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

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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, ZOU Xuexiao¹^,²^,^*()

¹ College 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
² Yuelushan Laboratory，Changsha 410128，China

Received:2025-03-11 Revised:2025-04-24 Online:2025-05-08 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

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.

Figures/Tables 5

Table 1 Stage-specific culture medium formulations for genetic transformation

培养基 Culture medium	g · L^-1			乙酰丁香酮/（μmol · L^-1） AS	mg · L^-1					小肽/（nmol · L^-1）CaREF1	特美汀/（mg · L^-1） Timentin
培养基 Culture medium	MS	蔗糖 Sucrose	琼脂 Agar	乙酰丁香酮/（μmol · L^-1） AS	细胞分裂素6-BA	生长素 IAA	赤霉素GA₃	卡那霉素Kan	硝酸银 AgNO₃	小肽/（nmol · L^-1）CaREF1	特美汀/（mg · L^-1） Timentin
播种Sowing	2.22	30	7	/	/	/	/	/	/	/	/
预培养Pre-culture	2.22	30	7	/	3	0.5	/	/	/	/	/
侵染液 Inoculation	2.22	30	/	200	2	0.1	/	/	/	/	/
共培养 Co-culture	4.44	30	7	200	2	0.1	/	/	/	/	/
分化Shoot regeneration	4.44	30	7	/	5	1.2	/	50	5	10	300
壮芽Shoot elongation	4.44	30	7	/	5 ~ 10	0.5	3	50	5	10	300
生根Rooting	2.22	30	7	/	/	0.1	/	/	/	/	200

Fig 1. Schematic representation of the pKSE-RUBY vector and transient expression validation A：Schematic diagram of the pKSE-RUBY vector；B：Phenotypic observation of agroinfiltration-mediated co-expression in tobacco leaves at 48 hpi （hours post-injection）；C：Transient expression dynamics in pepper cotyledons at 48 hpi and 72 hpi

Fig. 2 Screening of pepper germplasm with high-efficiency gene deliver

Fig. 3 Genetic transformation workflow and transformation efficiency analysis in pepper A-H：Schematic illustration of the pepper genetic transformation workflow；I and J：CaREF1 peptide（10 nmol · L-1） elevates regeneration efficiency and shoot proliferation. t-Test，*** P < 0.001；**** P < 0.0001

Fig. 4 Molecular identification（A-D）and phenotypic characterization（E-F）of gene-edited pepper plants

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

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