Hybrid embryo rescue method and method for greatly creating interspecific hybrid seedlings of black poplar pie by using hybrid embryo rescue method
By using the hybrid embryo rescue method, the problems of long seed maturation cycle and premature fruit drop in interspecific hybridization of Populus tomentosa have been solved, resulting in a significant increase in seed germination rate and rapid acquisition of hybrid seedlings, providing technical support for the breeding of superior varieties of Populus tomentosa.
Patent Information
- Application Number
- CN202511060645.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-30
- Publication Date
- 2025-10-31
AI Technical Summary
In existing technologies, the seed maturation cycle during interspecific hybridization of black poplar is too long and premature fruit drop is likely to occur, resulting in limited quantity and quality of hybrid seedlings and making it difficult to select superior varieties.
The hybrid embryo rescue method was adopted. The embryo rescue system was carried out by cutting the fruit inflorescence two months after pollination. Embryo rescue was carried out using 1/2MS medium and medium with specific hormone concentrations. The optimal medium was screened to be 1/2MS + 0.06 mg/L naphthaleneacetic acid + 0.08 mg/L indolebutyric acid, which achieved physiological after-ripening of young embryos and improved seed germination rate.
The seed maturation time was shortened from three months to two months, and the germination rate of hybrid seeds was increased to 95%. Nearly 2,000 hybrid seedlings were obtained in a short period of time, providing technical support for the breeding of superior varieties of black poplar.
Smart Images
Figure CN120858872A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant conservation and breeding optimization, and in particular to a hybrid embryo rescue method and a method for mass-producing interspecific hybrid seedlings of Populus tomentosa using the hybrid embryo rescue method. Background Technology
[0002] Poplar ( Populus As an indispensable tree species in my country's plantation system, poplar plays a crucial role in my country's forest ecological security and timber industry due to its significant advantages such as rapid growth, strong resistance, high timber yield, and short rotation period. These advantages make poplar irreplaceable in ecological protection and timber production. To effectively enhance the ecological protection value and timber yield of plantations, the selection and promotion of superior varieties are particularly important, and hybridization breeding is one of the main methods for selecting superior poplar varieties.
[0003] Black poplar hybrids, represented by varieties such as Populus 107, Populus 109, and Populus Zhonglin 46, have become mainstream timber and afforestation plants widely used in the Yangtze River Basin, Huai River Basin, and North China due to their ease of propagation by cuttings, excellent wood quality, and extremely fast growth rate. Populus 107, in particular, a hybrid of European and American poplars introduced to my country by Professor Zhang Qiwen in 1984, has been widely applied in production and promotion, making a significant contribution to my country's timber industry and ecological construction.
[0004] However, after more than forty years of cultivation and development, we have still failed to cultivate a new germplasm that surpasses Populus 107 in terms of timber volume and production, which has significantly limited the progress of poplar breeding in my country. Furthermore, the uniformity of germplasm also poses a potential threat to the long-term stability and sustainable development of the timber industry. The main reason for this is that when hybridizing European and American poplars using the cut branch hydroponics method, it takes nearly three months from pollination to seed maturity. During this process, an abscission layer forms prematurely between the inflorescence and the branch, thus blocking the normal transport of nutrients. This phenomenon leads to premature seed drop and poor development, greatly limiting the quantity and quality of hybrid seedlings. Due to the small size of the offspring population, it is difficult to select superior varieties with outstanding traits.
[0005] Therefore, there is an urgent need to develop and improve a technical system to solve the breeding problems of excessively long seed maturation cycles and premature fruit drop during interspecific hybridization of black poplar. By overcoming this technical bottleneck, we can expect to cultivate more high-quality and efficient new poplar varieties, thereby promoting the further development of my country's timber industry and ecological construction.
[0006] On July 23, 2025, a search was conducted in the China Patent Publication Database using "poplar and hybridization and embryo and rescue and culture and pollination" as the abstract keywords and the option to allow synonym expansion was selected. No relevant literature was found.
