Method for creating novel high-anthocyanin tomato germplasm by transferring lycium barbarum eccDNA (ectocDNA) through pollen Mongolian method
By transferring wolfberry eccDNAs into tomatoes using pollen-guided transfer, the reproductive isolation problem of distant hybridization was solved, achieving efficient and low-cost multi-gene transfer, creating a new tomato germplasm with high anthocyanin content, and improving the stress resistance and anthocyanin content of tomatoes.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2026-03-27
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies cannot efficiently and safely achieve cross-species transfer of natural multigene vectors (eccDNAs) in distant hybridization of tomatoes and wolfberries. Furthermore, traditional methods are complex, costly, and difficult to improve the anthocyanin content and stress resistance of tomatoes.
Using the pollen-guided method, wolfberry eccDNAs were combined with tomato for co-pollination. By adjusting the growth environment to synchronize flowering, the pollen from the maternal tomato was mixed and transferred. Repeated pollination was carried out to achieve large-scale and stable transfer of wolfberry eccDNAs, creating a new high-anthocyanin tomato germplasm.
It has achieved efficient and large-scale transfer of wolfberry eccDNAs, simplified the operation process, reduced costs by more than 50%, avoided the controversy of genetic modification, and significantly improved the anthocyanin content and overall stress resistance of tomatoes.
Smart Images
Figure CN122004123A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant molecular breeding technology, specifically relating to a method for creating new high-anthocyanin tomato germplasm by transferring wolfberry eccDNA through pollen guidance. Background Technology
[0002] tomato( Solanum lycopersicum Tomatoes, belonging to the genus *Solanum* of the family Solanaceae, are one of the most widely cultivated vegetable crops globally, playing a crucial role in ensuring food security and balanced dietary nutrition. During their growth and development, tomato plants often face various abiotic stresses such as drought, salinity, low temperatures, and diseases, directly impacting yield and stability. Anthocyanins are natural secondary metabolites widely involved in plant coloring, antioxidant activity, and stress responses. High anthocyanin content in plants is usually accompanied by stronger antioxidant capacity and overall stress resistance, playing a vital role in resisting abiotic stresses and mitigating abiotic damage. Therefore, increasing the anthocyanin content of tomato plants and enhancing their overall stress resistance is of great significance for improving tomato adaptability and achieving stable and high yields. Currently, there are limitations to methods for improving tomato anthocyanin levels and stress resistance: traditional hybridization breeding has a long cycle, and molecular techniques such as gene editing and genetic transformation still face difficulties in industrial application. Therefore, there is an urgent need to explore efficient, stable, and green new approaches to improve anthocyanin levels and stress resistance traits in tomato plants. Lycium, belonging to the Solanaceae family, is rich in anthocyanins and exhibits outstanding resistance to drought, salinity, and low temperatures, making it an excellent close relative for improving tomato stress resistance and anthocyanin traits. However, tomatoes and lycium belong to different genera, and conventional distant hybridization results in severe reproductive isolation, making it difficult to efficiently utilize superior genes through traditional methods. This has become a significant bottleneck in improving tomato stress resistance and anthocyanin content.
[0003] In recent years, extrachromosomal circular DNA (ECCDNA) has gradually attracted attention. It has been confirmed that ECCDNA is a type of covalently closed circular DNA molecule widely present in eukaryotic cells, independent of the nucleus and chromosomes. It can carry complete functional genes or regulatory elements and can autonomously replicate and express in recipient cells, making it an important natural genetic element regulating plant genome plasticity and phenotypic variation. Studies have shown that plant ECCDNAs can carry clusters of genetic elements regulating complex traits (such as nutrient synthesis and stress response), improving crop nutritional quality and stress resistance through multi-gene synergy, overcoming the limitations of traditional single-gene improvement techniques. If eccDNAs from wolfberry carrying genetic information related to anthocyanin synthesis and regulation can be effectively introduced into tomatoes, it is expected that genetic modules related to target traits can be introduced in a more intensive manner, simultaneously improving the anthocyanin nutritional quality and overall stress resistance of tomatoes.
