A method for creating germplasm of a distant cross between species of randia

Abstract

The application discloses a kind of mountain yellow skin inter-specific distant hybridization germplasm creation method, belong to plant hybridization breeding technical field.The method includes: (1) father pollen collection and drying;(2) father pollen is first stored at 4~5 ℃ for 16~36 h, then-20~-30 ℃ is frozen for 36~60 h, finally stored at-196 ℃;(3) before pollination, father pollen is recovered, and restores vitality;(4) mother flower is emasculated and bagged isolation;(5) mother stigma pretreatment, the mixed solution of sucrose and boric acid is infiltrated stigma;(6) inactivated mountain yellow skin homologous pollen is mixed with heterologous yellow skin pollen;(7) artificial pollination;(8) after pollination management.The application realizes long-term preservation of pollen by three-step gradient cooling, then combines stigma pretreatment measures, inactivated homologous pollen is mixed with heterologous father pollen to assist pollination, selects appropriate pollination opportunity, and the combined effect greatly improves hybridization success rate, and fruit setting rate reaches more than 42%, effectively solves the problems, such as traditional mountain yellow skin parent flowering does not meet, female and male gametophytes develop asynchronously, inter-specific hybridization rejection and poor compatibility.

Description

Technical Field

[0001] This invention belongs to the field of plant hybridization breeding technology, specifically relating to a method for creating germplasm through interspecific distant hybridization of *Phellodendron amurense*. Background Technology

[0002] Mountain wampee (Clausena anisum-olens) belongs to the Rutaceae family and is a tropical and subtropical spice fruit tree unique to southern my country (especially Guangxi, Guangdong, and Hainan). Also known as fine-leaved wampee or chicken skin fruit, its fruit has a unique and rich aroma, a sweet and sour taste, and is rich in volatile oils, flavonoids, vitamins, and minerals. It can be eaten fresh, used as a condiment, processed into candied fruit, and fruit wine. It also has medicinal value, such as regulating qi, strengthening the spleen, and aiding digestion. It is a unique fruit tree resource with economic, nutritional, and medicinal value.

[0003] Common wampee fruits are sweeter and have thicker skin, primarily intended for fresh consumption; while mountain wampee fruits are smaller, weighing only 2–3g per fruit, with an edible rate of 70%–75% and a total acidity as high as 2.28%. Through distant hybridization between mountain wampee and wampee, it is possible to selectively breed new wampee germplasm resources that possess high aroma, suitable sweetness and acidity, and high yield, which is of great significance for enriching the wampee genus variety system and enhancing the value of specialty fruit tree industries.

[0004] The technical challenges currently encountered in the breeding of *Cinnamomum camphora* are as follows: (1) There is a problem of poor compatibility between the parents in interspecific hybridization of *Cinnamomum camphora*. When using heterologous *Cinnamomum camphora* pollen, the stigma of *Cinnamomum camphora* has a repulsive effect on heterologous *Cinnamomum camphora* pollen, resulting in severe fertilization obstacles and extremely low fruit set rate. (2) Conventional room temperature and short-term cold storage methods cannot maintain the viability of *Cinnamomum camphora* pollen for a long time. A large amount of pollen is inactivated in a short period of time, resulting in low effective pollination utilization. Even if the pollination operation is completed, the pollen tube elongation, fertilization and fruit setting process cannot be completed normally, resulting in great breeding losses. (3) The natural flowering period of mountain yellow peel and yellow peel is 12–18 days apart. Conventional artificial control methods such as pruning, water control, and shading can only shorten the flowering period difference to 5–7 days, and cannot achieve synchronous development of male and female gametes. The half-life of yellow peel pollen viability at room temperature is less than 4 hours. After 48 hours of refrigeration at 4℃, the pollen germination rate is only 28%, and the pollen viability decays very quickly. At the same time, the receptive window of the stigma of mountain yellow peel is very narrow, only 12–24 hours after flowering, and there is a lack of targeted stigma affinity enhancement technology.

[0005] Current research primarily focuses on intraspecific hybridization within the Citrus genus, lacking a comprehensive technical approach that integrates pollen preservation, stigma regulation, precise pollination, and reproductive isolation breaking for interspecific distant hybridization within the Citrus genus. Conventional hybridization results in a fruit set rate of less than 1.2%, leading to extremely low germplasm creation efficiency. Therefore, research on in vitro pollen preservation and mating, stigma receptivity regulation, and pollination timing will be of great significance for achieving germplasm creation through interspecific distant hybridization within the Citrus genus. Summary of the Invention

[0006] The purpose of this invention is to provide a method for germplasm creation through distant hybridization of *Phellodendron chinense* species. This method utilizes a three-stage gradient cooling process to achieve ultra-low temperature preservation of the male parent pollen, extending its storage period. Combined with stigma pretreatment, inactivation of homologous pollen mixed with heterologous male parent pollen to assist pollination, and selection of appropriate pollination timing, the combined effects significantly improve the hybridization success rate. This achieves germplasm resource innovation and variety improvement for *Phellodendron chinense*, effectively solving problems such as inconsistent flowering periods of the parent plants, asynchronous development of male and female gametes, interspecific hybridization rejection, and poor compatibility in traditional *Phellodendron chinense* species.

[0007] This invention is achieved through the following technical solution: A method for creating germplasm through interspecific distant hybridization of *Phellodendron amurense* includes the following steps: (1) Collection and drying of male parent pollen The yellow-skinned variety is used as the male parent. On a sunny morning, unopened flowers of the male parent during the large bud stage are collected, and the intact anthers are removed. The anthers are dried in a drying oven, and pure pollen is screened to ensure that the pollen viability is ≥85%.

[0008] (2) Preservation of male parent pollen by three-step gradient cooling The pollen from step (1) was placed in cryovials and subjected to a gradient cooling method to avoid low-temperature damage. First, it was refrigerated at 4 ~ 5℃ for 16 ~ 36 hours, then frozen at -20 ~ -30℃ for 36 ~ 60 hours, and finally stored in an ultra-low temperature environment of -196℃. (3) Resurgence of male pollen viability Before pollinating the female parent plant, the male parent pollen preserved in step (2) is placed in an environment of 4 ~ 5℃ for 2 ~ 3 hours to restore its vitality, with a vitality recovery rate of ≥90%.

