Method for improving hybridization breeding success rate of chimonanthus praecox

Abstract

The invention discloses a method for improving the success rate of chimonanthus praecox crossbreeding. The method comprises the following steps: step (1), enlarging the cultivation area of chimonanthus praecox in a piece; step (2), multi-variety interval mixed planting; and (3) keeping bees moderately. According to the method, through the comprehensive technical means of enlarging the chimonanthus praecox cultivation area, multi-variety interval mixed planting, moderate bee keeping and the like, the technical bottlenecks of low strength and success rate of artificial hybridization and natural hybridization and difficulty in obtaining a new variety through hybridization breeding at present are overcome, the chimonanthus praecox hybridization probability is increased, the hybrid seed obtaining amount is increased, and the yield of chimonanthus praecox breeding is increased. Meanwhile, the method is a simple, convenient, feasible and effective method for replacing artificial pollination, a large amount of artificial emasculation and pollination cost can be reduced, the chimonanthus praecox cross breeding efficiency is comprehensively improved, and the method is suitable for large-scale popularization and application. A brand-new way which is simple, easy to operate, labor-saving, cost-saving and efficient is opened up for chimonanthus praecox crossbreeding, and the chimonanthus praecox crossbreeding process can be effectively promoted.

Description

Technical Field

[0001] The present invention relates to the field of forest tree cross-breeding, and particularly to a method for improving the success rate of wintersweet cross-breeding. Background Art

[0002] Wintersweet [Chimonanthus praecox (Linn.) Link], as a unique traditional precious ornamental flower native to China, has a unique charm, rich fragrance, and strange flavor, with rich ornamental characteristics, precious cultural value, high medicinal value, and economic value (Song Xingrong et al., 2018). In terms of new variety breeding, breakthroughs have been made in the genus Calycanthus. Lasseigne et al. (2001) successfully hybridized Calycanthus chinensis and Calycanthus floridus to breed the fine variety 'Hartlage Wine'. Ranney and Eaker (2005) hybridized the hybrid offspring of Calycanthus chinensis and Calycanthus floridus, and Calycanthus chinensis and Calycanthus occidentalis to breed the new variety 'Venus'. Among Calycanthus floridus, varieties such as 'Athens' (flowers are yellow, with a floral fragrance), 'Michael Lindsey' (flowers are purplish-red, with a floral fragrance), and 'Purpureus' with purplish-red leaf backs have also been bred. Domestically, Yao Qingju et al. also obtained the intergeneric hybrid 'Hongyun' with Calycanthus chinensis as the female parent and Calycanthus floridus as the male parent through cross-breeding methods such as repeated pollination. This hybrid combines the advantages of both parents, is heat-resistant, has strong light tolerance, and has a strong growth advantage.

[0003] However, the progress of breeding varieties in the genus Chimonanthus has been slow. With the increasing attention paid to wintersweet in the past two decades, the resource investigation of wintersweet varieties has gradually expanded and deepened nationwide, and the recorded wintersweet varieties have also increased day by day. However, the phenomenon of "different names for the same species" and "the same name for different species" has always existed due to the chaotic classification traits and non-standard recording criteria of wintersweet varieties. Due to the lack of a unified and standardized name and a scientific quantitative selection standard for wintersweet varieties, it has seriously affected the development and utilization of excellent wintersweet varieties and the breeding of new varieties. Currently, the breeding methods are single, mainly using methods such as seedling selection and bud mutation, and few new varieties have been bred (Shen Zhiguo et al., 2019; Ding Xin et al., 2020), which cannot meet the production needs and restricts the rapid development of the industry. So far, there are almost no reports or successful cases of obtaining new varieties through cross-breeding in the genus Chimonanthus. And carrying out the innovation of wintersweet germplasm resources and breeding excellent new varieties is an important link to promote the development of the wintersweet industry.

