By combining traditional plant growth regulators with a novel peptide, CaREF1, the regeneration efficiency of the widely cultivated Zunla-1 variety was nearly doubled—from 27.2% to 55.0%. This advance addresses one of the major barriers in pepper biotechnology, offering new opportunities for genetic transformation and trait improvement in one of the world’s most important spice and vegetable crops.
Chili peppers are globally valued for their culinary, nutritional, and economic importance, with species such as C. annuum, C. frutescens, and C. chinense forming dietary and cultural staples worldwide. Rich in vitamins, carotenoids, and capsaicinoids, peppers are also linked to health benefits. Yet, their genetic improvement has been hindered by the plants’ recalcitrance to in vitro regeneration, a process essential for modern breeding and genetic engineering. Regeneration bottlenecks—such as low shoot formation, abnormal morphologies, and strong genotype dependence—have long slowed progress in pepper transformation systems. Addressing these challenges requires novel growth regulators and carefully tailored culture protocols to unlock peppers’ full potential for crop improvement.
A study (DOI: 10.48130/seedbio-0025-0012) published in Seed Biology on 11 August 2025 by Huamao Wu’s & Lihao Wang’s team, Chinese Academy of Agricultural Sciences, demonstrates that the CaREF1 peptide can effectively overcome the regeneration recalcitrance of pepper, doubling plant regeneration efficiency and providing a valuable tool for genetic improvement of Capsicum crops.
The study established a stepwise tissue-culture workflow to optimize regeneration in pepper by systematically testing callus induction, shoot formation, elongation, and rooting, followed by overall regeneration efficiency and histological validation. Callus induction was first assessed in Zunla-1 and CM334 using five auxin/peptide treatments, revealing Tc2 (1 mg/L IAA + 1 nM CaREF1) as the most effective, with Zunla-1 cotyledons achieving 97.2% callus and CM334 90.0%, whereas higher CaREF1 concentrations or 2,4-D led to poor responses. Hypocotyls also responded well to Tc2, but root explants failed in all treatments, underscoring tissue-specific recalcitrance. Extending Tc2 to seven additional genotypes showed genotype-dependent variation, with cotyledons of ZJ6, 243, and 354 reaching over 70% callus induction, while hypocotyls lagged and none advanced to shoot formation. Organogenesis trials demonstrated that Tsf2 (5 mg/L AgNO₃ + 1 nM CaREF1) maximized shoot regeneration, producing nearly 80% shoots in Zunla-1 and CM334 cotyledons, compared to reduced efficiency with AgNO₃ alone or at higher concentrations, while controls failed. Shoot elongation further highlighted genotype specificity, as Zunla-1 achieved 74.6% elongation under Tse2 (0.5 mg/L GA₃ + CaREF1), whereas CM334 showed minimal growth. Rooting experiments in Zunla-1 identified Tr2 (IBA + CaREF1) as optimal, reaching 78% compared with 52% using IBA alone, while IAA + CaREF1 proved ineffective. Overall regeneration success in Zunla-1 doubled with CaREF1, increasing from 27.2% to 55.0%. Microscopic analyses corroborated these gains, showing that CaREF1 enhanced cellular density, organization, and meristematic activity, thereby facilitating the transition from callus to shoot primordia. Collectively, these results demonstrate the critical role of CaREF1 in overcoming pepper’s recalcitrance and emphasize the necessity of genotype- and explant-specific strategies for efficient regeneration.
The optimized regeneration protocol offers a major advance for pepper biotechnology. Reliable regeneration systems are essential for introducing traits such as disease resistance, stress tolerance, and nutritional enhancement through genetic transformation. By integrating CaREF1 peptide into standard tissue culture protocols, breeders and researchers can bypass long-standing bottlenecks in pepper regeneration. Beyond peppers, the approach could be adapted for other transformation-recalcitrant crops, expanding opportunities for accelerating crop improvement across the Solanaceae family and beyond.
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References
DOI
10.48130/seedbio-0025-0012
Original Source URL
https://doi.org/10.48130/seedbio-0025-0012
Funding information
This work was funded by the Graduate School of Chinese Academy of Agricultural Sciences (Grant No. GSCAAS), the Nanfan Special Project, CAAS (Grant Nos. YBXM2522, YBXM2418); the Agricultural Science and Technology Innovation Program (ASTIP) (Grant No. Y2024QC06); the National Natural Science Foundation of China for Youth Scholar (Grant No. 32302557); Basic Research Center, Innovation Program of Chinese Academy of Agricultural Sciences (Grant No. CAAS-BRC-HS-2025-01); the National Key Research and Development program of China (Grant No. 2023YFD1200101); Hainan Seed Industry Laboratory and China National Seed Group (Grant No. B23CQ15KP); the Major Project of the National Natural Science Foundation of China (Grant No. 32494780); China Agriculture Research System (Grant No. CARS-23-A15); the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences (Grant No. CAAS-ASTIP-IVFCAAS); the General Program of National Natural Science Foundation of China (Grant No. 32372712); and Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (Vegetables), Ministry of Agriculture and Rural Affairs.
About Seed Biology
Seed Biology (e-ISSN 2834-5495) is published by Maximum Academic Press in partnership with Yazhou Bay Seed Laboratory. Seed Biology is an open access, online-only journal focusing on research related to all aspects of the biology of seeds, including but not limited to: evolution of seeds; developmental processes including sporogenesis and gametogenesis, pollination and fertilization; apomixis and artificial seed technologies; regulation and manipulation of seed yield; nutrition and health-related quality of the endosperm, cotyledons, and the seed coat; seed dormancy and germination; seed interactions with the biotic and abiotic environment; and roles of seeds in fruit development. Seed biology publishes a wide range of research approaches, such as omics, genetics, biotechnology, genome editing, cellular and molecular biology, physiology, and environmental biology. Seed Biology publishes high-quality original research, reviews, perspectives, and opinions in open access mode, promoting fast submission, review, and dissemination freely to the global research community.