Establishment and Optimization of Agrobacterium-mediated Transient Transformation System of Toona sinensis Leaves

doi:10.13560/j.cnki.biotech.bull.1985.2025-0449

Abstract

Abstract:

Objective Establishing a fast and efficient genetic transformation system in Toona sinensis may provide technical support for gene function research and molecular breeding. Method This study utilized tissue-cultured seedlings of T. sinensis as the experimental materials, employed Agrobacterium-mediated vacuum infiltration, and adopted the red RUBY reporter gene to monitor transformation events and gene expression in the plants. Research was conducted to optimize conditions such as the type of Agrobacterium strain, infection concentration, and vacuum infiltration duration. Finally, validation and evaluation were conducted using five tissue-cultured seedlings from different provenances of T. sinensis. Result Simultaneously, a transient transformation system suitable for T. sinensis has been established. The Agrobacterium strains suitable for transient transformation in T. sinensis were screened out, namely K599 and GV3101 strains. The optimal transformation conditions obtained by comparing transformation efficiency were 150 μmol/L acetosyringone, OD₆₀₀ at 0.5, vacuum infiltration for 30 min, and co-cultivation for 3–4 d. Ultimately, the results indicated that the transformation system has high transformation efficiency. Conclusion This system provides technical support for rapidly and effectively conducting functional identification of genes in T. sinensis.

Key words: Toona sinensis, Agrobacterium, transient transformation, system establishment, system optimization

ZHANG Yao-yi, YIN Heng-fu, LIU Jun, HAN Xiao-jiao, FAN Yan-ru, WANG Min-yan, CAO Shou-jin. Establishment and Optimization of Agrobacterium-mediated Transient Transformation System of Toona sinensis Leaves[J]. Biotechnology Bulletin, 2025, 41(11): 221-227.

Figures/Tables 6

Table 1 Fluorescence quantitative PCR primer sequences

引物名称 Primer name	引物序列 Primer sequence （5'-3'）	碱基数 Base pairs （bp）
Ts-acting-F	TGATTGGGATGGAAGCAGCA	20
Ts-acting-R	GAACATGGTTGAACCGCCAC	20
Ts-RUBY-F	AACAGCATCCTTGAGTCTCTTCG	23
Ts-RUBY-R	TTCTCTTTGGAGATCTCGCCTTC	23

Fig. 1 Establishment of transient transformation system of T. sinensisA: Uninfected tissue-cultured seedlings of T. sinensis, scale bar=2 cm; B: transiently transformed T. sinensis seedlings with RUBY, scale bar=2 cm; C: analysis of RUBY gene expression, where EV indicates empty vector-infected T. sinensis seedlings, and RUBY-OE indciates RUBY-overexpressing T. sinensis seedlings infected with RUBY. *** P<0.001

Fig. 2 Comparison of transformation efficiency of T. sinensis bydifferent treatmentsA: T. sinensis seedlings infected with different Agrobacterium strains containing RUBY plasmids, scale bar=2 cm. B: T. sinensis seedlings treated with different concentrations of Agrobacterium, scale bar=2 cm. C: T. sinensis seedlings infected with different durations of vacuum infiltration treatment, scale bar=2 cm

Fig. 3 Effects of different treatments on the transformation efficiency of T. sinensis leavesA: The effect of Agrobacterium strain types on the transient transformation efficiency of T. sinensis leaves. B: The effect of Agrobacterium concentration on the transient transformation efficiency of T. sinensis leaves. C: The effect of vacuum infiltration time on the transient transformation efficiency of T. sinensis leaves. Different letters indicate significant differences at the 0.05 level. + is red area ≤20% of total area of variegated leaves; ++ is red area 20%-50% of total area of variegated leaves; +++ is red area 50%-80% of total area of variegated leaves

Fig. 4 Infection of five T. sinensis tissue culture seedling materials from different regionsIn the figure， C-1 and C-2 are T. sinensis material originating from the Guangxi region. C-3 is a T. sinensis material originating from the United Kingdom. C-4 is a T. sinensis material originating from Anhui. C-5 is a T. sinensis material originating from Sichuan. All used materials were sub-cultured for 30 d in the tissue culture room of research group on forest tree germplasm resources of Research Institute of Tropical Forestry， Chinese Academy of Forestry. The scale bar in the figure is 2 cm

Fig. 5 Transformation efficiency of five T. sinensis tissue culture seedling materials from different regionsDifferent letters indicate significant differences at the 0.05 level

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