[0007] On July 23, 2025, an abstract search was conducted on CNKI (China National Knowledge Infrastructure) using the keywords "European and poplar and hybridization and embryo and rescue and culture and pollination". The results showed that Geng Xiangyu published "Research on the Creation and Propagation Technology of Grape Specific Germplasm" in Northwest A&F University on May 1, 2022. Chu Yannan published "Research on Seedless Cold-Resistant Grape Embryo Rescue Breeding Technology" in Northwest A&F University on June 1, 2021. Yu Saisai published "Creation of New Seedless Cold-Resistant Grape Germplasm and Application of Embryo Rescue Breeding" in Northwest A&F University on May 1, 2021. Wang Zhirong, Li Xiaodong, Huang Zejun, Gao Jianchang, and Guo Yanmei published "Screening of Resource Materials Resistant to Tomato Chlorosis Virus and Creation of Resistant Germplasm" in the "Abstracts of Papers of the Chinese Society for Horticultural Science in 2017" on October 19, 2017. Wang Gang published "Creation of Seedless Grape Germplasm Resistant to Downy Mildew and Optimization of Embryo Rescue System" in Northwest A&F University on May 1, 2014. Tian Lili published "Embryo rescue breeding and germplasm innovation of disease-resistant seedless grapes" in Northwest A&F University on May 1, 2007. Zhang Guoqing published "Interspecific hybridization and plant regeneration of several Brassica species and their genetic evaluation" in Zhejiang University on April 1, 2005.
[0008] The methods for rescuing the above embryos are different from those in this patent. Summary of the Invention
[0009] Purpose of the invention: To provide a more effective method for rescuing hybrid embryos and a method for mass-producing interspecific hybrid seedlings of Populus tomentosa using the method for rescuing hybrid embryos. Specific objectives are detailed in the specific implementation section, which describes several substantial technical effects.
[0010] To achieve the above objectives, the present invention adopts the following technical solution: The hybrid embryo rescue method is characterized by the following steps: Approximately two months after pollination, an abscission layer is observed between the inflorescence and the branch. A portion of the inflorescence is cut to develop the embryo rescue system. The capsules are rinsed with running water for 120 minutes, then the pericarp is gently wiped with 75% alcohol. The capsules are then rinsed 5-6 times with sterile water in a clean bench. The surface moisture is gently absorbed with sterile filter paper. The capsules are then cut open with a sterile scalpel, and the inflorescence is completely removed. The seeds are carefully picked up with sterile forceps and placed on 1 / 2 MS medium containing auxin analogues for embryo rescue. Using the petri dish germination method, with 1 / 2 MS as the medium, the optimal medium was determined to be 1 / 2 MS + 0.06 mg / L naphthaleneacetic acid (NAA) + 0.08 mg / L indolebutyric acid (IBA), pH = 5.8-6.0.
[0011] The method for mass-producing interspecific hybrid seedlings of Populus tomentosa using the hybrid embryo rescue method is characterized by, Hybrid breeding was carried out using Populus I-69 as the female parent and Populus nigra as the male parent. The hybrid embryo rescue method was employed, characterized by the following steps: Approximately two months after pollination, an abscission layer was observed between the inflorescence and branches. A portion of the inflorescence was cut to develop the embryo rescue system. The capsules were rinsed with running water for 120 minutes, then the pericarp was gently wiped with 75% alcohol. The capsules were then rinsed 5-6 times with sterile water in a laminar flow hood. The surface moisture was gently absorbed with sterile filter paper. The capsules were then cut open with a sterile scalpel, and the inflorescences were thoroughly removed. The seeds were carefully picked up with sterile forceps and placed on 1 / 2 MS medium containing auxin analogues for embryo rescue. Germination was performed using a petri dish method. The optimal medium was determined to be 1 / 2 MS + 0.06 mg / L naphthaleneacetic acid (NAA) + 0.08 mg / L indolebutyric acid (IBA), pH 5.8-6.0. Immature embryos are extracted from early-developing ovules and cultured on a culture medium to achieve physiological after-ripening, which can significantly improve seed germination rate.
[0012] A further technical solution of the present invention comprises the following steps: Developing a hybrid breeding system for black poplar using the branch cutting hydroponics method combined with embryo rescue: 1. Branch Collection: Select robust Populus I-69 and Populus nigra var. ... 2: Pollen collection: When the pollen of the European black poplar matures, gently collect the pollen with a brush. The best time to collect pollen is between 11:00 and 15:00 every day. Place the collected pollen in a petri dish, add an appropriate amount of silica gel desiccant, seal it, and store it in a refrigerator at -20℃. 3. Artificial pollination: After the female flowers of the I-69 poplar mother tree are cultivated in the greenhouse for about one month, they will sprout. The first pollination will be carried out when the stigma protrudes about 2-3 mm from the flower bud. The pollination time is from 11:00 to 14:00. Use a brush to dip a small amount of pollen and lightly flick it evenly onto the stigma. The pollen should be slightly visible to the naked eye. Because the poplar inflorescence is long and the flowers in different parts open at different times, the pollination time should last for 2-3 days. If it is cloudy or rainy, it should be extended by 1-2 days. 4. Post-pollination management: When the stigma of the female flower withers and the ovary gradually swells, it indicates that pollination has been successful and the fruit has entered the development stage. During the period from pollination to maturity, the temperature and humidity conditions should be the same as those for hydroponic flower branch cultivation. Change the water every 2 days after pollination. After one month of continuous cultivation, thin the fruit by cutting off the smaller and poorly developed fruit clusters. Leave about 3-5 fruit clusters on each branch to reduce nutrient competition between fruit clusters and ensure sufficient nutrient supply to the remaining fruit clusters. 5: Hybrid embryo rescue method; 6: Using the embryo rescue technology developed in this patent, the black poplar hybrid population 4F53 was obtained. After successful pollination, the seeds were collected when the fruit clusters and branches were found to be prone to abscission and fall off. After disinfection, the seeds were peeled in a clean bench and placed on the best culture medium at 25°C for 14 hours of light and 10 hours of darkness for 5 days.
