粉红溲疏组培再生优化及遗传转化体系构建

doi:10.11707/j.1001-7488.LYKX20260041

Abstract

Abstract:

Objective: On the basis of two previously established tissue culture regeneration systems of Deutzia species, the regeneration systems of Deutzia were screened and optimized, and a genetic transformation system was constructed with the optimal regeneration system as the recipient system in this study, aiming to provide technical support for molecular breeding of important landscape tree species in Deutzia, and a reference paradigm for the de novo domestication of woody plants. Method: The tissue culture rooted plantlets of six wild species in Deutzia were used as research materials. The leaves, petioles and internodes from the 1st to 5th node along the morphological axis were taken as explants. In the tissue culture regeneration process, the genotype, explant type and maturity were screened to optimize the tissue regeneration system of Deutzia. Subsequently, the optimized system used as the recipient system to construct an Agrobacterium tumefaciens-mediated leaf-disc genetic transformation system, and simultaneously screen the optimal concentration of Timentin. Result: Among the six tested wild Deutzia species, the genotype with the best regeneration was Deutzia rubens. The optimum explant for the tissue culture regeneration of D. rubens was the first expanded leaf of 30-day-old rooted plantlets, with a differentiation rate of 100%. The rooting rate of regenerated plantlets reached 100% on 1/2 MS medium supplemented with 0.5 mg·L^–1 IBA, showing the best growth of plantlets and root systems. The rooted plantlets grew best in the acclimatization and transplantation substrate with perlite∶nutrient soil = 3∶7. The maximum plant height was 15.68 cm after 30 days of acclimatization. A. tumefaciens successfully infected the leaves of D. rubens and regenerated plantlets. The differentiation rate reached the maximum of 83.33% at 50 mg·L^–1 Timentin, and the genetic transformation efficiency was 16.7%. Conventional PCR detection verified that exogenous Ruby and NtGRF4-NtGIF1 genes were integrated into the genome of regenerated D. rubens plants, and the transgenic plants showed a visible red phenotype. Conclusion: This study has established an efficient and stable tissue culture regeneration system of Deutzia represented by D. rubens, and an Agrobacterium-mediated leaf-disc genetic transformation system is for the first time constructed. The result fills the technical gap in genetic transformation of Deutzia, and provides core technical support for gene function analysis and targeted molecular improvement of this genus, as well as a practical reference for the utilization and de novo domestication breeding of wild woody plant resources.

Key words: Deutzia rubens, tissue culture regeneration, leaf-disc method, genetic transformation, de novo domestication

CLC Number:

S722.3⁺7

Yingnan Hao,Yuting Yuan,Yuxin Tu,Xinyi Feng,Yuzhou Zhang,Huihui Wang,Puxia Wu,Chunxiang Fu,Jinyu Li,Xin Zhang. Optimization of Tissue Culture Regeneration and Construction of Genetic Transformation System of Deutzia rubens[J]. Scientia Silvae Sinicae, 2026, 62(9): 152-164.

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引物名称 Primer name	引物序列 Primer sequences（5′→3′）	引物用途 Primer purpose
Ruby-F	CCACTGCGGATGGAACAGCATC	基因克隆与验证 Gene cloning and verification
Ruby-R	CTTGACGAACGTTGTCGAAACCG
NtGRF4-nF1	ATGAGTGCGACTACGATGAGGA
NtGRF4-nR1	CTTCGTTCCAGTGCTTCCAT

处理 Treatment	基因型 Genotype	外植体类型 Explant type	外植体成熟度 Explant maturity/node	分化率 Differentiation rate（%）	出芽数 Number of sprouts
I	粉红溲疏 D.rubens	叶片Leaf	1	100.00±0.00a	9.63±1.30b
			2	35.00±0.49cd	2.25±2.43f
			3~5	0.00±0.00f	0.00±0.00f
		叶柄Petiole	1	75.00±0.44b	5.38±2.45d
			2	25.00±0.44de	1.38±1.92fg
			3	10.00±0.31ef	0.38±1.92gh
			4	10.00±0.31ef	0.25±0.46gh
			5	0.00±0.00f	0.00±0.00h
		节间Internode	1~5	0.00±0.00f	0.00±0.00h
II	钩齿溲疏 D.baroniana	叶片Leaf	1	75.00±0.44b	16.5±0.73a
			2	10.00±0.32ef	4.15±0.88e
			3~5	0.00±0.00f	0.00±0.00h
		叶柄Petiole	1	5.23ef±0.23c	6.05±0.94cd
			2	5.88ef±0.32b	5.90±0.97cd
			3~5	0.00±0.00f	0.00±0.00h
		节间Internode	1	4.76ef±0.23c	6.75±0.64c
		节间Internode	2~5	0.00±0.00f	0.00±0.00h
III	褐毛溲疏 D.pilosa	叶片Leaf	1	75.00±0.45b	3.64±0.92e
		叶片Leaf	2	50.00±0.52c	1.18±0.40fgf
		叶柄Petiole	1~5	0.00±0.00f	0.00±0.00h
		节间Internode	1~5	0.00±0.00f	0.00±0.00h
IV	长江溲疏 D.schneideriana	叶片Leaf 叶柄Petiole 节间Internode	1~5	0.00±0.00f	0.00±0.00h
V	白溲疏 Deutzia albida	叶片Leaf 叶柄Petiole 节间Internode	1~5	0.00±0.00f	0.00±0.00h
VI	光萼溲疏 D.glabrata	叶片Leaf 叶柄Petiole 节间Internode	1~5	0.00±0.00f	0.00±0.00h

处理 Treatment	吲哚丁酸IBA/ （mg·L^–1）	生根率 Rootability （%）	根长 Root length/cm	株高 Plant height/cm
I	0.25	100a	4.23±0.50b	5.75±0.86b
II	0.5	100a	5.38±0.79a	7.09±0.93a
III	1	100a	5.19±0.92ab	5.93±0.78b
IV	2	100a	3.27±0.76c	4.50±0.85c

处理 Treatment	基质配比 Substrate ratio	成活率 Survival rate （%）	株高 Plant height/cm	植株状态 Healthy states of plant
Ⅰ	珍珠岩∶营养土=3∶7 Perlite∶nutrient soil =3∶7	100.00±0.00a	15.68±0.96a	叶片绿、长势好 Leaves remained green with robust growth
Ⅱ	珍珠岩∶营养土=6∶4 Perlite∶nutrient soil = 6∶4	93.75±0.25a	13.10±0.84b	叶片黄、长势较好 Leaves turned yellow and grew fairly well
Ⅲ	珍珠岩∶营养土=9∶1 Perlite∶nutrient soil = 9∶1	81.25±0.40a	5.92±0.53c	叶片黄、长势差 Leaves were yellow and growth was poor

处理 Treatment	特美汀 Timentin/ （mg·L^–1）	污染率 Contamination rate （%）	分化率 Differentiation rate （%）
I	50	0.00±0.00a	83.33±0.38a
II	100	0.00±0.00a	72.22±0.46a
III	200	0.00±0.00a	68.75±0.47a

Optimization of Tissue Culture Regeneration and Construction of Genetic Transformation System of Deutzia rubens

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