[0009] (4) Male flowers of the mother plant were removed and bagged for isolation. Healthy, disease-free *Cinnamomum camphora* plants were selected as the mother plants. Flowers in the large bud stage that were about to open the next day were selected, and all stamens were completely removed by manual emasculation. After emasculation, the plants were immediately bagged and sealed to prevent contamination by exogenous pollen. (5) Pretreatment of the stigma of the mother plant When the flowers of the mother plant are half-open, the stigma is fully exposed and free of secretions, spray the stigma evenly with a mixed aqueous solution of 5-6% sucrose and 1-2% boric acid, and let it stand for 15-20 minutes to enhance the activity of the stigma and the affinity of the pollen, and reduce the rejection reaction of the stigma.

[0010] (6) Pollen mixing and blending Collect pollen from healthy *Phellodendron chinense* flowers of the same variety during the large bud stage. Inactivate the pollen by dry heat at 121℃ for 15-20 minutes to destroy its reproductive activity and obtain inactivated homologous pollen from *Phellodendron chinense*. Mix the inactivated homologous pollen from *Phellodendron chinense* with heterologous pollen from *Phellodendron chinense* flowers revived in step (3) at a mass ratio of 3:7-5:5 to obtain composite pollen.

[0011] Adding homologous inactivated pollen of the same species of *Phellodendron amurense* can deceive the immune recognition system of the flower stigma, effectively breaking down interspecific reproductive isolation and thus achieving successful pollination.

[0012] (7) Artificial pollination During the peak pollination window after flowering, the compound pollen from step (6) was applied to the stigma of the pretreated *Phellodendron chinense* parent plant in step (5). After pollination, an isolation bag was immediately placed on top. (8) Post-pollination management After pollination, maintain an air humidity of 60% to 70% in the field and avoid strong sunlight exposure and heavy rain. Remove the isolation bag 7 to 10 days after pollination, and cultivate the hybrid germplasm according to conventional water and fertilizer management and pest and disease control until the fruit matures.

[0013] Furthermore, in step (1), the pollen collection of the male parent is carried out on a sunny and windless morning from 9:00 to 11:00; the drying oven is controlled at a temperature of 20 to 25°C and a relative humidity of 30 to 40% for constant temperature drying for 2 to 3 hours.

[0014] Furthermore, the three-stage gradient cooling of the paternal pollen in step (2) involves placing the pollen from step (1) into cryopreservation tubes, refrigerating it at 4 ℃ for 24 h, then freezing it at -20 ℃ for 48 h, and finally storing it in a liquid nitrogen ultra-low temperature environment at -196 ℃.

[0015] Furthermore, in step (3), the preserved paternal pollen is placed in an environment of 4 ℃ for 2 h to revive.

[0016] Furthermore, in step (4), the manual removal of male reproductive organs is performed gently to avoid damaging the stigma; the bagging is done using a breathable sulfuric acid paper bag.

[0017] Furthermore, in step (5), the mixed aqueous solution is made of 5% sucrose and 1% boric acid, and the treatment time is 18 min.

[0018] Furthermore, in step (6), the inactivated homologous pollen of *Cinnamomum camphora* and the revived heterologous pollen of *Cinnamomum camphora* are mixed uniformly at a mass ratio of 3:7.

[0019] Furthermore, in step (6), the peak pollination window period is 10 to 14 days after flowering of the *Phellodendron amurense* parent plant.

[0020] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: 1. This invention utilizes a three-stage gradient cooling method: first maintaining the temperature at 4℃ for 24 hours, then lowering it to -20℃ for 48 hours, and finally storing it in an ultra-low temperature environment of -196℃. This allows pollen to slowly adapt to the low-temperature environment, avoiding pollen frost damage, maximizing the preservation of pollen physiological activity, ensuring almost no decrease in pollen viability, and extending the effective preservation period of yellow-skinned pollen to 6–12 months. The pollen recovery viability is ≥90%, completely offsetting the natural 12–18-day difference in flowering period between mountain yellow-skinned and yellow-skinned varieties, achieving precise matching of male and female gamete timing, effectively solving the problem of parental flowering mismatch, significantly improving the hybridization success rate, and achieving a hybridization fruit set rate of over 42%.

[0021] 2. This invention uses a pollen cryopreservation method, which not only extends the pollen preservation period but also ensures that the pollen retains high viability after revival. This solves the problem of rapid pollen inactivation caused by conventional direct collection of pollen from anthers and conventional drying and temperature storage methods. It increases the effective utilization rate of pollen from less than 30% to over 90%, greatly reducing breeding costs and working time costs.

[0022] 3. This invention uses homologous inactivated pollen of the same variety of *Cinnamomum camphora* to mix with pollen from the male parent of *Cinnamomum camphora*, resulting in mixed pollen for pollination of the female parent plant. The addition of homologous inactivated pollen deceives the immune recognition system of the flower stigma, effectively weakening reproductive isolation in distant hybridization, thereby increasing the success rate of hybridization pollination and raising the fruit set rate from the traditional 1.2% to over 42%.

[0023] 4. Before pollination, the stigmas of the female parent flowers are pretreated by soaking them in a mixed aqueous solution of 5-6% sucrose and 1-2% boric acid. This enhances the activity of the stigmas and their affinity with pollen, improves their receptivity, and reduces their rejection reaction. In subsequent mixed pollen pollination, the stigmas can better contact the pollen of the male parent, thereby increasing the hybrid fruit set rate.

[0024] 5. This invention effectively solves the problems of mismatched flowering periods and hybridization incompatibility in *Pyringa spp.*, significantly improving the fruit set rate of distant hybridization and filling the technological gap in efficient distant hybridization breeding among species of the *Pyringa* genus. The invention team conducted multiple rounds of field trials from 2022 to 2025 to verify the stability of the method, providing a basis for subsequent standardized work on the creation of distant hybrid germplasm in the *Pyringa* genus.