[0013] A further technical solution of the present invention is as follows: 7: Transplanting: The seeds on the petri dish are transplanted into the seedling tray and cultured under natural light for about 20 days before being transplanted to the ground to obtain multiple I-69 poplar × European black poplar hybrid seedlings.
[0014] The present invention, employing the above technical solution, has the following beneficial effects compared to the prior art: This patent uses Populus I-69 as the female parent and Populus nigra as the male parent to carry out the embryo rescue method to create a large number of interspecific hybrid seedlings of Populus nigra. It has developed a new technical method suitable for Populus nigra embryo rescue, shortening the time from pollination to seed maturity from three months to two months, and increasing the germination rate of hybrid seeds to 95%. Nearly 2,000 hybrid seedlings were obtained in a short period of time, providing a technical system that can be referenced for the breeding of superior varieties of Populus nigra. Attached Figure Description
[0015] To further illustrate the present invention, the following description is provided in conjunction with the accompanying drawings: Figure 1 The image shows the process of placing collected pollen in a petri dish, adding an appropriate amount of silica gel desiccant, sealing it, and storing it in a -20°C refrigerator. Figure 2A photograph showing the ovary gradually swelling as the stigma of the female flower withers; Figure 3 The germination rate of seeds on the optimal culture medium; Figure 4 This is a diagram showing the transplanting of seeds from a petri dish into a seedling tray. Figure 5 Images of seedlings transplanted after approximately 20 days of cultivation under natural light; Figure 6 Screening for the optimal culture medium for in vitro embryo germination as shown in Table 1; Figure 7 This study compares embryo rescue methods with traditional breeding methods. Detailed Implementation
[0016] The present invention will be further illustrated below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.
[0017] Therefore, this patent uses Populus I-69 as the female parent and Populus nigra as the male parent to carry out hybridization breeding work, and develops a new technology and method suitable for Populus nigra embryo rescue, which greatly improves the germination rate and survival rate of hybrid seeds, and obtains a large number of hybrid seedlings in a short period of time, providing a technical system that can be used as a reference for the breeding of superior Populus nigra varieties.
[0018] In the hybridization breeding of black poplar using the cut branch hydroponics method, the seeds take nearly 3 months to mature. However, the fruit clusters and branches are prone to abscission and fall off, leading to embryo abortion, which greatly limits the quantity and quality of hybrid seedlings and prolongs the hybridization breeding cycle.
[0019] Therefore, this patent has developed a new poplar embryo rescue technology, which extracts the young embryo from the early-stage ovules, places it on a culture medium for cultivation, realizes the physiological after-ripening of the young embryo, significantly improves the seed germination rate, and provides a technical system that can be referenced for black poplar hybridization breeding.
[0020] Therefore, this patent employs a branch hydroponics method combined with embryo rescue to develop a black poplar hybrid breeding system: 1. Branch Collection: Select robust Populus I-69 and Populus nigra var. ...
[0021] 2. Pollen Collection: The European black poplars of this patent were collected from the Manas Plain Forest Farm in Xinjiang on January 10, 2025. When the pollen matured on February 5, 2025, the pollen was gently collected with a brush. The optimal time for pollen collection was between 11:00 AM and 3:00 PM daily. The collected pollen was placed in a petri dish with an appropriate amount of silica gel desiccant added, sealed, and stored in a -20°C refrigerator. Figure 1 ).