[0004] Pollen-guided assisted reproduction, a classic technique for distant hybridization, utilizes compatible pollen to assist exogenous pollen in fertilization and overcome fertilization barriers. However, current applications primarily focus on overcoming hybridization incompatibility and have not yet been combined with natural multi-gene vectors such as eccDNAs. This invention, for the first time, deeply couples pollen-guided assisted reproduction technology with the targeted transfer of wolfberry eccDNAs. By constructing a pollen-guided complex between wolfberry and tomato, it achieves efficient and large-scale transfer of multi-gene (including anthocyanin synthesis-related functions) wolfberry eccDNAs to tomato, breaking through the efficiency bottleneck of traditional genetic engineering's "one gene at a time" approach. Furthermore, it only transfers naturally occurring wolfberry eccDNAs, without any exogenous artificial vector sequences, fundamentally avoiding the biosafety controversies surrounding transgenic organisms and achieving large-scale transfer of exogenous genes via non-transgenic pathways. In summary, addressing the technological gap in the safe and large-scale cross-species transfer of natural multi-gene vectors (eccDNAs) from distantly related species, this invention establishes for the first time a technical system for the transfer of wolfberry eccDNAs mediated by pollen guidance, providing a novel technical paradigm for germplasm innovation in high-anthocyanin tomatoes and even the utilization of distant genetic resources in other crops. Summary of the Invention
[0005] The purpose of this invention is to solve the problems of reproductive isolation barriers, low transgenic efficiency, and complex traditional methods in existing tomato-goji berry distant hybridization, and to provide a pollen-guided method that is simple to operate, low in cost, and highly efficient in transfer, so as to achieve large-scale and stable transfer of goji berry eccDNAs in tomatoes and create new high-anthocyanin tomato germplasm.
[0006] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a method for creating new high-anthocyanin tomato germplasm by transferring Lycium barbarum eccDNA via pollen guidance, characterized by comprising the following steps: Step S1: Cultivate the male parent wolfberry and the female parent tomato. By adjusting the growth environment, the Chinese wolfberry plants will enter the flowering period simultaneously when the female parent tomato plants enter the full flowering period. Step S2: Obtain pollen from the male parent wolfberry and pollen from the female parent tomato as a pollinating agent. Mix the pollen from the female parent tomato and the pollen from the male parent wolfberry at a volume ratio of 1~1.5:1 to obtain pollen for pollination. After emasculating the female parent tomato flowers, pollinate the stigmas with the pollen for pollination. The amount of pollination per female parent tomato flower is 0.5-1mg. Step S3: Repeat step S2 at least once within 1-5 days after the first pollination, and obtain a new high anthocyanin tomato germplasm after water and fertilizer cultivation.
[0007] Preferably, in step S1, the female parent tomato is the Condine Red tomato variety, and the male parent wolfberry is the Chinese wolfberry. Lycium chinense .
[0008] Preferably, in step S2, the time for obtaining pollen is from 7:30 to 10:00 daily.
[0009] Preferably, in step S2, before mixing the pollen from the female tomato parent and the pollen from the male wolfberry parent, the pollen needs to be placed in a drying box containing silica gel desiccant and dried at 25°C for 4-6 hours, and then the residue is removed by passing it through a 60-mesh sieve.
[0010] Preferably, in step S3, the water and fertilizer cultivation specifically involves: watering every 5 days before fruit set and every 3 days after fruit set, maintaining a soil moisture content of 60-70%; spraying a 0.2% potassium dihydrogen phosphate solution weekly; and applying a compound fertilizer with an N:P:K ratio of 1:1:2 every 2 weeks, stopping fertilization 10 days before maturity; the compound fertilizer is added at a rate of 15g per plant. Compared with the prior art, the beneficial effects of this invention are: The pollen-guided complex provided by this invention enables large-scale transfer of eccDNA, far exceeding genetic engineering methods; it is simple and low-cost to operate: no complex equipment is required, the process is simple, and the cost is reduced by more than 50%, making it suitable for large-scale application; it has high biosafety: only natural eccDNA is transferred, without exogenous vector sequences, avoiding the controversy of transgenic technology; it creates new tomato germplasm and optimizes overall quality. Attached Figure Description
[0011] The invention will now be described in further detail with reference to the accompanying drawings, but this is not intended to limit the invention.