[0025] 6. The method of the present invention has the characteristics of standardized process, strong repeatability, and suitability for large-scale breeding. It can be applied to related industrial breeding work such as the creation of germplasm resources of Phellodendron amurense, the targeted improvement of superior new varieties, and the cultivation of high-quality breeding materials of Phellodendron amurense. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 is the collected Clausena lansium pollen; Figure 2 is the operation diagram for removing the stamens of the female parent flower; Figure 3 is the pollination bagging for the hybrid germplasm; Figure 4 is the operation diagram for dot pollination of the female parent using a pollination stick; Figure 5 is for recovering and measuring the hybrid germplasm; Figure 6 is the comparison diagram of the seedlings from seeds and the hybrid germplasm seedlings after being planted for one and a half years. SPECIFIC EMBODIMENTS

[0027] The following further elaborates on the present invention through examples. These examples are only used to illustrate the present invention and do not limit the protection scope of the present invention.

[0028] Example 1 Research on the Influence of the Pollen Preservation Process of the Male Parent on the Fruit Setting Rate A method for creating interspecific distant hybrid germplasm of Clausena lansium includes the following steps: (1) Collection and drying treatment of the pollen of the Clausena lansium male parent The female parent Clausena lansium: Guiyan No. 20, and the male parent Clausena lansium: Sweet Clausena lansium are crossed.

[0029] In the middle and late March 2022, which is the full bloom period of Clausena lansium, select a sunny and windless day from 9:00 - 11:00 in the morning, collect the unopened flowers at the large bud stage of the Clausena lansium male parent, and strip the complete anthers; place the anthers in a constant temperature and humidity drying oven, control the temperature at 22°C and the relative humidity at 35%, and after constant temperature drying for 3 hours, screen the pure pollen to ensure that the pollen viability ≥ 85%.

[0030] (2) Gradient comparison of the pollen preservation process of the male parent Pack the qualified dried pollen of the Clausena lansium male parent obtained in step (1) into sterile cryopreservation tubes, with 4 g in each cryopreservation tube. Divide them into three groups, with 3 cryopreservation tubes in each group.

[0031] Group A: Non-gradient cooling group. Directly store the cryopreservation tubes in the ultra-low temperature environment of -196°C liquid nitrogen.

[0032] Group B: Two-step gradient cooling group. First, store the cryopreservation tubes in a refrigerator at 4°C for 24 hours, and then directly place them in the ultra-low temperature preservation of -196°C liquid nitrogen.

[0033] Group C: Three-step gradient cooling group. First, store the cryopreservation tubes in a refrigerator at 4°C for 24 hours, then freeze them at -20°C for 48 hours, and finally place them in the ultra-low temperature environment of -196°C liquid nitrogen for preservation.

[0034] (3) Revival of the pollen viability of the male parent Depending on the annual phenological period of *Phellodendron amurense*, the peak flowering period of *Phellodendron amurense* should be selected before considering the revitalization of the male parent's pollen.

[0035] April 2022 was the peak flowering season for the mother plant, Shanhuangpi Guiyan No. 20.

[0036] Before pollination, pollen from groups A, B, and C that had been cryopreserved was placed in a 4°C environment for 2 hours to allow the pollen to regain its vitality.

[0037] (4) Remove male flowers from the mother plant of Phellodendron amurense, bag and isolate them. Select healthy, disease-free *Cinnamomum camphora* mother plants, choose large buds that are about to open the next day, and manually remove all stamens gently to avoid damaging the stigma. Immediately after removing the stamens, use breathable sulfuric acid paper bags to seal and isolate the plants to prevent external pollen contamination.

[0038] (5) Pretreatment of the stigma of the mother plant When the flowers of the mother plant are half open, the stigma is fully exposed and there is no secretion contamination, spray the stigma evenly with a mixed aqueous solution of 5% sucrose and 1% boric acid, and let it stand for 18 minutes. (6) Pollen mixing and blending Pollen from healthy mountain yellow peel flowers in the large bud stage was collected and inactivated at 121℃ for 20 minutes to completely destroy the reproductive activity of the pollen and ensure that there was no risk of self-pollination. The inactivated mountain yellow peel homologous pollen was mixed with the yellow peel pollen (heterologous pollen) from the three treatment groups after recovery in step (3) at a mass ratio of 3:7 to obtain three different composite pollen groups.

[0039] (7) Artificial pollination The mother plant of *Cinnamomum camphora* enters its peak pollination window 12 days after it begins to flower (starting on April 6, 2022).

[0040] Apply the composite pollen from step (6) evenly and gently to the stigma of the pretreated *Phellodendron amurense* parent plant. Immediately after pollination, bag the plant again for isolation and mark the pollination date.

[0041] (8) Post-pollination management After pollination, maintain air humidity of 60%–70% in the field and avoid strong sunlight exposure and heavy rain. Remove the isolation bag 7 days after pollination and follow the usual water and fertilizer and pest and disease control procedures.

[0042] Three 7-year-old mother trees of the *Cinnamomum camphora* species with uniform growth were selected for each treatment and artificially pollinated. Three bunches of fruit were selected from each tree in each of the four cardinal directions (east, south, west, and north), for a total of 12 bunches per tree. The fruit set rate was recorded 15 days after pollination, and the data was collected every 15 days. The fruit set rate 30 days after pollination was recorded as the hybridization fruit set rate and recorded in Table 1.

[0043] Table 1 Effects of Different Low-temperature Treatment and Preservation Methods of Male Parent Pollen on Fruit Set Rate

[0044] As can be seen from Table 1, the treatment method of the male parent pollen in Group C can better preserve the pollen viability, and the cross fruit set rate can reach 43.8%. It shows that the three-step gradient cooling can slowly adapt to the low-temperature environment, avoid low-temperature frost damage to pollen, retain the pollen physiological activity to the greatest extent, and make the pollen viability hardly attenuate.

