CN-122004123-A - Method for transferring Lycium barbarum eccDNA to create novel germplasm of tomato with high anthocyanin by pollen Mongolian method

Abstract

The invention discloses a method for transferring matrimony vine eccDNA to create new germplasm of tomato with high anthocyanin by pollen Mongolian method, belonging to the technical field of plant molecular breeding. The method forms a Mongolian complex of medlar pollen containing medlar eccDNAs and tomato pollen by a pollen Mongolian method, pollinates tomato female flowers in a targeted manner, realizes efficient transfer of medlar eccDNAs, and obtains new germplasm of the tomato with high anthocyanin. The invention has simple operation, low cost and high biological safety, and provides a high-efficiency technical means for improving tomato traits.

Inventors

• CHEN LIPING
• WAN XIAOYANG
• WANG TINGJIN
• HU ZHONGYUAN

Assignees

• 浙江大学

Dates

Publication Date

20260512

Application Date

20260327

Claims (5)

1. 1. A method for creating new germplasm of tomato with high anthocyanin by transferring matrimony vine eccDNA through pollen Mongolian method, which is characterized by comprising the following steps: step S1, cultivating male parent medlar and female parent tomatoes, and enabling Chinese medlar plants to synchronously enter a flowering period when female parent tomato plants enter a full-bloom period by adjusting growth environment conditions; Step S2, obtaining male parent matrimony vine pollen and female parent tomato pollen serving as Mongolian guide material, uniformly mixing the female parent tomato pollen and the male parent matrimony vine pollen according to the volume ratio of 1-1.5:1 to obtain Mongolian guide pollen, emasculating female parent tomato flowers, and pollinating the Mongolian guide pollen to stigma, wherein the pollination amount of each female parent tomato flower is 0.5-1mg; and step S3, repeating the step S2 at least once within 1-5 days after the primary pollination, and culturing the tomato with high anthocyanin content by using a water fertilizer to obtain new germplasm of the tomato.
2. 2. The method of claim 1, wherein in step S1, the female parent tomato is tomato variety Condine Red and the male parent wolfberry is chinese wolfberry Lyciumchinense.
3. 3. The method according to claim 1, wherein in step S2, the pollen is obtained for a time period of 7:30-10:00 a day.
4. 4. The method according to claim 1, wherein in step S2, the female parent tomato pollen and the male parent wolfberry pollen are put into a drying box containing silica gel drying agent before being mixed, and after drying at 25 ℃ for 4-6 h, the residue is removed by passing through a 60-mesh screen.
5. 5. The method according to claim 1, wherein in the step S3, the water and fertilizer culture is specifically that watering is carried out every 5 days before fruit setting, watering is carried out every 3 days after fruit setting, the water content of soil is kept to be 60-70%, a potassium dihydrogen phosphate aqueous solution with the mass concentration of 0.2% is sprayed every week, a compound fertilizer with the mass ratio of N: P: K=1:1:2 is applied every 2 weeks, fertilizer is stopped 10 days before ripening, and the addition amount of the compound fertilizer is 15 g/plant.

Description

Method for transferring Lycium barbarum eccDNA to create novel germplasm of tomato with high anthocyanin by pollen Mongolian method Technical Field The invention belongs to the technical field of plant molecular breeding, and particularly relates to a method for creating new germplasm of a tomato with high anthocyanin by transferring medlar eccDNA through a pollen Mongolian method. Background Tomato (Solanum lycopersicum) belongs to Solanaceae (Solanaceae) and is one of vegetable crops with the widest cultivation area worldwide, and occupies a key position in guaranteeing food safety and balanced dietary nutrition. In the growth and development process of tomato plants, the tomato plants are often subjected to various adversity stresses such as drought, saline alkali, low temperature, diseases and the like, and the yield and the stable yield are directly influenced. Anthocyanin is a natural secondary metabolite widely involved in plant coloration, antioxidant and stress response. The anthocyanin content in the plant body is high, and the anthocyanin plays an important role in resisting abiotic stress and relieving stress injury along with stronger oxidation resistance and comprehensive stress resistance. Therefore, the method has important significance in improving the anthocyanin content of the tomato plants, enhancing the comprehensive stress resistance of the tomato plants and realizing stable and high yield. At present, the way for improving the anthocyanin and stress resistance of tomatoes still has a certain limitation, and the traditional molecular means such as long hybridization breeding period, gene editing, genetic transformation and the like still have difficulty in industrialized application. Therefore, there is a need to explore new efficient, stable, green approaches for improving anthocyanin levels and stress resistance traits in tomato plants. The Lycium (Lycium) belongs to Solanaceae, has high anthocyanin content, and has outstanding stress resistance characteristics such as drought resistance, salt and alkali resistance, low temperature resistance, etc., and is an excellent kindred resource for improving tomato stress resistance and anthocyanin properties. However, tomato and medlar belong to different genus, the conventional distant hybridization has serious reproductive isolation, and excellent genes are difficult to be efficiently utilized in a traditional way, so that the tomato stress resistance and anthocyanin improvement are important bottlenecks. In recent years, extrachromosomal circular DNA (extrachromosomal circular DNA, eccna) has received attention, which has been demonstrated to be a class of covalently closed circular DNA molecules that are widely present in eukaryotic cells, independent of nuclear chromosomes, can carry complete functional genes or regulatory elements, and can autonomously replicate and express in recipient cells, and is an important natural genetic element that regulates plant genomic plasticity and trait variation. Researches show that plants eccDNAs can be clustered to carry genetic elements for regulating and controlling biological complexity characters (such as nutrition synthesis, stress resistance response and the like), so that the nutrition quality and stress resistance of crops are improved in a polygenic synergistic mode, and the technical limitation of traditional single-gene improvement is broken through. If the medlar source eccDNAs carrying anthocyanin synthesis and regulation related genetic information can be effectively introduced into tomatoes, genetic modules related to target traits are expected to be introduced in a more intensive mode, and the traits such as anthocyanin nutrition quality, comprehensive stress resistance and the like of the tomatoes are synchronously improved at one time. Pollen Mongolian method is used as a classical distant hybridization auxiliary technology, and can utilize affinity pollen to assist exogenous pollen to complete fertilization and break fertilization disorder, but the existing application focuses on overcoming hybridization incompatibility and is not combined with natural polygenic vectors such as eccDNAs. According to the invention, the pollen Mongolian technology is coupled with the oriented transfer depth of the medlar eccDNAs for the first time, and the high-efficiency and large-scale transfer of the medlar eccDNAs with multiple genes (containing anthocyanin synthesis related functions) to the tomato is realized by constructing the Mongolian pollen complex of the medlar and the tomato, so that the efficiency bottleneck of transferring one gene at a time in the traditional genetic engineering is broken. And only eccDNAs of the medlar which is naturally existed is transferred, no exogenous artificial vector sequence exists, the safe dispute of the transgenic organism is fundamentally avoided, and the large-scale transfer of exogenous genes in a non-transgenic way is realized. In summary, aiming at