[0012] Figure 1 A graph showing the number of eccDNA sequencing analyses after tomato hybridization. Figure 2 Phenotypic diagrams of tomato GT and the female parent tomato CR, hybrid offspring 20 days after transplanting; Figure 3 Statistical graphs of plant height for tomato GT and maternal tomato CR, hybrid offspring of tomato and wolfberry, 10 days and 20 days after transplanting; Figure 4 A comparison of anthocyanin content in the leaves of tomato GT (a hybrid offspring of tomato and wolfberry) and tomato CR (the parent tomato); Figure 5 A comparison of stem lignin content between tomato GT (a hybrid offspring of tomato and wolfberry) and the parent tomato CR (a hybrid of tomato and wolfberry). Detailed Implementation
[0013] The invention will be further illustrated below with reference to examples.
[0014] Example 1: Creating high-anthocyanin tomato progeny by transferring functional eccDNA from wolfberry to tomato 1. Experimental materials and equipment Materials: Mother parent: Condine Red tomato variety; Father parent: Chinese wolfberry Lycium chinense ; Equipment: LED supplemental lighting (30W), temperature and humidity controller, silica gel desiccant 2. Implementation Steps 2.1 Parent breeding: Sowing and seedling substrate consist of humus: garden soil: perlite = 3:2:1 (pH 6.0-6.5). Transplanting is done 15 days later. Greenhouse environment is controlled at 26℃ during the day and 19℃ at night, with 65% humidity and 13h / d light. Tomatoes enter their peak flowering period 30 days after transplanting. Goji berry plants are controlled to flower synchronously by adjusting the sowing time.
[0015] 2.2 Pollen Collection and Processing: Flowers are harvested daily from 7:30 to 10:00, when pollen viability is highest. After the anthers are removed, they are placed in a drying box containing silica gel desiccant (humidity 35%) and dried at 25℃ for 5 hours. Residue is removed through a 60-mesh sieve. Tomato and goji berry pollen are mixed in a 1:1 volume ratio in centrifuge tubes and gently stirred with a sterile soft brush to avoid damage.
[0016] 2.3 Targeted pollination and repeated pollination Pollination time: When the tomato flower petals are open and the stigma secretes mucus (8:00-10:00 AM); Procedure: Peel the corolla and anthers of the mother tomato plant, apply 0.5-1mg of Montmorillonite pollen (per flower) to the stigma with a soft brush, and cover with a transparent paper bag to prevent contamination; Repeat pollination: Repeat pollination once each on days 1, 2, and 5 after the first pollination, and remove the bag 3 days after the second pollination.
[0017] 2.4. Field Management of Hybrid Plants Water and fertilizer management: Water every 5 days before fruit set (soil moisture content 60-70%), water every 3 days after fruit set, spray an appropriate volume of 0.2% potassium dihydrogen phosphate solution every week (spray until the plant leaves are wet but no liquid drips), apply compound fertilizer with N:P:K=1:1:2 (15g / plant) every 2 weeks, and stop fertilizing 10 days before maturity; Pruning: Retain 2-3 main branches, remove old leaves and deformed fruits, and leave 4-6 fruits per plant.
[0018] Example 2 Harvesting and testing: After harvesting the seeds, the offspring of the seeds are screened for hybrid seeds of wolfberry and tomato.
[0019] eccDNA verification: eccDNA sequencing analysis was performed on the selected tomato and wolfberry hybrid germplasm.