[0004] The main technical bottlenecks in wintersweet cross-breeding are as follows:

[0005] (1) The pistils and stamens mature at different times, and there is a tendency for them to bloom at different times. The breeding system of Wintersweet is facultative outcrossing. Zhou Lihua and others have conducted research on the pollination biology of Wintersweet, and have conducted systematic research on its floral traits, pollen vitality, pollinating insects, etc. The results show that the stamens and pistils of Wintersweet have a tendency to bloom at different times and in different places, as shown in Table 1. The pistils mature first (the receptivity of the stigma is highest one day before the flower opens, followed by when the flower first opens. As the flower unfolds, the stigma gradually loses water and wilts, the stigma turns black, and the receptivity gradually decreases. On the fourth day of flowering, the receptivity of the stigma drops to 0. The pollen vitality is highest on the second day of flowering, when the anthers crack, reaching about 80%. Subsequently, the pollen vitality gradually decreases. When the pollen is completely dispersed, that is, when the flower is 3-4 days old, the pollen vitality drops to about 15%, and it is almost inactive after 5 days of flowering). When the stamens mature, they change from being separated and scattered for four weeks to being aggregated together, wrapping the pistil in it and making it difficult to contact the pollen, limiting the mechanism of self-pollination. Under natural conditions, the low temperature when wintersweet blooms greatly inhibits the activity of insects and reduces the frequency of insect visits to flowers, resulting in the general fruit-bearing capacity of wintersweet being <10% (there are large differences among different varieties, some with a fruit-bearing rate of less than 0.1%, while others are higher. Generally, good varieties have low fruit-bearing capacity, while traditional poor varieties have high fruit-bearing capacity). Insect pollination may infect the plant itself or the same variety, or it may infect other varieties. The probability of pollination nearby is higher, and the probability of pollination at a long distance is lower. Under natural conditions, the proportion of insect-pollination hybridization is also relatively small.

[0006] Table 1: Changes in stigma receptivity and pollen vitality during flowering of Chimonanthus chinensis

[0007]

[0008]

[0009] Note: "0" means the stigma is not receptive; the more "+" there are, the stronger the receptivity of the stigma; "-" means it has not been tested

[0010] (2) Low success rate of artificial hybridization. Artificial assisted pollination can increase the pollination rate. However, due to the different maturity times of male and female in Chimonanthus praecox, most artificial hybridizations need to emasculate during the period from the flower loosening stage to the early blooming stage. At this time, the pistil is mature while the stamen is not, the pistil has high receptivity, the flower buds and flowers are small, and the operation is not easy, which easily causes incomplete emasculation and is likely to damage the stigma, ovary, etc. of the female parent during emasculation, resulting in hybridization failure. At the same time, the pistils of Chimonanthus praecox are separate or aggregated, ranging from 5 to 20, the ovary is superior, the lower part of the ovary is triangular spindle-shaped (like garlic cloves), and the upper part of the style and stigma are linear. It is difficult to exactly encounter the best pollination period (when the stigma of the pistil secretes bright, transparent nectar and is moist and shiny) during pollination, it is easy to damage the small linear stigma during operation, and it is not easy for pollen to adhere to the stigma, etc., which are also likely to cause pollination failure. In addition, due to the influence of wind, rain, etc., the bagging may also damage the pollinated flowers or fruits during the bagging process, resulting in a decrease in fruit set. Moreover, even for the pollen attached to the linear stigma, most pollen tubes stop growing before reaching the bottom of the stigma, and the number of pollen tubes that can truly penetrate to the micropylar end to complete fertilization is limited, as shown in Figure 4 shown. Therefore, based on the hybridization results of the project team over the years, even if pollination is repeated once 1 day after the first pollination, the fruit set rate of artificial hybridization of Chimonanthus praecox is still low, generally <15%, which may also be the main reason why the hybridization breeding of Chimonanthus praecox has been stagnant for a long time and almost no artificial hybrid varieties have been introduced so far.

[0011] (3) Few flowers and low chance of attracting insect pollination. Chimonanthus praecox is mostly produced by individual households, the cultivated area is scattered, the single cultivated area is small, the cultivated varieties are all relatively excellent or the varieties are single, and at the same time, most of them need to cut flowers for sale, resulting in insufficient overall flower quantity left on the trees, fewer opportunities to attract various insects to collect nectar and pollinate, less possibility of obtaining insect hybridization, and even causing many cultivators to mistakenly think that excellent Chimonanthus praecox varieties do not bear fruit.

[0012] Therefore, it is an urgent problem for those skilled in the art to provide a method to overcome the low fruit set and success rate of current artificial hybridization and natural hybridization, increase the hybridization probability of Chimonanthus praecox, effectively improve the success rate of artificial hybridization of Chimonanthus praecox, greatly increase the success rate of new variety breeding and new material creation, and promote the process of Chimonanthus praecox hybridization breeding. Summary of the Invention

[0013] In view of this, the present invention provides a method for improving the success rate of Chimonanthus praecox hybridization breeding.

