed the test tube plantlets with different subculture times by ISSR and MSAP. The results showed that 10 samples with different generations had low genetic diversity and high genetic stability. Among them, the genetic similarity coefficient between the first generation and the tenth generation samples was the lowest, and the genetic relationship was far away. From the fourth generation of subculture, the bands of different primer amplification patterns changed in individual loci, and the change was more obvious with the increase of subculture times. After long-term subculture, the methylation level of test tube plantlet of A. lancea decreased, and the demethylation mode and methylation mode coexisted, but the demethylation mode was the main mode. The mutation phenomenon after long-term subculture might be caused by different culture conditions and culture time, resulting in gene reactivation and expression or inhibition, and demethylation mode was higher than methylation mode. The results of this study provide a theoretical basis for germplasm conservation and industrial production of A. lancea.

Key words: Atractylodes lancea (Thunb.) DC, subculture, inter-simple sequence repeat, methylation sensitive amplification polymorphism, DNA methylation