Pennisetum americanum and pennisetum purpureum interspecific hybridization method and application thereof
Through a new hybridization method, including isolation bags in the inflorescence of the American Wolftail, collecting pollen and pollinating, and subsequent net bag replacement, the efficiency of hybrid breeding between the American Wolftail and Elephant Grass is improved, and the problem of difficulty and low efficiency in the traditional de-hero hybridization method is solved.
Patent Information
- Application Number
- CN202510416868.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-03
- Publication Date
- 2025-06-06
AI Technical Summary
The hybrid breeding efficiency between the species of Wolftail and Elephant Grass is low. It is difficult to operate with de-heroic hybridization method, takes a long time, has great inflorescence damage, and has extremely low hybridization efficiency.
A new hybridization method is adopted, including the following steps: (1) Set the isolation bags for the inflorescences of the sacred wolves at 4 to 5 pm, and the inflorescences of the target inflorescences are packed with powder bags; (2) Collect pollen from 9 to 10 am the next day, remove the isolation bags, and pollinate the inflorescences of the sacred wolves; (3) 5 to 7 days after pollination, use the net bag to replace the pollination bag until the hybrid seeds mature.
The efficiency of hybrid breeding between the species of Wolftail and Elephant Grass is improved, and the problems of difficulty in operation, long time-consuming and large inflorescence damage in the dehumation hybridization method are solved, which significantly improves the hybridization efficiency.
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Figure CN120092704A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to the technical field of plant hybrid breeding, and in particular to a method for interspecific hybridization of Pennisetum americanum and Elephant Grass and an application thereof. Background Art
[0002] Pennisetum and elephant grass are two important species in the genus Pennisetum. Both of them occupy an important position in feed production due to their excellent forage properties. Among them, Pennisetum is an annual diploid plant with good palatability, drought resistance and adaptability, and is widely used in livestock feed production. Elephant grass is a perennial tetraploid plant with a wide range of adaptability under different soil and ecological conditions, especially under limited irrigation conditions. Elephant grass has huge biomass yield and rapid regeneration ability. The interspecific triploid hybrid produced by hybridization of the two - hybrid Pennisetum, combines the good palatability and high nutritional value of Pennisetum with the high-yield traits of elephant grass. It provides a wider range of choices for forage production in different regions, and also improves its application potential in forage production.
[0003] In view of the excellent quality and high forage yield of hybrid Pennisetum, it plays an important role in the development of the forage industry. However, the hybrid breeding between Pennisetum and elephant grass mainly depends on the application of the sterile line of Pennisetum. The improvement of the sterile line of Pennisetum is limited by the improvement of the maintenance line, and its breeding cycle is long, which is not conducive to the extensive hybridization between Pennisetum and elephant grass. Conventional emasculation hybridization is difficult to operate on Pennisetum, takes a long time, causes great damage to the inflorescence, and has extremely low hybridization efficiency. In order to make full use of the diversity of Pennisetum and elephant grass germplasm resources for hybrid breeding and carry out the breeding of new varieties of high-yield and high-quality hybrid Pennisetum, a reasonable and effective hybrid breeding method is needed to replace emasculation hybridization. Summary of the invention
[0004] The object of the present invention is to provide a method for improving the interspecific hybridization breeding efficiency of Pennisetum truncatum and Elephant Grass, and in particular to a method for interspecific hybridization of Pennisetum truncatum and Elephant Grass and an application thereof.
[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
[0006] The present invention provides a method for interspecific hybridization between Pennisetum americanum and Elephant Grass, comprising the following steps:
[0007] (1) At 4-5 pm on the first day, put an isolation bag on the Pennisetum inflorescence and a pollen collection bag on the target grass inflorescence;
[0008] (2) Collect pollen from the pollen collection bag at 9 to 10 a.m. the next day, remove the isolation bag, and use the collected pollen to pollinate the Pennisetum inflorescence;
[0009] (3) Put a pollination bag on the pollinated inflorescence and replace the pollination bag with a net bag 5 to 7 days after pollination until the hybrid seeds mature.
[0010] Preferably, the Pennisetum inflorescence in step (1) is an inflorescence whose pistil has not yet been exposed; and the isolation bag is a transparent bag.
[0011] Preferably, the elephant grass inflorescence in step (1) is an inflorescence that is shedding pollen.
[0012] Preferably, the Pennisetum americanum inflorescence described in step (2) is an inflorescence with exposed pistils.
[0013] Preferably, the pollination in step (2) is performed 1 to 2 times.
[0014] Preferably, the pollination bag in step (3) is a transparent bag.
[0015] The invention also provides application of the method in improving the efficiency of interspecific hybridization breeding between Pennisetum americanum and Elephant Grass.
