Establishment of an Efficient in planta Transformation Method for Camellia sinensis

doi:10.13560/j.cnki.biotech.bull.1985.2021-0635

Abstract

Abstract:

To address the lack of an efficient and stable genetic transformation system for tea trees,an in planta transformation transgenic method based on Agrobacterium tumefaciens-mediated transformation of tea trees was established,laying a solid foundation for gene function research and germplasm innovation of tea tree. The seedlings of tea cultivar ‘Tieguanyin’,‘Baiye1’,and ‘Longjing43’ were used as receptor plants,and the apical bud and axillary bud were removed. Using the A. tumefaciens broth infecting the wounds of the seedling,regenerative buds were obtained through resistance screening. After molecular biological identification,transgenic plants were obtained by short-ear cutting method. The results demonstrated that the GUS（-glucuronidase）and HYG（hyglymycin）labeled genes were tested to be positive with multiple PCR,and the PCR product sequence was consistent with the labeled gene sequence. The transformation efficiency of ‘Tieguanyin’,‘Baiye1’,and ‘Longjing43’ was 8.14%,2.99%,and 2.53%,respectively. This study establishes an in planta transformation method of tea plant that does not rely on tissue culture,and the transformation protocol characterizes of simple operation,high transformation rate,low cost,and short experiment cycle.

Key words: Camellia sinensis, in planta transformation, agrobacterium tumefaciens

ZHOU Cheng-zhe, CHANG Xiao-jun, ZHU Chen, CHENG Chun-zhen, CHEN Yu-kun, LAI Zhong-xiong, LIN Yu-ling, GUO Yu-qiong. Establishment of an Efficient in planta Transformation Method for Camellia sinensis[J]. Biotechnology Bulletin, 2022, 38(2): 263-268.

Figures/Tables 3

Table 1 Primer sequenle information

引物名称Primer name	引物序列Primer sequence（5'-3'）	退火温度Annealing temperature/℃	引物用途Purpose
GUS-F	ACGTCCTGAAGAAACCCCAACC	55.0	阳性转基因植株筛选 Screening of positive transgenic plants
GUS-R	TCCCGGCAATAACATACGGCGT	55.0
HYG-F	CTATTTCTTTGCCCTCGGACGAG	55.2
HYG-R	GAATCGGTCAATACACTACATGGC	53.7

Fig. 1 Process of obtaining transgenic tea plants A：Tea seedlings. B：Tea seedlings without apical and axillary buds. C：Agrobacterium tumefaciens-mediated transformation through infection of wounds. D：Wound moisturized using sealing film. E-H：Resistant buds sprouted from axillary buds after HYG resistance screening. I and J：Cuttings of transgenic plants

Fig. 2 Detection of marker genes in the transgenic plants A：GUS marker gene（675 bp）detection in transgenic plants. B：HYG marker gene（675 bp）detection in transgenic plants. M;DNA marker. CK：Positive control. H2O：Negative control

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