[0045] For the two-step gradient cooling in Group B, after only 24 hours of pretreatment at 4°C, it was directly transferred to -196°C ultra-low temperature preservation without a -20°C transitional cooling stage. Although it can relieve low-temperature damage to a certain extent and the pollen activity is better than that in Group A (the non-gradient cooling group), the cooling transition is insufficient, and there is still slight low-temperature stress damage. Therefore, the cross fruit set rate is 42.1%, and the overall effect is slightly worse than that in Group C.

[0046] This experiment proves that the three-step gradient cooling process of this invention is an important step to maintain the high viability of ultra-low temperature pollen, can effectively solve the problems of low-temperature frost damage and viability attenuation of pollen, and can significantly improve the cross fruit set rate of distant hybridization.

[0047] Example 2 Study on the Influence of Female Parent Stigma Pretreatment Method on Fruit Set Rate A method for germplasm creation of distant hybridization of Clausena lansium includes the following steps: (1) Collection and drying treatment of Clausena lansium male parent pollen The female parent is Clausena lansium cv. Guiyan 20, and the male parent is Clausena lansium cv. Tianhuangpi for hybridization.

[0048] In the middle and late March 2022, which is the full bloom period of Clausena lansium, on a sunny and windless day from 9:00 to 11:00 in the morning, unopened flowers at the large bud stage of the Clausena lansium male parent were collected, and the complete anthers were peeled off; the anthers were placed in a constant temperature and humidity drying oven, controlling the temperature at 22°C and the relative humidity at 35%, and after constant temperature drying for 3 hours, pure pollen was screened to ensure that the pollen viability ≥ 85%.

[0049] (2) Three-step gradient cooling preservation of male parent pollen The qualified Clausena lansium male parent pollen dried in step (1) was sub-packed into sterile cryopreservation tubes, with 2 - 4 g in each cryopreservation tube. The cryopreservation tubes were first refrigerated at 4°C for 24 hours, then frozen at -20°C for 48 hours, and finally stored in a -196°C liquid nitrogen ultra-low temperature environment.

[0050] (3) Recovery of male parent pollen viability Before pollination, the stored male parent pollen was placed in a 4°C environment to recover for 2 hours to restore the pollen viability.

[0051] (4) Removal of stamens and bagging isolation of Clausena lansium female parent flowers Select healthy, disease-free *Cinnamomum camphora* mother plants, choose large buds that are about to open the next day, and manually remove all stamens gently to avoid damaging the stigma. Immediately after removing the stamens, use breathable sulfuric acid paper bags to seal and isolate the plants to prevent external pollen contamination.

[0052] (5) Different pretreatment methods for the stigma of the mother plant Within the same orchard of *Lonicera japonica*, nine 7-year-old mother trees with uniform growth and a relatively high number of flowers were selected, with three trees per treatment group (D, E, and F). Different treatments were administered when the flowers were partially open, the stigmas were fully exposed, and there was no discharge contamination. Specifically: Group D: Blank control group, the column head was moistened by uniform spraying of water. Group E: Use a mixed aqueous solution of 5% sucrose and 1% boric acid to spray and wet the column head evenly, until the column head is completely wet and no water drips. After treatment, let it stand for 10 minutes before proceeding with subsequent artificial pollination. Group F: The column head was uniformly sprayed with a mixed aqueous solution of 5% sucrose and 1% boric acid. After treatment, it was left to stand for 18 minutes before subsequent artificial pollination was carried out. (6) Pollen mixing and blending Collect pollen from healthy mountain yellow peel during the large bud stage, and inactivate it by dry heat at 121℃ for 20 minutes to completely destroy the reproductive activity of the pollen and ensure that there is no risk of self-pollination and fruit setting. Mix the inactivated mountain yellow peel homologous pollen with the yellow peel pollen (heterologous pollen) revived in step (3) at a mass ratio of 3:7 to obtain composite pollen.

[0053] (7) Artificial pollination The peak pollination window period of the mother plant of *Phellodendron amurense* begins 12 days after flowering (starting on April 6, 2022). The compound pollen from step (6) is evenly and gently applied to the stigma of the pretreated mother plant of *Phellodendron amurense*. After pollination, the plant is immediately bagged and isolated, and the pollination date is marked.

[0054] (8) Post-pollination management After pollination, maintain air humidity of 60%–70% in the field and avoid strong sunlight exposure and heavy rain. Remove the isolation bag 7 days after pollination and follow the usual water and fertilizer and pest and disease control procedures.

[0055] Three bunches of fruit were selected from each of the four cardinal directions (east, south, west, and north) of the mother tree for each treatment, for a total of 12 bunches per tree. The fruit set rate was calculated 15 days after pollination, and the calculation was repeated every 15 days. The fruit set rate 30 days after pollination was recorded as the hybridization fruit set rate and recorded in Table 2.

[0056] Table 2. Effects of different pretreatment methods on the fruit set rate of the stigma of the maternal parent plant.

[0057] As can be seen from Table 2, in Group D (without pretreatment), the natural stigma receptivity of Clausena lansium is weak and the heterologous rejection is strong, so the fruit setting rate is relatively low, only 36.2%. In Group E, after a short 10-minute treatment with the pretreatment agent, the pretreatment agent can be infiltrated, but the nutrient supplementation is insufficient and the activation effect of the stigma is limited. Therefore, although the fruit setting rate has increased, it is not obvious, and the fruit setting rate is 39.5%; in Group F, the treatment time for the stigma is 18 minutes, which is sufficient, enabling the stigma to fully absorb the agent, and the activity and affinity are maximally improved, and the fruit setting rate reaches 44.3%.

[0058] Example 3 Research on the Influence of the Ratio of Inactivated Pollen on the Fruit Setting Rate A method for creating germplasm for interspecific distant hybridization of Clausena lansium includes the following steps: (1) Collection and drying treatment of the pollen of the wampee male parent The female parent Clausena lansium: Guiyan No. 20, and the male parent wampee: Sweet Wampee are crossed.

[0059] In the middle and late March 2022, which is the full bloom period of wampee, select a sunny and windless day from 9:00 to 11:00 in the morning, collect the unopened flowers at the big bud stage of the wampee male parent, and strip the complete anthers; place the anthers in a constant temperature and humidity drying oven, control the temperature at 22°C and the relative humidity at 35%, and screen the pure pollen after constant temperature drying for 3 hours to ensure that the pollen viability is ≥ 85%.