[0014] To solve the above technical problems, the present invention adopts the following technical solutions:

[0015] A method for improving the success rate of Chimonanthus praecox hybridization breeding, comprising the following steps:

[0016] Step (1): Expand the contiguous cultivated area of Chimonanthus praecox

[0017] Increase the cultivated area and control the amount of cut flowers according to the area size;

[0018] Step (2): Intercropping of multiple varieties at intervals

[0019] Each variety is planted alternately or randomly at intervals, and each plant is surrounded by other different varieties; for two varieties, "interval + dislocation" cultivation is adopted;

[0020] Step (3): Appropriately keep bees

[0021] During the flowering period of wintersweet every year, bees are released in the wintersweet breeding base, with 1 - 1.2 boxes configured per mu; alternatively, directional hybridization can be carried out through isolation in a net house, and 1 box is configured for every standard plot of 270m 2 Configure 1 box.

[0022] Preferably, in the step (1), the cultivation area is increased to at least more than 2 mu, and the effect is better for hundreds or thousands of mu.

[0023] Preferably, in the step (1), the amount of cut flower harvesting is controlled according to the area size: when the area is less than 5 mu, only the extremely high-quality flower branches are harvested or not harvested; when the area is 5 - 10 mu, only the high-quality flower branches are harvested; when the area is 10 - 20 mu, the high-quality and first-class flower branches are harvested; when the area is 20 - 50 mu, the high-quality, first-class and second-class flower branches are harvested; when the area is more than 50 mu, all cut flower branches are harvested, but the flower branches with serious disconnection, residual branches, extremely short and unsaleable or unable to be used as cut flowers are retained.

[0024] Preferably, in the step (2), for two varieties, isolation in a plot net house is adopted, and the plants are isolated by a net house around to form a separate plot to achieve directional hybridization.

[0025] Preferably, in the step (3), the flowering period of wintersweet every year is from late November to the end of February.

[0026] Preferably, in the step (3), 1 - 1.2 boxes of bees are configured per mu, and its length × width × height are: the outer size of the hive is 41cm × 51cm × 26cm; the inner size of the hive is 37cm × 47cm × 26cm; the size of the hive cover is 45cm × 55cm × 8cm.

[0027] Preferably, in the step (3), it is explored that it is appropriate to configure 1 box of bees for every standard plot of 270m 2 If the area is less than the standard plot, 1 box of bees is also configured; if the area is larger than the standard plot, the number of bee boxes is increased.

[0028] The present invention has achieved the following technical effects compared with the prior art:

[0029] (1) The method of the present invention increases the hybridization probability of wintersweet, improves the yield of hybrid seeds, and greatly increases the success rate of new variety breeding and new material creation;

[0030] (2) The method of the present invention is a simple, easy-to-implement and effective method to replace artificial pollination, which can reduce a large amount of manual emasculation and pollination costs, comprehensively improve the efficiency of wintersweet cross-breeding, and open up a new path for wintersweet cross-breeding that is simple, easy to operate, labor-saving, cost-saving and highly efficient, effectively promoting the process of wintersweet cross-breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 It is a schematic diagram of mixed planting of multiple varieties at intervals of the present invention (taking 8 varieties as an example); among them, the numbers represent different varieties;

[0032] Figure 2 It is a schematic diagram of the cultivation of two varieties in the form of "interval + dislocation" per standard plot of the present invention (the plant spacing is 1.5m x 2.5m, and each standard plot is 270m 2 )

[0033] Figure 3 It is a diagram of new specific materials of wintersweet of the present invention; among them, (a) is extremely early flowering; (b) is extremely late flowering; (c) is a specific flower type; (d) is a rare flower color (green); (e) is a rare flower color (red); (f) is a spike-shaped densely branched flower branch; (g) is flowers clustering;

[0034] Figure 4 It is a diagram of the research on fertilization and embryo development of wintersweet of the present invention; among them, 1. Pollen begins to germinate on the stigma; 2. Two pollen tubes enter the ovary simultaneously; 3. The pollen tube winds around the outer wall of the ovule to reach the micropylar end, and then enters the upper and lower ovules in the ovary from the micropylar end, and the upper ovule develops poorly; 4. Growth in a forward pushing manner with a callose plug; 5. The entire stigma can receive pollen, and callose is unevenly deposited in the pollen tube; 6. The pollen tube grows in the stigma;

[0035] Figure 5 It is a planting diagram of multi-variety rotation (mixed) planting of wintersweet of the present invention; among them, the numbers represent rows and columns; the capital letters represent different varieties;

