Construction of a High-Efficiency Genetic Transformation System in 
Pepper Leveraging RUBY and CaREF1

doi:10.16420/j.issn.0513-353x.2025-0368

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (4): 1093-1104.doi: 10.16420/j.issn.0513-353x.2025-0368

• New Technology and New Methods • Previous Articles Next Articles

Construction of a High-Efficiency Genetic Transformation System in Pepper Leveraging RUBY and CaREF1

WANG Zhongyi1,2，LIU Yi1，HU Bowen1，ZHU Fan1，LIU Feng1，YANG Sha1，XIONG Cheng1，OU Lijun1，DAI Xiongze1，and ZOU Xuexiao1,2,* （zouxuexiao428@163.com）

¹College of Horticulture，Hunan Agricultural University，International Collaborative Joint Laboratory for Germplasm Innovation and Molecular Breeding of Horticultural Crops，Ministry of Education，Engineering Research Center of Germplasm Innovation and New Variety Breeding for Horticultural Crops，Ministry of Education，Changsha 410128， China ²Yuelushan Laboratory，Changsha 410128，China

Received:2025-03-11 Revised:2025-04-24 Online:2025-04-25 Published:2025-04-27
Contact: ZOU Xuexiao

Abstract

Abstract: Using the novel visual reporter system RUBY as a screening marker，combined with the regeneration-promoting peptide CaREF1，a pepper genetic transformation system were constructed and identified three efficient recipient materials for gene delivery. The transient expression efficiency of RUBY was significantly higher in the pepper genotypes‘L27’（line pepper），‘Zunla-1’（chili pepper），and ‘1-51’（bead pepper）compared to other materials. Agrobacterium rhizogenes K599，carrying the pKSE401-RUBY vector，was used for transformation. After 60 days of culturing on the adventitious bud regeneration medium（MS + 5 mg · L^-1 6-BA + 1.2 mg · L^-1 IAA + 10 nmol · L^-1 CaREF1）and shoot proliferation medium[MS + （5–10） mg · L^-1 6-BA + 0.5 mg · L^-1 IAA + 3 mg · L^-1 GA3 + 10 nmol · L^-1 CaREF1]，the average regeneration efficiency of cotyledon explants was 84% ± 7%，with 5.52 regenerated shoots per explant and a positive transformation rate about 5‰. This system enabled the first Agrobacterium-mediated visual genetic transformation in pepper and facilitated CRISPR/Cas9-mediated gene editing in multiple pepper germplasms，with editing efficiency 100% in T0 generation plants.

Key words: pepper, genetic transformation, RUBY, regeneration factor REF1, gene editing

CLC Number:

S 641.3

WANG Zhongyi, LIU Yi, HU Bowen, ZHU Fan, LIU Feng, YANG Sha, XIONG Cheng, OU Lijun, DAI Xiongze, ZOU Xuexiao. Construction of a High-Efficiency Genetic Transformation System in Pepper Leveraging RUBY and CaREF1[J]. Acta Horticulturae Sinica, 2025, 52(4): 1093-1104.

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Construction of a High-Efficiency Genetic Transformation System in Pepper Leveraging RUBY and CaREF1

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