[0060] (2) Three-step gradient cooling preservation of the pollen of the male parent Pack the qualified dried pollen of the wampee male parent in step (1) into sterile cryopreservation tubes, with 2 - 4 g in each cryopreservation tube. First, refrigerate the cryopreservation tubes at 4°C for 24 hours, then freeze them at -20°C for 48 hours, and finally store them in a -196°C liquid nitrogen ultra-low temperature environment.

[0061] (3) Resuscitation of the pollen viability of the male parent Before pollination, the stored pollen of the male parent is placed in a 4°C environment for 2 hours to restore the pollen viability.

[0062] (4) Removal of stamens and bagging isolation of the flowers of the Clausena lansium female parent Select strong-growing Clausena lansium female parent plants without diseases and pests, select the big bud stage flowers that will bloom the next day, manually and thoroughly remove all stamens, and perform the emasculation operation gently to avoid damaging the stigma; immediately after emasculation, use a breathable sulfuric acid paper bag to bag and seal for isolation to block the contamination of exogenous pollen.

[0063] (5) Different pretreatment methods for the stigma of the female parent When the flowers of the female parent are half-open, the stigma is completely exposed and there is no secretion contamination, spray and infiltrate the stigma evenly with a mixed aqueous solution of 5% sucrose and 1% boric acid, and continuously treat for 18 minutes; (6) Pollen doping and compounding Collect the pollen of healthy Clausena lansium at the large bud stage, inactivate it at 121 °C by dry heat for 20 min to completely destroy the reproductive activity of the pollen and ensure no risk of self-pollination and fruit setting. Mix the inactivated homologous pollen of Clausena lansium with the resuscitated pollen of Clausena wampi (heterologous pollen) in step (3) evenly at different mass ratios of 1:9, 3:7, and 5:5 respectively to obtain different composite pollen groups G, H, and I.

[0064] (7)Artificial pollination The female parent of Clausena lansium enters the peak pollination window period 12 days after the start of flowering (starting from April 6, 2022). Gently dot the composite pollen in step (6) evenly on the stigmas of the pre-treated female parent of Clausena lansium. Immediately after pollination, bag it again for isolation and mark the pollination date.

[0065] (8)Management after pollination After pollination, keep the air humidity in the field at 60% - 70% to avoid strong light exposure and heavy rain scouring; remove the isolation bag 7 days after pollination and carry out conventional water, fertilizer, and pest control.

[0066] For the experiment, three plants of the 7-year-old female parent Clausena lansium trees with consistent growth were selected for artificial pollination for each treatment. Three clusters of fruits were selected in each of the four directions (east, south, west, and north) of each tree, with a total of 12 clusters per tree. 15 days after pollination, the fruit setting rate was statistically analyzed, and it was statistically analyzed once every 15 days. The fruit setting rate 30 days after pollination was recorded as the hybrid fruit setting rate and recorded in Table 3.

[0067] Table 3 Effects of different doping ratios of inactivated pollen on the fruit setting rate

[0068] As can be seen from Table 3, in the low ratio 1:9 group (Group G), due to the insufficient proportion of inactivated homologous pollen, the effects of stigma barrier breaking and affinity guidance were not sufficient, and the fruit setting rate was 39.6%. The optimal ratio 3:7 group (Group H) achieved the peak fruit setting rate of 43.2% because it balanced the breaking of reproductive isolation and the pollination base of active pollen without self-pollination interference. In the high ratio 5:5 group (Group I), however, due to the too high proportion of inactivated pollen, which diluted the effective pollination quantity of active Clausena wampi pollen, the fruit setting rate decreased to 41.2%.

[0069] Therefore, the best compounding method is to compound the inactivated homologous pollen of Clausena lansium with the pollen of Clausena wampi (heterologous pollen) in a ratio of 3:7.

[0070] Example 4 Fruit setting rate under the optimal experimental conditions A method for creating germplasm of interspecific distant hybridization of Clausena lansium includes the following steps: (1)Collection and drying treatment of pollen of Clausena wampi male parent The female parent of Clausena lansium: Guiyan No. 20, and the male parent of Clausena wampi: Sweet Clausena wampi are hybridized.

[0071] The peak flowering period of Wampee is in mid-to-late March 2023. On a sunny and windless morning between 9:00 and 11:00, unopened flowers of the male parent Wampee during the large bud stage were collected, and the intact anthers were removed. The anthers were placed in a constant temperature and humidity drying oven, with the temperature controlled at 22℃ and the relative humidity at 35%. After drying at a constant temperature for 3 hours, the pure pollen was screened to ensure that the pollen viability was ≥85%.

[0072] (2) Preservation of male parent pollen by three-step gradient cooling The dried pollen from the male parent plant (in step (1)) was aliquoted into sterile cryovials, each containing 2-4g. The cryovials were first refrigerated at 4℃ for 24h, then frozen at -20℃ for 48h, and finally stored in a liquid nitrogen cryogenic environment at -196℃.

[0073] Before pollination, the preserved male pollen is placed in a 4℃ environment for 2 hours to allow the pollen to regain its viability.

[0074] (3) Remove the male flowers of the mother plant of Phellodendron amurense and isolate them by bagging. Select healthy, disease-free *Cinnamomum camphora* mother plants, choose large buds that are about to open the next day, and manually remove all stamens gently to avoid damaging the stigma. Immediately after removing the stamens, use breathable sulfuric acid paper bags to seal and isolate the plants to prevent external pollen contamination.

[0075] (4) Pretreatment of the stigma of the mother plant with pesticide When the flowers of the mother plant are half open, the stigma is fully exposed and there is no secretion contamination, spray the stigma evenly with a mixed aqueous solution of 5% sucrose and 1% boric acid for 18 minutes. (6) Pollen mixing and blending Collect pollen from healthy mountain yellow peel during the large bud stage, and inactivate it by dry heat at 121℃ for 20 minutes to completely destroy the reproductive activity of the pollen and ensure that there is no risk of self-pollination and fruit setting. Mix the inactivated mountain yellow peel homologous pollen with the yellow peel pollen (heterologous pollen) revived in step (3) at a mass ratio of 3:7 to obtain composite pollen.