[0016] The invention also provides application of the method in hybrid breeding.
[0017] The present invention provides a method for interspecific hybridization between American Pennisetum and elephant grass and its application. The method of the present invention comprises the following steps: (1) at 4 to 5 p.m. on the first day, an isolation bag is placed on the inflorescence of American Pennisetum, and at the same time, a pollen collection bag is placed on the inflorescence of the object grass; (2) at 9 to 10 a.m. on the second day, pollen in the pollen collection bag is collected, the isolation bag is removed, and the inflorescence of American Pennisetum is pollinated with the collected pollen; (3) a pollination bag is placed on the pollinated inflorescence, and 5 to 7 days after pollination, the pollination bag is replaced with a net bag until the hybrid seeds are mature.
[0018] Pennisetum often cross-pollinates, and the flowering period of stamens and pistils is different, which is manifested as the pistil matures first and the stamen matures later, with a difference of 3 to 4 days. Taking advantage of the characteristic of staggered pollination of Pennisetum, Pennisetum is pollinated when the stigma of the pistil matures and the stamen is not yet mature, so as to achieve the effect of hybridization. In order to prevent Pennisetum from being contaminated by pollen from different flowers within the species after the pistil is exposed, transparent isolation bags are placed on the inflorescences of Pennisetum whose pistil stigma has not yet been exposed in the breeding field at 4 to 5 pm, which is convenient for subsequent observation and prevents the contamination of pollen from different flowers within the species. When the stigma of the pistil of the inflorescence of Pennisetum begins to be slightly exposed or fully exposed, it can be pollinated. The hybridization method can broaden the scope of utilization of Pennisetum and elephant grass germplasm resources and improve the efficiency of interspecific hybridization breeding between Pennisetum and elephant grass. It solves the problems of difficult operation, long time consumption, large inflorescence damage and extremely low hybridization efficiency caused by emasculation hybridization. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1This is a diagram of the development process of the pistil and stamen of Pennisetum americanum;
[0020] Figure 2 This is the result of the root tip chromosome counting method (the left picture shows the number of maternal chromosomes, the middle picture shows the number of paternal chromosomes, and the right picture shows the number of true hybrid progeny chromosomes);
[0021] Figure 3 is the molecular marker identification result of hybrid progeny seedlings (M is marker, P is 1 Pennisetum americanum, P 2 represents purple elephant grass, black numbers represent true hybrid offspring, and red numbers represent false hybrid offspring);
[0022] Figure 4 This is a field phenotype diagram (the left picture is American foxtail grass, the middle picture is purple elephant grass, and the right picture is a true hybrid progeny). DETAILED DESCRIPTION
[0023] The present invention provides a method for interspecific hybridization between Pennisetum americanum and Elephant Grass, comprising the following steps:
[0024] (1) At 4-5 pm on the first day, put an isolation bag on the Pennisetum inflorescence and a pollen collection bag on the target grass inflorescence;
[0025] (2) Collect pollen from the pollen collection bag at 9 to 10 a.m. the next day, remove the isolation bag, and use the collected pollen to pollinate the Pennisetum inflorescence;
[0026] (3) Put a pollination bag on the pollinated inflorescence and replace the pollination bag with a net bag 5 to 7 days after pollination until the hybrid seeds mature.
[0027] In the present invention, the Pennisetum inflorescence described in step (1) is an inflorescence whose pistil has not yet been exposed; and the isolation bag is a transparent bag. The Pennisetum is "Ningza No. 4", and the Pennisetum is used as the female parent. This grass often cross-pollinates, and the flowering periods of the stamens and pistils are not coincident, which is manifested as the pistil matures first and the stamens mature later, with a difference of 3 to 4 days. By utilizing the characteristic of staggered pollination of Pennisetum, the Pennisetum is pollinated when the stigma of the pistil is mature and the stamen is not yet mature, thereby achieving the effect of hybridization. In order to prevent the Pennisetum from being contaminated by pollen from other flowers within the species after the pistil is exposed, transparent isolation bags are placed in the breeding field at 4 to 5 pm for those Pennisetum inflorescences whose pistil stigma has not yet been exposed, so as to facilitate subsequent observation and prevent contamination from pollen from other flowers within the species. When the stigma of the pistil of the Pennisetum inflorescence is observed to begin to be slightly exposed or fully exposed, it can be pollinated. The development process of the stamens and pistils of Pennisetum is as follows Figure 1 shown.
[0028] The pollination time is chosen between 9 and 10 in the morning, when the stigma is more active and easier to receive pollen.