[0022] 3. Artificial pollination: The flowering branches of Populus I-69 used in this patent were collected on February 7, 2025, at the Weihe Experimental Station in Zhouzhi, Shaanxi Province. After being cultivated in a greenhouse for about one month, the female flowers broke through the buds, and the stigma protruded about 2-3 mm from the flower bud. The first pollination was carried out between 11:00 and 14:00. A small amount of pollen was lightly and evenly sprinkled onto the stigma with a brush. The pollen was just barely visible to the naked eye. Because the Populus inflorescence is long and the flowers in different parts open at different times, the pollination period should last for 2-3 days. If it is cloudy or rainy, the pollination period should be extended by 1-2 days.
[0023] 4. Post-pollination management: When the stigma of the female flower withers and the ovary gradually swells, it indicates that pollination has been successful and the fruit has entered the development stage. Figure 2 During the pollination and maturity period, the temperature and humidity conditions should be the same as those for hydroponic flower branch cultivation. Change the water every 2 days after pollination. After one month of continuous cultivation, thin the fruit by removing smaller and underdeveloped fruit clusters, leaving about 3-5 fruit clusters on each branch to reduce nutrient competition between fruit clusters and ensure sufficient nutrient supply to the remaining fruit clusters.
[0024] 5. Establishment of embryo rescue system: About 2 months after pollination, an abscission layer was observed between the inflorescence and the branch. A portion of the inflorescence was cut off to develop the embryo rescue system. After rinsing the capsule with running water for 120 minutes, the pericarp was gently wiped with 75% alcohol. The capsule was then rinsed 5-6 times with sterile water in a laminar flow hood. The surface moisture of the capsule was gently absorbed with sterile filter paper. The capsule was then cut open with a sterile scalpel, and the inflorescence was completely removed. The seeds were carefully picked up with sterile forceps and placed on 1 / 2 MS medium containing different hormones for embryo rescue. The effects of different concentrations (0.04, 0.06, 0.08 mg / L) of NAA and (0.06, 0.08, 0.1 mg / L) of IBA on seed germination were investigated using a petri dish germination method with 1 / 2 MS medium (Table 1). The optimal medium was determined to be 1 / 2 MS + 0.06 mg / L NAA + 0.08 mg / L IBA, pH 5.8-6.0. Germination rate (%) was calculated as (number of germinated seeds on day 5 / total number of seeds) × 100%. On the optimal medium, the seed germination rate reached 95%. Figure 3 ).
[0025] Table 1 Screening of optimal culture media for in vitro embryo germination NAA (mg / L) IBA (mg / L) Germination rate % 0 0.06 50.7 0 0.08 86.4 0 0.1 70.5 0.04 0 53.7 0.06 0 82.5 0.08 0 67.7 0.06 0.08 95.0 Figure 3 6. Using the embryo rescue technology developed in this patent, the 4F53 hybrid population of Populus tomentosa was obtained: After successful pollination, the seeds were collected when the fruit clusters and branches were found to be prone to abscission and fall off. After disinfection, the seeds were peeled in a clean bench and placed on the best culture medium at 25°C for about 5 days under light for 14 hours and darkness for 10 hours.
[0026] 7. Transplanting: Transplant the seeds from the petri dish to the seedling trays. Figure 4 After approximately 20 days of cultivation in natural light, the plants are transplanted to the ground. Figure 5 Approximately 2,000 seedlings of I-69 poplar × European black poplar hybrid were obtained.
[0027] This patent uses Populus I-69 as the female parent and Populus nigra as the male parent to develop a large number of interspecific hybrid seedlings of Populus nigra using the embryo rescue method. It has developed a new technology and method applicable to Populus nigra embryo rescue, which shortens the time from pollination to seed maturity from three months to two months and increases the germination rate of hybrid seeds to 95%. Nearly 2,000 hybrid seedlings were obtained in a short period of time, providing a technical system that can be used as a reference for the breeding of superior varieties of Populus nigra (Table 2).
[0028] Table 2 Comparison of embryo rescue methods and traditional breeding methods Comparison Projects Cut stem hydroponics This patented embryo rescue method Time from pollination to seed maturity 2-3 months 2 months Seed germination rate 38.28%-41.97% 95.00% Innovatively, each of the above effects exists independently, yet a single structure can be used to combine the results.
[0029] It should be noted that the multiple solutions provided in this patent include their own basic solutions, which are independent of each other and do not restrict each other. However, they can also be combined with each other without conflict to achieve multiple effects.
[0030] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claims.