[0076] (7) Artificial pollination The peak pollination window period of the 7-year-old *Phellodendron amurense* parent plant begins 12 days after flowering (starting on April 7, 2023). The compound pollen from step (6) is evenly and lightly applied to the stigma of the pretreated 7-year-old *Phellodendron amurense* parent plant. After pollination, the plant is immediately bagged and isolated, and the pollination date is marked.

[0077] (8) Post-pollination management After pollination, maintain air humidity of 60%–70% in the field and avoid strong sunlight exposure and heavy rain. Remove the isolation bag 7 days after pollination and follow the usual water and fertilizer and pest and disease control procedures.

[0078] Select three plants from the number of female parents for artificial pollination, and select three clusters of fruits from each of the four directions (east, south, west, and north) of each female tree. Take the fruit setting rate 30 days after pollination as the hybrid fruit setting rate, and the fruit setting rate is 43.8%. Cultivate until the fruit is mature and harvest the hybrid germplasm.

[0079] Example 5 A method for creating germplasm of interspecific distant hybridization of Clausena lansium includes the following steps: (1) Collection and drying treatment of pollen from Clausena lansium male parents The female parent is Clausena lansium: Guiyan No. 20, and the male parent is Clausena lansium: Sweet Clausena lansium for hybridization.

[0080] In the middle and late March 2024, which is the full bloom period of Clausena lansium, select a sunny and windless day from 9:00 to 11:00 in the morning, collect the unopened flowers at the big bud stage of the Clausena lansium male parent, and strip the complete anthers; place the anthers in a constant temperature and humidity drying oven, control the temperature at 22 °C and the relative humidity at 35%, and screen the pure pollen after 3 hours of constant temperature drying, ensuring that the pollen viability ≥ 85%.

[0081] (2) Three-step gradient cooling preservation of male parent pollen Pack the qualified dried pollen of the Clausena lansium male parent in step (1) into sterile cryopreservation tubes, with 2 - 4 g in each cryopreservation tube. First, refrigerate the cryopreservation tubes at 4 °C for 24 hours, then freeze them at -20 °C for 48 hours, and finally store them in a -196 °C liquid nitrogen ultra-low temperature environment.

[0082] Before pollination, the stored male parent pollen is placed at 4 °C for 2 hours to recover the pollen viability.

[0083] (3) Removal of stamens and bagging isolation of Clausena lansium female parent flowers Select healthy Clausena lansium female parent plants without diseases and pests, select the big bud stage flowers that will bloom the next day, manually and thoroughly remove all stamens, and perform the stamen removal operation gently to avoid damaging the stigma; immediately use a breathable sulfuric acid paper bag to bag and seal for isolation to block exogenous pollen pollution.

[0084] (4) Pretreatment of female parent stigma with药剂 When the female parent flowers are semi-open, the stigma is completely exposed and there is no secretion pollution, spray and moisten the stigma evenly with a mixed aqueous solution of 5% sucrose and 1% boric acid, and continue the treatment for 18 minutes; (6) Pollen doping and compounding Collect the pollen of healthy Clausena lansium at the big bud stage, inactivate it at 121 °C by dry heat for 20 minutes to completely destroy the pollen reproductive activity and ensure no risk of self-pollination and fruiting; evenly mix the inactivated homologous pollen of Clausena lansium with the pollen of Clausena lansium (heterologous pollen) recovered in step (3) according to a mass ratio of 3:7 to obtain composite pollen.

[0085] (7) Artificial pollination The Clausena lansium female parent enters the peak pollination window period 12 days after the start of flowering (starting on April 3, 2024). The compound pollen from step (6) is evenly and gently applied to the stigmas of the 7-year-old Clausena lansium female parent after pretreatment. Immediately after pollination, it is bagged and isolated again, and the pollination date is marked.

[0086] (8) Management after pollination After pollination, the air humidity in the field is maintained at 60% - 70%, avoiding strong light exposure and heavy rain washing; the isolation bag is removed 7 days after pollination, and conventional water, fertilizer, and pest control are carried out.

[0087] Select three plants from the number of female parents with artificial pollination, and select three clusters of fruits in each of the four directions (east, south, west, and north) of each female tree. The fruit setting rate 30 days after pollination is recorded as the hybrid fruit setting rate, and the fruit setting rate is 44.2%. It is cultivated until the fruit is mature, and the hybrid germplasm is harvested.

[0088] Example 6 A method for creating germplasm of interspecific distant hybridization of Clausena lansium includes the following steps: (1) Collection and drying treatment of pollen from Clausena lansium male parent The female parent of Clausena lansium: Guiyan 20, and the male parent of Clausena lansium: Sweet Clausena lansium are hybridized.

[0089] In the middle and late March 2025, which is the full bloom period of Clausena lansium, select a sunny and windless day from 9:00 - 11:00 in the morning, collect the unopened flowers at the large bud stage of the Clausena lansium male parent, and strip the complete anthers; place the anthers in a constant temperature and humidity drying oven, control the temperature at 22°C and the relative humidity at 35%, and screen the pure pollen after constant temperature drying for 3 hours, ensuring that the pollen viability ≥ 85%.

[0090] (2) Three-step gradient cooling preservation of pollen from male parent The qualified pollen of the Clausena lansium male parent dried in step (1) is sub-packed into sterile cryopreservation tubes, and each cryopreservation tube is 2 - 4 g. The cryopreservation tube is first refrigerated at 4°C for 24 hours, then frozen at -20°C for 48 hours, and finally stored in a -^196°C liquid nitrogen ultra-low temperature environment.

[0091] Before pollination and use, the stored pollen of the male parent is placed in a 4°C environment to recover for 2 hours to restore the pollen viability.

[0092] (3) Removal of stamens and bagging isolation of flowers of Clausena lansium female parent Select strong and healthy Clausena lansium female parent plants without diseases and pests, select the large bud stage flowers that will bloom the next day, and manually and thoroughly remove all stamens. The operation of removing stamens is gentle to avoid damaging the stigma; immediately after removing stamens, it is sealed and isolated with a breathable sulfuric acid paper bag to block exogenous pollen pollution.