[0036] Figure 6 It is the female parent and excellent mutant of 'Biying Ziban' of the present invention;

[0037] Figure 7 It is the female parent and excellent mutant of 'Baochun' of the present invention;

[0038] Figure 8 It is the female parent and excellent mutant of 'Happy' of the present invention;

[0039] Figure 9 It is the female parent and excellent mutant of 'Happy Scent' of the present invention;

[0040] Figure 10 It is the female parent and excellent mutant of 'Golden' of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0041] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] The invention discloses a method for improving the success rate of wintersweet hybrid breeding, comprising the following steps:

[0043] Step (1): Expand the area of ​​wintersweet cultivation

[0044] The cultivation area should be increased to at least 2 mu, and the effect will be better if it is hundreds or thousands of mu. The amount of cut flowers should be controlled according to the size of the area: if the area is less than 5 mu, it is recommended to only cut the best branches or not cut them; if the area is 5-10 mu, it is recommended to only cut the best branches; if the area is 10-20 mu, it is recommended to cut the best and first-level branches; if the area is 20-50 mu, it is recommended to cut the best, first- and second-level branches; if the area is more than 50 mu, all cut branches can be cut, but the branches that are seriously disconnected, broken, very short, and not attractive or cannot be used as cut flowers can be retained. In this way, the overall flowering volume of the remaining trees of wintersweet can be effectively increased during the entire flowering period of 3-4 months, which can more effectively attract more species and a larger number of insects to come, and increase the frequency of insect pollination;

[0045] Step (2): Mixed planting of multiple varieties

[0046] In 2010, a 20-mu Chimonanthus germplasm resource garden and breeding base of Sichuan Academy of Agricultural Sciences was established. Due to the need to observe the flowering period every year, the amount of cut flowers is small, and more than 80% of the flowers are retained. At present, more than 200 resource varieties have been collected. With the increase in the number of collected varieties year by year, the fruiting power of each plant and each variety has increased significantly. Due to the relatively large area and the large amount of flowers retained, the ability to attract insects for pollination is strong, as shown in Table 2. From the observation of flowering and fruiting of sown plants for many years, it was found that the mutation rate also increased significantly. The reason is that when the original number of varieties is small, insects mostly pollinate the same variety. After multiple varieties are mixed, the proportion of the same variety decreases, and the probability of insects pollinating the same variety decreases, while the probability of pollinating other varieties increases, which increases the probability of insect hybridization. More hybrid mutations appeared after seed sowing, as shown in Table 3. At the same time, this result further confirms the previous research results that "the success rate of self-pollination of Chimonanthus is lower than the success rate of cross-pollination". In addition, the project team has also conducted multiple experiments to show that: when multiple varieties are mixed with intervals, such as Figure 1As shown in the figure, that is, all around the same plant of the same variety are other varieties, which can further increase the probability of insect hybridization and reduce the probability of self-pollination. However, it is a bit difficult to find the male parent of the hybrid in the later stage. Most can only be achieved through molecular means, and a small number can be inferred through the observation of flowering morphology; the cultivation of "spacing + dislocation" of two varieties + isolation in a small plot with a net room, that is, two varieties are like Figure 2 As shown in the figure, after cultivation, the plants are isolated with a net room around to form a separate small plot, which can promote directed hybridization (A×B or B×A), laying a foundation for the directed hybridization breeding of wintersweet.

[0047] Table 2: Statistical table of the seed-setting rate of wintersweet in different years

[0048]

[0049]

[0050] Note: The letters in the table represent the significance of the difference in the seed-setting rate at the 0.05 level in different cultivation areas of the same variety.

[0051] Table 3: Statistical table of the variation types and variation rates of different wintersweet varieties

[0052]

[0053]

[0054] Note: The letters in the table represent the significance of the difference in the variation rate at the 0.05 level in different cultivation areas of the same variety.

[0055] Step (3): Appropriately raise bees

[0056] During the flowering period of wintersweet every year (from late November to the end of February), assign beekeepers who raise Chinese honeybees, Caucasian bees, yellow-legged hornets, etc. to release bees in the wintersweet breeding base. Each mu is configured with 1 - 1.2 boxes (length × width × height: the outer dimensions of the hive are 41cm × 51cm × 26cm; the inner dimensions of the hive are 37cm × 47cm × 26cm; the dimensions of the box cover are 45cm × 55cm × 8cm), which greatly makes up for the disadvantages of limited insect activities and insufficient pollination in winter under natural conditions. The number of insect visits to single flowers during the peak period is increased from about 2 times per hour to more than 5 times. It can be seen that raising bees can greatly improve the pollination and hybridization success rate. In addition, through directed hybridization in the net room, since other pollinating insects are isolated by the net room, it is explored that for each standard small plot of 270m 2 (18m × 15m), it is appropriate to configure 1 box of bees. If the area is smaller than the standard small plot, 1 box of bees is also configured; if the area is larger than the standard small plot, the number of beehives is increased as appropriate.