Claims
1. A hybrid embryo rescue method, characterized in that, The method involves establishing an embryo rescue system: approximately two months after pollination, an abscission layer was observed between the inflorescence and the branch. A portion of the inflorescence was cut to develop the embryo rescue system. The capsules were rinsed with running water for 120 minutes, then the pericarp was gently wiped with 75% alcohol. The capsules were then rinsed 5-6 times with sterile water in a laminar flow hood. The surface moisture of the capsules was gently absorbed with sterile filter paper. The capsules were then cut open with a sterile scalpel, and the inflorescences were thoroughly removed. The seeds were carefully picked up with sterile forceps and placed on 1 / 2 MS medium containing auxin analogues for embryo rescue. Using the petri dish germination method, with 1 / 2 MS as the medium, the optimal medium was determined to be 1 / 2 MS + 0.06 mg / L naphthaleneacetic acid (NAA) + 0.08 mg / L indolebutyric acid (IBA), pH = 5.8-6.
0.
2. A method for mass-producing interspecific hybrid seedlings of Populus tomentosa using the hybrid embryo rescue method, characterized in that, With I-69 Yang ( Populusdeltoides ) as the parent plant, European black poplar ( Populus nigra Using hybridization breeding with the male parent as the parent, the embryo rescue method is employed. The method is characterized by the following steps: Approximately two months after pollination, an abscission layer is observed between the inflorescence and the branch. A portion of the inflorescence is cut to develop the embryo rescue system. The capsules are rinsed with running water for 120 minutes, then the pericarp is gently wiped with 75% alcohol. The capsules are then rinsed 5-6 times with sterile water in a clean bench. The surface moisture of the capsules is gently absorbed with sterile filter paper. The capsules are then cut open with a sterile scalpel, and the inflorescence is completely removed. The seeds are carefully picked up with sterile forceps and placed on 1 / 2 MS medium containing auxin analogues for embryo rescue. The germination method is used in petri dishes. Using 1 / 2 MS as the medium, the optimal medium is selected as 1 / 2 MS + 0.06 mg / L naphthaleneacetic acid (NAA) + 0.08 mg / L indolebutyric acid (IBA), pH = 5.8-6.
0. Immature embryos are extracted from early-developing ovules and cultured on a culture medium to achieve physiological after-ripening, which can significantly improve seed germination rate.
3. The method for mass-producing interspecific hybrid seedlings of Populus tomentosa using the hybrid embryo rescue method as described in claim 2, characterized in that, It includes the following steps: Developing a hybrid breeding system for black poplar using the branch cutting hydroponics method combined with embryo rescue:
1. Branch Collection: Select robust Populus I-69 and Populus nigra var. ... 2: Pollen collection: When the pollen of the European black poplar matures, gently collect the pollen with a brush. The best time to collect pollen is between 11:00 and 15:00 every day. Place the collected pollen in a petri dish, add an appropriate amount of silica gel desiccant, seal it, and store it in a refrigerator at -20℃.
3. Artificial pollination: After the female flowers of the I-69 poplar mother tree are cultivated in the greenhouse for about one month, they will sprout. The first pollination will be carried out when the stigma protrudes about 2-3 mm from the flower bud. The pollination time is from 11:00 to 14:
00. Use a brush to dip a small amount of pollen and lightly flick it evenly onto the stigma. The pollen should be slightly visible to the naked eye. Because the poplar inflorescence is long and the flowers in different parts open at different times, the pollination time should last for 2-3 days. If it is cloudy or rainy, it should be extended by 1-2 days.
4. Post-pollination management: When the stigma of the female flower withers and the ovary gradually swells, it indicates that pollination has been successful and the fruit has entered the development stage. During the period from pollination to maturity, the temperature and humidity conditions should be the same as those for hydroponic flower branch cultivation. Change the water every 2 days after pollination. After one month of continuous cultivation, thin the fruit by cutting off the smaller and poorly developed fruit clusters. Leave about 3-5 fruit clusters on each branch to reduce nutrient competition between fruit clusters and ensure sufficient nutrient supply to the remaining fruit clusters. 5: Hybrid embryo rescue method; 6: Using the embryo rescue technology developed in this patent, the black poplar hybrid population 4F53 was obtained. After successful pollination, the seeds were collected when the fruit clusters and branches were found to be prone to abscission and fall off. After disinfection, the seeds were peeled in a clean bench and placed on the best culture medium at 25°C for 14 hours of light and 10 hours of darkness for 5 days.
4. The method for mass-producing interspecific hybrid seedlings of Populus tomentosa using the hybrid embryo rescue method as described in claim 3, characterized in that, Step 6 is followed by the following steps:
7. Transplanting: Transplant the seeds from the petri dish into the seedling tray and culture them under natural light for about 20 days before transplanting them to the ground to obtain multiple I-69 poplar × European black poplar hybrid seedlings.