[0020] This embodiment achieves efficient transfer of wolfberry eccDNAs and stable inheritance of eccDNAs, significantly improving the tomato gene background. The effectiveness of the method is verified by detecting the types and quantities of eccDNAs in wolfberry and hybrid tomato leaves using techniques such as circular DNA extraction and rolling circle amplification. Figure 1 (Table 1).
[0021] Table 1: Transferred Lycium barbarum eccDNA in hybrid offspring The GT phenotype and growth potential of the hybrid offspring tomatoes were significantly slower than those of the maternal parent tomatoes (CR). Figure 2 , Figure 3 G1, G2, and GT represent different individual goji berries and their hybrid offspring, respectively. Leaves of the hybrid offspring (tomato GT) and the parent goji berry (goji) were taken approximately 30 days after transplanting. Circular DNA extraction and rolling circle amplification techniques were used to detect the types and quantities of eccDNAs. The leaves of the hybrid offspring tomato GT contained a large amount of eccDNAs derived from goji berries. Figure 1 Table 1 illustrates that this embodiment achieves efficient transfer of Lycium barbarum eccDNAs.
[0022] Leaves and stems of the hybrid offspring (tomato GT) and the parent tomato (tomato CR) were collected approximately 30 days after transplanting. Anthocyanin content in leaves was determined using a pH differential method, and lignin content was determined using a 72% sulfuric acid acid hydrolysis-titration method. The anthocyanin content in leaves and the lignin content in stems were then analyzed. The test results are as follows: Figure 4 and Figure 5 As shown, the hybrid offspring, tomato GT, significantly increased the anthocyanin content in leaves and the lignin content in stems compared to the parent tomato CR (P < 0.001).
[0023] The conclusions drawn from the above results demonstrate that the pollen-guided method can achieve large-scale transfer of eccDNAs from wolfberry to tomatoes, resulting in new tomato germplasm with high anthocyanin and lignin content.
[0024] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.
Claims
1. A method for creating new high-anthocyanin tomato germplasm by transferring Lycium barbarum eccDNA via pollen guidance, characterized in that, Includes the following steps: Step S1: Cultivate the male parent wolfberry and the female parent tomato. By adjusting the growth environment, the Chinese wolfberry plants will enter the flowering period simultaneously when the female parent tomato plants enter the full flowering period. Step S2: Obtain pollen from the male parent wolfberry and pollen from the female parent tomato as a pollinating agent. Mix the pollen from the female parent tomato and the pollen from the male parent wolfberry at a volume ratio of 1~1.5:1 to obtain pollen for pollination. After emasculating the female parent tomato flowers, pollinate the stigmas with the pollen for pollination. The amount of pollination per female parent tomato flower is 0.5-1mg. Step S3: Repeat step S2 at least once within 1-5 days after the first pollination, and obtain a new high anthocyanin tomato germplasm after water and fertilizer cultivation.
2. The method according to claim 1, characterized in that, In step S1, the maternal tomato parent is the Condine Red tomato variety, and the paternal wolfberry parent is the Chinese wolfberry variety. Lyciumchinense .
3. The method according to claim 1, characterized in that, In step S2, the pollen is collected between 7:30 and 10:00 daily.
4. The method according to claim 1, characterized in that, In step S2, before mixing the pollen from the female tomato parent and the pollen from the male wolfberry parent, the pollen needs to be placed in a drying box containing silica gel desiccant and dried at 25°C for 4-6 hours, and then the residue is removed by passing it through a 60-mesh sieve.
5. The method according to claim 1, characterized in that, In step S3, the water and fertilizer cultivation specifically involves: watering every 5 days before fruit set and every 3 days after fruit set, maintaining soil moisture content at 60-70%, spraying a 0.2% potassium dihydrogen phosphate solution weekly, and applying compound fertilizer with a mass ratio of N:P:K=1:1:2 every 2 weeks, stopping fertilization 10 days before maturity; the amount of compound fertilizer added is 15g / plant.