[0057] In summary, by comprehensively applying the three major measures of "expanding the cultivated area of wintersweet in large patches + intercropping multiple varieties at intervals + appropriately raising bees", the hybridization success rate has been greatly improved, and a small number of breakthrough new varieties have been successfully obtained. Among them, in September 2023, two new varieties with application numbers 20231149 and 20231153 were declared, and a large number of specific new materials such as early flowering, late flowering, rare flower colors, different flower shapes, different flower fragrances, characteristic tree shapes, and spike-shaped flower branches were obtained, as Figure 3 shown.

[0058] Example 1: Comparison of the seed-setting rates of wintersweet under different treatments

[0059] Taking the 'Baochun' variety with a relatively high seed-setting rate in the Xindu base as an example, the seed-setting rates under different treatments are shown in Table 4. The seed-setting rate under natural conditions is 28.21%, the seed-setting rate with bagging without emasculation is 3.45%, the seed-setting rate with emasculation without bagging is 15.38%, and the seed-setting rate with bagging and cross-pollination of different varieties (i.e., artificial hybridization) is 18.80%. The data comparison between the natural control and the treatment with bagging without emasculation is extremely different, with the former being 8.18 times that of the latter, indicating that self-pollination and cross-pollination of wintersweet coexist simultaneously, and the breeding system type is facultative outcrossing. Moreover, the role of cross-pollination (hybridization) of different varieties is much greater than that of self-pollination (selfing) of the same variety; the seed-setting rate with bagging without emasculation (including self-pollination and cross-pollination of the same variety) is extremely low. The reason may be that when the self-pollen develops and matures, the stigma receptivity has been greatly reduced, resulting in a low success rate of self-pollination, or the stigma receptivity is low and the receptive time is short after the flower opens, making it difficult or even impossible for the mature cross-pollen to be transferred to the stigma; the seed-setting rate with emasculation without bagging is lower than the natural seed-setting rate, with the former being about 1 / 2 of the latter, probably because the attractiveness to insects is reduced after emasculation and the source of its own pollen is lost, so the seed-setting rate is much lower; comparing the two treatments of emasculation without bagging and bagging without emasculation, the seed-setting rate of the latter is significantly lower than that of the former, only 22.43% of the former, indicating that after bagging isolates the visitors, the pollination success rate is greatly reduced; under the condition of artificial assisted pollination, the cross-pollination seed-setting rate is relatively high, 1.22 times that of emasculation without bagging, but it is lower than the natural seed-setting rate, probably because although assisted pollination improves the pollination rate, it may also cause damage to the flower organs and other effects on the pollination success rate.

[0060] In addition, through the investigation and comparison of the wintersweet seed-setting rates in the Xindu base over the years, the natural seed-setting rates of different varieties vary greatly, ranging from 0.63% to 70.56%, and there are also certain differences in the natural seed-setting rates in different years.

[0061] Table 4: Statistical table of the seed-setting rates of wintersweet under different treatments

[0062] Different treatments Number of treated flowers (flowers) Number of fruits set (fruits) Fruit set rate (%) Natural state 234 66 28.21 Bagging without emasculation in natural state 145 5 3.45 Emasculation without bagging 65 10 15.38 Bagging and cross-pollination with different varieties 133 25 18.80

[0063] Emasculation: Select the flower buds about to open, and it can be carried out at any time period after the dew evaporates during the day.

[0064] Pollination: Conducted between 12:00 and 16:00 on sunny days, i.e., during the period of relatively high temperature in a day (the optimal temperature is 5 - 15°C); Select the male parent flower that is about to shed pollen or has just shed pollen, take out the pollen grains and place them in a clean petri dish. If the outdoor temperature is low and the pollen grains do not shed, the method of heating with a hot water bag can be adopted to heat the petri dish containing the pollen grains on-site for 4.5 - 5.5 minutes (the temperature of the hot water bag is 30 - 35°C), and the viable pollen grains will crack and release pollen; Use tweezers to pick out the just-released pollen grains and gently smear the pollen on the villi of the exposed stigma of the female parent flower, and the pollination is successful.