[0029] In the present invention, the elephant grass inflorescence in step (1) is an inflorescence that is shedding powder. The elephant grass is purple elephant grass.
[0030] In the present invention, the Pennisetum inflorescence described in step (2) is an inflorescence with the pistil exposed. The method for collecting pollen is: lightly flicking the pollen collection bag, waiting for the pollen to fall into the bag for pollen collection.
[0031] In the present invention, the pollination in step (2) is performed 1 to 2 times. The pollination method is: the collected pollen is evenly shaken onto the pistil of Pennisetum americanum.
[0032] In the present invention, the pollination bag in step (3) is a transparent bag.
[0033] After 28 to 32 days of pollination, hybrid seeds are harvested.
[0034] The invention also provides application of the method in improving the efficiency of interspecific hybridization breeding between Pennisetum americanum and Elephant Grass.
[0035] The invention also provides application of the method in hybrid breeding.
[0036] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.
[0037] In the embodiment of the present invention, the American Pennisetum is "Ningza No. 4", which is used as the female parent; the elephant grass is purple elephant grass, which is used as the male parent.
[0038] Example 1
[0039] At 4-5 pm, the inflorescence of American Pennisetum whose pistil has not yet been exposed is covered with an isolation bag. The transparent isolation bag is used to observe the development of the pistil and prevent the contamination of pollen from different flowers within the species. At the same time, the inflorescence of the elephant grass that is shedding pollen is covered with a pollen collection bag to collect pollen the next morning. The method of collecting elephant grass pollen is to flick the pollen collection bag at 9-10 am on the second day after bagging, and wait for the pollen to fall into the bag. Then immediately remove the pollen collection bag and quickly transfer it to the isolation bag that has been covered before. Through the transparent isolation bag, it is observed that the inflorescence of American Pennisetum with the pistil slightly or fully exposed is observed. At this time, the transparent isolation bag is removed, and the pollen collection bag is covered. The collected pollen is evenly shaken onto the pistil of American Pennisetum by the shaking powder method. After pollination, a transparent pollination bag is covered for observation; at 9-10 am on the third day, the pollination is repeated in the same way to increase the fertilization and fruiting rate, and a transparent pollination bag is covered after pollination. 6 days after pollination, the stamens of Pennisetum fasciatus completely stop blooming, and the pollination bag is replaced with a net bag until the seeds mature. 30 days after pollination, the hybrid seeds in the net bag are harvested.
[0040] Example 2
[0041] The collected hybrid seeds are raised in seedbeds. The experiment was carried out in the greenhouse of the Institute of Grassland, Flowers and Landscape Ecology of Beijing Academy of Agricultural and Forestry Sciences from May to June 2024. A tidal irrigation seedbed with a specification of 1.7×20×0.7m was used to cover the surface with a layer of seedling cloth. Water was applied to make the seedling cloth completely wet, and then the hybrid seeds were evenly sown on the seedling cloth. Water was applied every day to keep it moist. After 2 to 3 days, the seeds began to germinate. When the seeds grew to the 2 to 3 leaf stage, the hybrid seedlings could be moved into a 50-hole tray to continue raising the seedlings.
[0042] Example 3
[0043] Verification of hybridization authenticity by root tip chromosome counts and molecular markers (SSR / SNP)
[0044] The root tip chromosome count was carried out on the seedlings of two parents and some hybrid seeds. The young root tips of about 0.5 cm were taken from each material after hydroponics. They were fixed in Carnoy's fixative (ethanol: glacial acetic acid = 3:1) for 24 hours and then placed in 75% ethanol and stored in a refrigerator at 4°C for later use. The root tip was then placed in distilled water for 20 minutes, and then placed in a lysis solution with cellulase and pectinase (1:1) for 150 minutes of dissociation at 37°C. After the dissociation was completed, 1 mL of distilled water was added to the lysis solution to promote water absorption and rupture of the cell membrane. After 20 minutes, the root tip was aspirated and placed in distilled water, and then aspirated and placed on a glass slide, the surrounding water was dried, and a drop of fixative was added to break the root tip, and the root tip was stained with gentian violet stain. After staining, the division phase was observed under a microscope and the number of chromosomes was counted.
[0045] Some hybrid seedlings were identified using the root tip chromosome counting method. Figure 2 As shown, the female parent, American foxtail grass, has 14 chromosomes, the male parent, purple elephant grass, has 28 chromosomes, and the true hybrid offspring has 21 chromosomes.
