Watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai) is an important fruit and vegetable cash crop in the world. Seedless watermelon has become one of the main types of watermelon at home and abroad because of its many advantages such as convenient eating, excellent quality, storage and transportation resistance. At present, more than 60% of the commodity market is obtained through the following two ways: (1) triploid seedless watermelon is produced by cross-breeding of traditional tetraploid maternal and diploid paternal parents; (2) The plant growth regulator CPPU was used to induce the formation of unfertilized seedless watermelon. The main limiting factors restricting the healthy increase of seedless watermelon industry are the long breeding cycle of tetraploid mother, low yield, high cost, difficult germination and quality decline of F1 seeds, and consumer food safety concerns. Therefore, the research and development of a diploid seedless watermelon breeding technology innovation system, accelerating the seedless watermelon breeding process, and improving production efficiency are major technical and industrial needs to help the industrialization of seedless watermelon to save costs and increase efficiency. In the early stage, our research group reported for the first time in the journal New Phytologist that a new and effective diploid seedless watermelon breeding technology system was developed by manipulating the SPOROCYTELESS (SPL) gene to block the development process of the female seed in watermelon inbred line materials. On this basis, how to break the limitation of inbred line materials, realize the major research and development of the diploid seedless watermelon technology system independent of genotype, and increase the range of shortening the creation cycle, is the technical and industrial focus of the seedless watermelon industry to expand and speed up. On October 4, 2024, Prof. Li Yuan's research group from Northwest A&F University published a paper titled "A sexually and vegetatively reproducible diploid seedless watermelon inducer via ClHAP2 mutation" online in Nature Plants. This study innovated and developed a self-propagating universal diploid seedless watermelon induction system for the first time, which can successfully realize the efficient creation of diploid seedless watermelon independent of genotype, which provides a new technical idea and industrial solution strategy for efficient biological breeding of seedless watermelon, and also provides a new entry point for the efficient creation of other seedless fruit crops. Double fertilization is a key process in seed formation. In this study, the key regulatory gene ClHAP2 (HAPLESS2) of watermelon double fertilization was analyzed and identified by bioinformatics methods. ClHAP2 was knocked out using CRISPR/Cas9 gene editing technology, and a mutant Clhap2 with deletion of histidine-rich domain (His-rich) was created. Phenotypic testing of morphological and physiological characteristics of mutants showed that their vegetative growth and pollen physiological characteristics were normal. Clhap2 was crossed between the female parent and wild-type WT, and the seeds developed normally. Clhap2, on the other hand, when crossed or self-crossed with the wild type as a paternal parent, showed an ideal phenotype in which the fruit produced only a very small number of seeds. The semi-thin paraffin section cytometry fine phenotypic detection confirmed that the seedlessness was caused by the inability of sperm and egg cells to fuse normally. The results of induction experiments of different varieties confirmed that Clhap2 could induce any watermelon variety to form seedless fruits, indicating that Clhap2 inducible lines could not only reproduce themselves, but also break the genotype dependence. In addition, in order to address consumer concerns about the food safety of individual grains, the T-DNA insertion in Clhap2 progeny was completely isolated.
Figure 1. Whether the quality of Clhap2-mediated diploid seedless watermelon inducible lines is affected is another core criterion for testing the success of inducible lines. In this study, three commercial watermelon varieties 'LLW', '8424' and 'Jingmu (JM)' were induced by the plant growth regulator CPPU, and the morphological and physiological indexes showed that the seedless fruits showed prominent problems such as crooked fruits, cracked fruits, and significantly increased pulp hardness, while there was no significant difference between the seedless fruits induced by Clhap2 and the inbred fruits of commercial varieties. The diploid seedless watermelon inducible line pioneered in this study provides a new technical perspective for accelerating the breeding process and large-scale application of diploid seedless watermelon.
Chen Xin'er, a graduate of the College of Horticulture, Northwest A & F University, is the first author of the paper, and Professor Yuan Li of the melon crop germplasm resources and genetic breeding team of the College of Horticulture, Northwest A & F University is the corresponding author. Academician Yang Weicai of Yazhou Bay National Seed Laboratory provided valuable suggestions. Prof. Xian Zhang, Assoc. Prof. Man Liu, Shujuan Tian and Prof. Jiafa Wang participated in this study. This research was supported by the National Key R&D Program of China (SQ2023YFE0201239), the National Young Talents Program (A279021801), the National Natural Science Foundation of China (32372734), the National Modern Agricultural Industry Technology System (CARS-25), the Key R&D Program of Guangdong Province (2022B0202060001), Supported by the Shaanxi Provincial Key R&D Program (2023-YBNY-008) and the Natural Science Foundation of Shaanxi Province (2022JM-112).
Fig. 2.Schematic diagram of the creation of seedless watermelon using the diploid seedless watermelon inducible line Clhap2
In the past five years, a series of key generic breeding technology innovation systems urgently needed by the industry, such as efficient and stable transformation of watermelon genotype, efficient gene editing and precise single-base editing, self-propagating diploid seedless watermelon inducible lines, haploid inducible lines, etc. As the first or corresponding author, he has published more than 30 papers in international authoritative academic journals such as Developmental Cell, Cell Research, Plant Cell, New Phytologist, Journal of Integrative Plant Biology, Horticulture Research, etc., created more than 100 excellent new materials and germplasm of melons, and cultivated 4 new varieties of melons. 12 national invention patents have been authorized/applied.
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