[0065] Example 2: Comparison of seed setting rates and variation rates in different cultivation areas

[0066] In 2007, the seed setting rates of the main cultivars 'Spring Herald' and 'Joyful' were investigated in 9 main wintersweet production areas around Chengdu. The corresponding seeds were harvested in the middle and late June of 2008, sown in plug trays in the plum grove base in March 2009, transplanted and planted before germination in February 2010, carefully managed, and first flowered at the end of 2012. All plants flowered stably at the end of 2014, and then their variation rates were counted. The results are as follows: Keeping bees can significantly improve the seed setting rate and variation rate of wintersweet; The larger the planting area, the more flowers left, and the more varieties, the higher the seed setting rate and the greater the variation rate after sowing the seeds; After reaching a certain scale and retaining a certain amount of flowers, the seed setting rate shows an upward trend with the increase in the number of varieties, while the variation rate increases significantly with the increase in the number of varieties, as shown in Table 5. At the same time, from the variant plants sown of the 'Joyful' variety in the plum grove base, an excellent strain 'Golden Disk with Curled Petals' with large flowers, bright colors, strong fragrance, easy flower formation on long branches, and high cut flower yield was selected. After years of observation and propagation, its traits are stable and it passed the fine variety approval of the Sichuan Forest Tree Variety Approval Committee in 2020 (No. 07, Sichuan Approval 2020).

[0067] Table 5: Investigation results of seed setting rates and variation rates in different cultivation areas

[0068]

[0069]

[0070] Note: The letters in the table represent the significant differences in seed setting rates and variation rates of the same variety in different cultivation areas at the 0.05 level.

[0071] Example 3: Comparison of seed setting rates and variation rates in different years with the increase in germplasm resources

[0072] In the modern agricultural science and technology innovation demonstration park of Sichuan Academy of Agricultural Sciences (with a total area of about 2,700 mu, abbreviated as Xindu Base), the wintersweet germplasm resource nursery and breeding base were established in early 2010, with an area of about 20 mu. At the beginning of its establishment, 43 varieties were planted row by row with 9 plants per variety for preservation, and the others were mainly planted varieties in Sichuan such as 'Spring Herald', 'Joyful', and 'Glistening Star', or sown seedlings planted in patches, with a row spacing of 1.5 m × 2.5 m. Winter sweet varieties were collected year by year inside and outside the province. Some were preserved by topworking, and some were preserved by plant replacement. Most varieties retained 9 plants (planted in rows), with slightly more excellent varieties and slightly fewer extremely poor varieties. From 2012 to 2016, through the investigation of the seed setting rate of 5 internationally registered varieties and the use of sowing seeds as soon as they were harvested (harvested after the epidermis of the fruit receptacle turned brown, and the seeds were sown immediately after being peeled out. This method has fast and neat emergence and high germination rate), the mutation rate was counted after 6 years of stable flowering. The results showed that: with the increase in the number of varieties collected year by year, especially the number of flowering varieties, both the seed setting rate and the mutation rate of wintersweet showed an increasing trend. The increase in the seed setting rate was relatively slow, while the increase in the mutation rate was relatively large, and the difference between them was obvious. The detailed results are shown in Table 6.

[0073] Table 6: Investigation results of seed setting rate and mutation rate in different years in Xindu Base

[0074]

[0075]

[0076] Note: ① Variety 1: 'Fragrant Happiness'; Variety 2: 'Joyful'; Variety 3: 'Early Red'; Variety 4: 'Golden Sunshine'; Variety 5: 'Glistening Star'. ② The letters in the table indicate the significance of the difference in the seed setting rate and mutation rate of the same variety in different years at the 0.05 level.