[0093] (4) Pretreatment of female parent stigma with药剂 When the flowers of the mother plant are half open, the stigma is fully exposed and there is no secretion contamination, spray the stigma evenly with a mixed aqueous solution of 5% sucrose and 1% boric acid for 18 minutes. (6) Pollen mixing and blending Collect pollen from healthy mountain yellow peel during the large bud stage, and inactivate it by dry heat at 121℃ for 20 minutes to completely destroy the reproductive activity of the pollen and ensure that there is no risk of self-pollination and fruit setting. Mix the inactivated mountain yellow peel homologous pollen with the yellow peel pollen (heterologous pollen) revived in step (3) at a mass ratio of 3:7 to obtain composite pollen.

[0094] (7) Artificial pollination The peak pollination window period of the 7-year-old *Phellodendron amurense* parent plant begins 12 days after flowering (starting on April 6, 2024). The compound pollen from step (6) is evenly and lightly applied to the stigma of the pretreated 7-year-old *Phellodendron amurense* parent plant. After pollination, the plant is immediately bagged and isolated, and the pollination date is marked.

[0095] (8) Post-pollination management After pollination, maintain air humidity of 60%–70% in the field and avoid strong sunlight exposure and heavy rain. Remove the isolation bag 7 days after pollination and follow the usual water and fertilizer and pest and disease control procedures.

[0096] Three female plants were selected for artificial pollination, and three bunches of fruit were selected from each of the four cardinal directions (east, south, west, and north). The fruit set rate 30 days after pollination was recorded as the hybrid fruit set rate. The fruit set rate was 43.6%. The plants were then cultivated until the fruit matured, and the hybrid germplasm was harvested.

[0097] Example 7 A method for creating germplasm through interspecific distant hybridization of *Phellodendron amurense* includes the following steps: (1) Collection and drying of pollen from the male parent of the yellow-skinned fruit The female parent, *Lonicera japonica* var. *guiyan* 20, and the male parent, *Lonicera japonica* var. *bingtang*, were hybridized. During the peak flowering period of *Lonicera japonica* in mid-to-late March 2024, on a sunny, windless morning between 9:00 and 11:00, unopened flowers of the male parent were collected during the large bud stage, and the intact anthers were removed. The anthers were placed in a constant temperature and humidity drying chamber at 22℃ and 35% relative humidity for 3 hours. After drying, pure pollen was screened, ensuring pollen viability ≥85%.

[0098] (2) Preservation of male parent pollen by three-step gradient cooling The dried pollen from the male parent plant (in step (1)) was aliquoted into sterile cryovials, each containing 2-4g. The cryovials were first refrigerated at 4℃ for 24h, then frozen at -20℃ for 48h, and finally stored in a liquid nitrogen cryogenic environment at -196℃.

[0099] Before pollination, the preserved male pollen is placed in a 4℃ environment for 2 hours to allow the pollen to regain its viability.

[0100] (3) Remove the male flowers of the mother plant of Phellodendron amurense and isolate them by bagging. Select healthy, disease-free *Cinnamomum camphora* mother plants, choose large buds that are about to open the next day, and manually remove all stamens gently to avoid damaging the stigma. Immediately after removing the stamens, use breathable sulfuric acid paper bags to seal and isolate the plants to prevent external pollen contamination.

[0101] (4) Pretreatment of the stigma of the mother plant with pesticide When the flowers of the mother plant are half open, the stigma is fully exposed and there is no secretion contamination, spray the stigma evenly with a mixed aqueous solution of 5% sucrose and 1% boric acid for 18 minutes. (6) Pollen mixing and blending Collect pollen from healthy mountain yellow peel during the large bud stage, and inactivate it by dry heat at 121℃ for 20 minutes to completely destroy the reproductive activity of the pollen and ensure that there is no risk of self-pollination and fruit setting. Mix the inactivated mountain yellow peel homologous pollen with the yellow peel pollen (heterologous pollen) revived in step (3) at a mass ratio of 3:7 to obtain composite pollen.

[0102] (7) Artificial pollination The peak pollination window period of the 7-year-old *Phellodendron amurense* parent plant begins 12 days after flowering (starting on April 6, 2024). The compound pollen from step (6) is evenly and lightly applied to the stigma of the pretreated 7-year-old *Phellodendron amurense* parent plant. After pollination, the plant is immediately bagged and isolated, and the pollination date is marked.

[0103] (8) Post-pollination management After pollination, maintain air humidity of 60%–70% in the field and avoid strong sunlight exposure and heavy rain. Remove the isolation bag 7 days after pollination and follow the usual water and fertilizer and pest and disease control procedures.

[0104] Three female plants were selected for artificial pollination, and three bunches of fruit were selected from each of the four cardinal directions (east, south, west, and north) on each plant. The fruit set rate 30 days after pollination was recorded as the hybrid fruit set rate. The fruit set rate was 43.9%. The plants were then cultivated until the fruit matured, and the hybrid germplasm was harvested.

[0105] Example 8 A method for creating germplasm through interspecific distant hybridization of *Phellodendron amurense* includes the following steps: (1) Collection and drying of pollen from the male parent of the yellow-skinned fruit The female parent, *Cinnamomum camphora* var. *guiyan* 20, and the male parent, *Cinnamomum camphora* var. *tianye*, were hybridized. During the peak flowering period of *Cinnamomum camphora* in mid-to-late March 2025, on a sunny, windless morning between 9:00 and 11:00, unopened flowers of the male parent were collected during the large bud stage, and the intact anthers were removed. The anthers were placed in a constant temperature and humidity drying chamber at 22℃ and 35% relative humidity for 3 hours. After drying, pure pollen was screened, ensuring pollen viability ≥85%.

[0106] (2) Preservation of male parent pollen by three-step gradient cooling The dried pollen from the male parent plant (in step (1)) was aliquoted into sterile cryovials, each containing 2-4g. The cryovials were first refrigerated at 4℃ for 24h, then frozen at -20℃ for 48h, and finally stored in a liquid nitrogen cryogenic environment at -196℃.