[0046] Molecular marker identification mainly uses SSR and SNP marker primer sequences. The primer information is shown in Table 1. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. (Beijing, China). First, DNA was extracted from the parental and hybrid seedlings to be identified. 0.1 g of young leaves of the above materials were selected and DNA was extracted according to the plant genome extraction kit of Nanjing Novozyme Biotechnology Co., Ltd. (Nanjing, China). The DNA purity and concentration were detected by ultraviolet spectrophotometer. Finally, part of the DNA stock solution was washed with ddH 2O was uniformly diluted to 20 ng / μL and stored at -20°C for later use. The PCR amplification system consisted of 10 μL 2× Taq MasterMix (Tiangen Biochemical Technology (Beijing) Co., Ltd.), 1 μL forward primer, 1 μL reverse primer, 2 μL DNA and 6 μL ddHO. 2 O. The PCR reaction procedure was: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, annealing for 30 s according to the primer annealing temperature minus 3°C, extension at 72°C for 1 min, 25 cycles of denaturation at 94°C for 30 s and extension at 72°C for 1 min, and extension at 72°C for 10 min. The PCR amplification products were mainly separated by 8.0% non-denaturing polyacrylamide gel or 1.5% agarose electrophoresis. 100 bp or 250 bp Marker (Beijing Quanshijin Biotechnology Co., Ltd.) was used for labeling, and the bands were observed by silver staining or bromophenol blue staining after the electrophoresis.
[0047] Table 1 Primer sequences
[0048]
[0049] The molecular marker identification results are as follows Figure 3 As shown, the seedlings with both maternal and paternal bands or paternal bands are true hybrid offspring, and those with only maternal bands are false hybrid offspring.
[0050] In summary, the method of Example 1 was used to carry out interspecific hybridization between Pennisetum americanum and Elephant Grass, and hybrid seeds were finally harvested. The hybrid seedlings bred from the hybrid seeds were identified by root tip chromosome counting and molecular markers, and the hybrid progeny seedlings were successfully identified.
[0051] The yield of true hybrid progeny was 21.54%.
[0052] Example 4
[0053] Field planting
[0054] The phenotypes of the seedlings identified as true hybrid progeny in the field also showed that they were true hybrid progeny ( Figure 4 ). It contains both the maternal phenotype and the paternal phenotype.
[0055] This indicates that the method of the present invention is feasible in the interspecific hybridization of Pennisetum americanum and Elephant Grass, and that true hybrid offspring can be obtained using this method.
[0056] As can be seen from the above embodiments, the present invention provides a method for interspecific hybridization between Pennisetum and elephant grass and its application. The method of the present invention comprises the following steps: (1) at 4-5 pm on the first day, the Pennisetum inflorescence is covered with an isolation bag, and at the same time, the inflorescence of the object grass is covered with a pollen collection bag; (2) at 9-10 am on the second day, the pollen in the pollen collection bag is collected, the isolation bag is removed, and the Pennisetum inflorescence is pollinated with the collected pollen; (3) the pollinated inflorescence is covered with a pollination bag, and 5-7 days after pollination, the pollination bag is replaced with a net bag until the hybrid seeds are mature. The hybridization method of the present invention can improve the interspecific hybridization breeding efficiency between Pennisetum and elephant grass, so that more germplasm resources can be fully utilized. The problems of difficult operation, long time consumption, large inflorescence damage, and extremely low hybridization efficiency caused by emasculation hybridization are solved.
[0057] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.
Claims
1. A method for interspecific hybridization between Pennisetum americanum and Elephant Grass, characterized in that: The steps include: (1) At 4-5 pm on the first day, put an isolation bag on the Pennisetum inflorescence and a pollen collection bag on the target grass inflorescence; (2) Collect pollen from the pollen collection bag at 9 to 10 a.m. the next day, remove the isolation bag, and use the collected pollen to pollinate the Pennisetum inflorescence; (3) Put a pollination bag on the pollinated inflorescence and replace the pollination bag with a net bag 5 to 7 days after pollination until the hybrid seeds mature.
2. The method according to claim 1, characterized in that In step (1), the Pennisetum inflorescence is an inflorescence whose pistil has not yet been exposed; and the isolation bag is a transparent bag.
3. The method according to claim 2, characterized in that The elephant grass inflorescence in step (1) is an inflorescence that is shedding pollen.
4. The method according to claim 3, characterized in that: The Pennisetum inflorescence described in step (2) is an inflorescence with the pistil exposed.
5. The method according to claim 4, characterized in that The pollination is performed 1 to 2 times in step (2).
6. The method according to claim 5, characterized in that The pollination bag in step (3) is a transparent bag.
7. Use of the method according to any one of claims 1 to 6 in improving the efficiency of interspecific hybrid breeding between Pennisetum fasciatum and Elephant Grass.
8. Use of the method according to any one of claims 1 to 6 in hybrid breeding.