[0077] Example 4: Comparison of seed setting rate and mutation rate between beekeeping and non-beekeeping bases

[0078] In 2017, two beekeeping wintersweet bases were respectively investigated. One was in Xinfan, Xindu, with an area of 10 mu, raising 28 boxes of Chinese bees, with an average of 2.8 boxes per mu. The other was the Amber Wintersweet Garden in Dujiangyan, with an area of 80 mu, raising 80 boxes of Chinese bees, with an average of 1 box per mu. The number of wintersweet varieties in both wintersweet bases was about 35. The seed setting rates of 5 same varieties, namely 'Zaohong', 'Baochun', 'Happy', 'Huangqing', and 'Huanglatuo', were investigated in comparison with the wintersweet resource nursery and breeding base in Xindu (referred to as the Xindu base, with an area of 20 mu, no beekeeping, and nearly 180 wintersweet varieties). And they were sown immediately after collection by variety in mid-to-late May. The variation rate of the sown plants was investigated at the end of 2023. The results are shown in Table 7. It can be seen that the seed setting rates of the two beekeeping bases were significantly higher than that of the Xindu base, and the variation rate was also relatively high. Except for the 'Zaohong' variety, they all reached significance, indicating that beekeeping can greatly improve the seed setting rate and variation rate of wintersweet and increase the probability of crossbreeding; there were also certain differences between the two beekeeping bases, generally the Xinfan base in Xindu was slightly higher than the Amber Wintersweet Garden in Dujiangyan. But through variance analysis, the difference between the two was not significant, indicating that allocating 1 box of bees per mu on average can basically complete the pollination of wintersweet flowers; comparing the two beekeeping bases with the Xindu base, although the number of varieties in the latter base was extremely different from that in the former, being 5 times that of the former, the seed setting rate and variation rate were not as high as those of the former. This further confirmed that in the natural state, due to the relatively low temperature, insufficient number and activity of insect pollinators during the winter flowering period of wintersweet, the pollination was limited, and the natural hybridization was relatively low; there were significant differences in the seed setting rate and variation rate among different varieties, and most of them showed significant and highly significant differences, indicating that the seed setting rate and variation rate were closely related to the characteristics of the varieties themselves. A high seed setting rate might be due to the relatively high pistil receptivity, longer receptive time, or stronger self-pollination ability after the flowers opened, etc. On the contrary, a low seed setting rate might be caused by low pistil receptivity, shorter receptive time, or weaker self-pollination ability after the flowers opened, etc. A high variation rate might be due to the significantly higher cross-pollination success rate of different varieties than self-pollination, or the low success rate of self-pollination and cross-pollination between the same varieties, that is, the high possibility of hybridization between different varieties.

[0079] Table 7: Investigation Results of Seed Setting Rates and Variation Rates in Three Bases

[0080]

[0081] Note: The letters in the table represent the significance of the difference in seed setting rates and variation rates of the same variety in different bases at the 0.05 level.

[0082] Example 5: Comparison of Seed Setting Rates and Variation Rates with Different Mixed Loading Degrees

[0083] At the end of 2016, a 10-mu wintersweet planting base (referred to as the new Xindu base) was expanded at the boundary of the Modern Agricultural Science and Technology Innovation Demonstration Park of the Sichuan Academy of Agricultural Sciences, about 800 m in a straight line from the original wintersweet base (the old Xindu base). 5 mu of this new base was used for producing potted wintersweet flowers (the variety was a well-bred grafted seedling), and 5 mu was used as a resource nursery. All the plants in the resource nursery were adult wintersweet plants, among which 1320 m2 (40m × 33m) Rotate (or mix) plant 18 excellent varieties such as 'Early Red', 'Happy Floral Scent', 'Joyful', 'Missing', 'Flying', 'Golden Sunshine' etc. according to the row and plant spacing of 1.5m × 2.5m, as Figure 5 shown. Plant the remaining area according to the original base planting method. In 2018, the ground-planted trees basically restored their vigor and the potted plants could also bloom. In late November, 8 boxes of Chinese honeybees were fostered in the new base. To reduce the interference of external plants, in the middle and late May 2019, the seed setting rates of 5 varieties of plants within 10m from the outside in the mixed planting plot (the plants in the blue area in the figure) were counted and sown immediately after collection, and carefully managed. After the flowering was stable at the end of 2024, the plant variation situation was counted. At the same time, use the old base as a comparison, raise 22 boxes of Chinese honeybees, and count the seed setting rates and variation situations of the same 5 varieties.

