Establishment of Genetic Transformation and Gene Editing System for a Potato Cultivar Chuanyu 50

doi:10.13560/j.cnki.biotech.bull.1985.2024-1178

Abstract

Abstract:

Objective To construct a genetic transformation system and gene editing system of potato Chuanyu 50. Method Aseptic histocultured seedling stems and leaves of Chuanyu 50, the main potato (Solanum tuberosum L.) variety promoted in Sichuan province, were used as explants. The potato explants were transformed with Agrobacterium-mediated transformation containing the plant CRISPR/Cas9 vector pJCV55-StU6-200-StUBI10-T#01. Four different media with various phytohormone ratios were analyzed and screened for callus induction and differentiation. CRISRR-Cas9-based editing method was applied for creating mutants. Result 1) The optimal medium among the tested ones for genetic transformation Chuanyu 50 was B system. The medium formulation for stem and leaf pre-cultivation and callus induction, respectively, were as follows: MS basic salt+20 g/L sucrose +1 mL/L 1 000× N&N vitamins+1.0 mg/L TZR(trans-zeatin-riboside,TZR)+0.027 8 mg/L GA3(gibberellin A3,GA3)+0.02 mg/L NAA(1-naphthaleneacetic acid,NAA)+2.0 g/L phytagel, and MS basic salt+20 g/L sucrose +1 mL/L 1 000× N&N vitamins+0.5 mg/L TZR+2.5 mg/L IAA(indole acetic acid,IAA)+2.0 g/L phytagel, the callus induction rates using stem and leaf were 93% and 88%, respectively. The medium for differentiation was MS basic salt+20 g/L sucrose+1 mL/L 1 000× N&N vitamins+2.0 mg/L TZR+10 mg/L GA3+2.0 g/L phytagel. 2) It is demonstrated that the two-round rooting screening method accurately identified transgenic-positive plants with a 100% accuracy. 3) An Agrobacterium-mediated CRISPR/Cas9 gene editing system for Chuanyu 50 with 63% editing rate was established. Conclusion The genetic transformation and gene editing system of potato Chuanyu 50 are initially established and multiple factors affecting the process of callus induction and regeneration are evaluated.

Key words: potato, Chuanyu 50, Agrobacterium tumefaciens, genetic transformation, gene editing, CRISPR/Cas9

WEN Bo-lin, WAN Min, HU Jian-jun, WANG Ke-xiu, JING Sheng-lin, WANG Xin-yue, ZHU Bo, TANG Ming-xia, LI Bing, HE Wei, ZENG Zi-xian. Establishment of Genetic Transformation and Gene Editing System for a Potato Cultivar Chuanyu 50[J]. Biotechnology Bulletin, 2025, 41(4): 88-97.

Figures/Tables 9

References 36

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培养基体系 Medium system	培养基 Medium	培养基成分 Medium composition	pH
A	A1	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+2.49 mg/L TZR+0.027 8 mg/L GA3+0.2 mg/L NAA+2 g/L Phytagel	5.7
A	A2	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+2.49 mg/L TZR+1.0 mg/L NAA+2.0 g/L Phytagel	5.7
B	BS（茎段）	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+1.0 mg/L TZR+0.027 8 mg/L GA3+0.02 mg/L NAA+2.0 g/L Phytagel	5.7
	BL（叶片）	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N 维生素+0.5 mg/L TZR+2.5 mg/L IAA+2.0 g/L Phytagel	5.7
	B2	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+2.0 mg/L TZR+10 mg/L GA3 +2.0 g/L Phytagel	5.7
C	C1	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+1.0 mg/L TZR+2.0 mg/L NAA+2.0 g/L Phytagel	5.7
C	C2	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+0.5 mg/L GA3+2.0 mg/L 6-BA +0.02 mg/L NAA+2.0 g/L Phytagel	5.7
D	D	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+4.78 mg/L TZR+0.2 mg/L GA3 +0.01 mg/L IAA+2.0 g/L Phytagel	5.7
	MS 液体 Liquid MS	4.33 g/L MS基础盐（Phyto NO.524）+ 30 g/L蔗糖	5.8
	生根培养基 Rooting medium	4.74 g/L MS培养基（不含琼脂和蔗糖）+30 g/L蔗糖+2.0 g/L植物凝胶（Phytagel）	5.6

培养基体系 Medium system	培养基 Medium	培养基成分 Medium composition	pH
A	A1	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+2.49 mg/L TZR+0.027 8 mg/L GA3+0.2 mg/L NAA+2 g/L Phytagel	5.7
A	A2	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+2.49 mg/L TZR+1.0 mg/L NAA+2.0 g/L Phytagel	5.7
B	BS（茎段）	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+1.0 mg/L TZR+0.027 8 mg/L GA3+0.02 mg/L NAA+2.0 g/L Phytagel	5.7
	BL（叶片）	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N 维生素+0.5 mg/L TZR+2.5 mg/L IAA+2.0 g/L Phytagel	5.7
	B2	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+2.0 mg/L TZR+10 mg/L GA3 +2.0 g/L Phytagel	5.7
C	C1	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+1.0 mg/L TZR+2.0 mg/L NAA+2.0 g/L Phytagel	5.7
C	C2	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+0.5 mg/L GA3+2.0 mg/L 6-BA +0.02 mg/L NAA+2.0 g/L Phytagel	5.7
D	D	MS基础盐+20 g/L 蔗糖+1 mL/L 1 000× N&N维生素+4.78 mg/L TZR+0.2 mg/L GA3 +0.01 mg/L IAA+2.0 g/L Phytagel	5.7
	MS 液体 Liquid MS	4.33 g/L MS基础盐（Phyto NO.524）+ 30 g/L蔗糖	5.8
	生根培养基 Rooting medium	4.74 g/L MS培养基（不含琼脂和蔗糖）+30 g/L蔗糖+2.0 g/L植物凝胶（Phytagel）	5.6

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