[0084] The seed setting rates and variation rates of the new and old bases are shown in Table 8. It can be seen that the seed setting rates and variation rates of the new base are generally higher than those of the old base. Among them, the differences in the seed setting rates of 'Spring Report', 'Joyful', and 'Glistening Gold' are significant, and the differences in the variation rates of 'Blue Shadow with Purple Spots' and 'Glistening Gold' are significant. Analyzing the reasons, although the number of varieties in the old base is 2 - 3 times more than that in the new base, the probability that the plants around the same plant are of different varieties is nearly 50% less. This result further confirms that the success rate of cross-pollination is higher than that of self-pollination, and the cross-pollination between different varieties is higher than that between the same variety. Mixed planting can significantly increase the probability of insect pollination and hybridization; 0.8 boxes and 1.1 boxes of bees are allocated per mu in the new and old bases respectively. Although there are differences, the potted plants in the new base are small and the ground-planted plants have not fully reached the plump state. The average flower quantity per mu is about 65 - 70% of that of the old base. This further shows that when the base is not too small, normally allocating 1 - 1.2 boxes of bees per mu can well complete the pollination task.

[0085] Table 8: Investigation Results of Seed Setting Rates and Variation Rates of New and Old Bases

[0086]

[0087] Note: The letters in the table represent the significance of the differences in the seed setting rates and variation rates of the same variety in different bases at the 0.05 level.

[0088] In addition, from the investigation results of plant variations, it is found that the excellent variations resulting from the mixed planting of excellent varieties account for a relatively large proportion, significantly more than that of the old base, indicating that the probability of excellent variation plants emerging from the hybridization between excellent varieties is greater, further verifying the importance of selecting the parents for hybridization. Hybridization can pass the excellent traits of the parents to the offspring. The excellent variation plants of 5 varieties that emerged in the new base are as Figures 6 - 10As shown, there are more than five excellent mutant strains in each variety, and the mutant strains have excellent quality, obvious differences from the female parent, and prominent specificity. In the future, after repeated observations, regional trials, and multi-generation propagation, once the traits are confirmed to be stable and stably inherited, new variety rights and improved variety certification can be applied for. This undoubtedly opens up a new and simple, easy-to-operate, labor-saving, cost-saving, and efficient path for wintersweet cross-breeding.

[0089] As described above, the foregoing is only a preferred embodiment of the present invention, and does not impose any limitation on the technical scope of the present invention. Therefore, any minor modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. A method for improving the success rate of wintersweet hybrid breeding, characterized in that, The steps include: Step (1): Expand the area of ​​wintersweet cultivation The cultivated area is increased, and the amount of cut flowers is controlled according to the size of the area; Step (2): Mixed planting of multiple varieties Each variety is planted alternately or randomly, with each plant surrounded by other varieties; two varieties are planted in "interval + staggered" manner; Step (3): Moderate beekeeping Every year during the wintersweet flowering season, bees are released into the wintersweet breeding base, with 1-1.2 bee boxes per mu. Directed hybridization can also be carried out through isolation in net rooms, with each standard plot of 270m 2 Configure 1 box.

2. A method for improving the success rate of wintersweet hybrid breeding according to claim 1, characterized in that, In the step (1), the cultivation area is increased to at least 2 mu, and the effect is better if it is increased to hundreds or thousands of mu.

3. A method for improving the success rate of wintersweet hybrid breeding according to claim 1, characterized in that, In the step (1), the amount of cut flowers to be cut is controlled according to the size of the area: if the area is less than 5 mu, only the finest flower branches are cut or no branches are cut; if the area is 5-10 mu, only the finest flower branches are cut; if the area is 10-20 mu, the finest and first-class flower branches are cut; if the area is 20-50 mu, the finest, first- and second-class flower branches are cut; if the area is more than 50 mu, all cut flower branches are cut, but the branches that are severely disconnected, broken, very short and unsaleable or cannot be used as cut flowers are retained.

4. A method for improving the success rate of wintersweet hybrid breeding according to claim 1, characterized in that, In the step (2), the two varieties are isolated by using a small plot net house, and the plants are isolated by net houses on all sides to form a separate plot to achieve directional hybridization.

5. A method for improving the success rate of wintersweet hybrid breeding according to claim 1, characterized in that, In the step (3), the flowering period of wintersweet is from late November to the end of February each year.

6. A method for improving the success rate of wintersweet hybrid breeding according to claim 1, characterized in that, In the step (3), 1-1.2 beehives are arranged per acre, and the length×width×height thereof are: the outer dimensions of the nest box are 41cm×51cm×26cm; the inner dimensions of the nest box are 37cm×47cm×26cm; the dimensions of the nest box cover are 45cm×55cm×8cm.

7. A method for improving the success rate of wintersweet hybrid breeding according to claim 1, characterized in that, In step (3), the network room is isolated for each standard cell of 270m 2 It is advisable to configure one beehive. If the area is smaller than the standard community, also configure one beehive. If the area is larger than the standard community, increase the number of beehives.

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