[0107] Before pollination, the preserved male pollen is placed in a 4℃ environment for 2 hours to allow the pollen to regain its viability.

[0108] (3) Remove the male flowers of the mother plant of Phellodendron amurense and isolate them by bagging. Select healthy, disease-free *Cinnamomum camphora* mother plants, choose large buds that are about to open the next day, and manually remove all stamens gently to avoid damaging the stigma. Immediately after removing the stamens, use breathable sulfuric acid paper bags to seal and isolate the plants to prevent external pollen contamination.

[0109] (4) Pretreatment of the stigma of the mother plant with pesticide When the flowers of the mother plant are half open, the stigma is fully exposed and there is no secretion contamination, spray the stigma evenly with a mixed aqueous solution of 5% sucrose and 1% boric acid for 18 minutes. (6) Pollen mixing and blending Collect pollen from healthy mountain yellow peel during the large bud stage, and inactivate it by dry heat at 121℃ for 20 minutes to completely destroy the reproductive activity of the pollen and ensure that there is no risk of self-pollination and fruit setting. Mix the inactivated mountain yellow peel homologous pollen with the yellow peel pollen (heterologous pollen) revived in step (3) at a mass ratio of 3:7 to obtain composite pollen.

[0110] (7) Artificial pollination The peak pollination window period of the 7-year-old *Phellodendron amurense* parent plant begins 12 days after flowering (starting on April 7, 2025). The compound pollen from step (6) is evenly and lightly applied to the stigma of the pretreated 7-year-old *Phellodendron amurense* parent plant. After pollination, the plant is immediately bagged and isolated, and the pollination date is marked.

[0111] (8) Post-pollination management After pollination, maintain air humidity of 60%–70% in the field and avoid strong sunlight exposure and heavy rain. Remove the isolation bag 7 days after pollination and follow the usual water and fertilizer and pest and disease control procedures.

[0112] Three female plants were selected for artificial pollination, and three bunches of fruit were selected from each of the four cardinal directions (east, south, west, and north) on each plant. The fruit set rate 30 days after pollination was recorded as the hybrid fruit set rate. The fruit set rate was 43.2%. The plants were then cultivated until the fruit matured, and the hybrid germplasm was harvested.

[0113] Comparative Example 1: Conventional Method Similar to Example 4, hybridization breeding of *Lonicera japonica* was carried out in the same orchard, using a method of timely pollination with fresh pollen from parental lines during the same flowering period. Specifically: (1) Select normal flowering plants of Guiyan No. 20 Mountain Yellow Skin (mother) and Sweet Yellow Skin (father).

[0114] (2) Select the flower buds of the mother plant, the mountain yellow peel, that are about to open, remove the stamens manually, and immediately cover them with bags for isolation.

[0115] (3) Pollen collection: During the flowering period of wampee, fresh pollen is directly picked from open flowers and stored in a cool and ventilated place.

[0116] (4) Artificial pollination: After the flowering of the mountain yellow peel, remove the isolation bag, use a brush to dip fresh yellow peel pollen, and lightly apply it to the stigma for pollination, and then put the bag back on.

[0117] (5) After pollination, maintain air humidity of 60%–70% in the field and avoid strong sunlight exposure and rain. Remove the isolation bag 7 days after pollination and follow the conventional water and fertilizer and pest and disease control. 30 days after pollination, the fruit setting rate is counted. The fruit setting rate is only 1.1%. Continue to cultivate until the fruit matures, but no stable hybrid fruit survives.

[0118] Experimental Example (1) Observe the germination of hybrid seeds Hybrid seeds were obtained from Example 4 and Comparative Example 1. 30 healthy and plump seeds from each example were selected, soaked in warm water, and then sown in a seedbed. The humidity of the seedbed was maintained at 70-90%. Germination was observed and recorded in Table 4 below.

[0119] Table 4 Comparison of germination rates of hybrid seeds obtained by different methods

[0120] As shown in Table 4, the germination rate of the hybrid seeds obtained by the method of the present invention in Example 4 is higher than that of the hybrid seeds obtained in Comparative Example 1. The germination rate after 20 days is 46.7% higher by the method of the present invention than by the traditional method (Comparative Example 1), indicating that the method of the present invention can effectively preserve the viability of the male parent pollen and improve the pollination success rate.

[0121] (2) Observe the growth of hybrid seedlings.

[0122] The germinated hybrid seeds in (1) were cultivated until the plants reached a height of 10-15cm, at which point they were ready for transplanting. However, during the experiment, it was found that the hybrid seeds in Comparative Example 1 could hardly develop normally. After germination, they stopped developing or grew weakly, and no plants survived after transplanting.

[0123] (3) Observe the growth of seedlings of hybrid seeds and common mountain yellow peel seeds obtained by this invention. Hybrid seeds obtained in 2022 from the cross between Sweet Yellow Peel and Guiyan 20 Mountain Yellow Peel, and seeds of Guiyan 20 Mountain Yellow Peel, were used to cultivate seedlings after germination in the spring of 2023. The growth of both types of seedlings was observed. The results are shown in Table 5. Figure 6 As shown.

[0124] Table 5 Comparison of germination rates of hybrid seeds obtained by different methods

[0125] From Table 5 and Figure 6 The comparison shows that the seedlings grown from the hybrid seeds of this invention and those grown from ordinary Guiyan 20 seeds are not significantly different in height. However, the seedlings grown from these hybrid seeds are significantly thicker and grow more vigorously than the seedlings grown from ordinary Guiyan 20 seeds.

Claims

1. A method for creating germplasm through interspecific distant hybridization of *Phellodendron amurense*, characterized in that, Includes the following steps: (1) Collection and drying of male parent pollen The yellow-skinned variety is used as the male parent. On a sunny morning, unopened flowers of the male parent during the large bud stage are collected, and the intact anthers are removed. The anthers are then dried in a drying oven, and the pure pollen is screened. (2) Preservation of male parent pollen by three-step gradient cooling The pollen from step (1) was placed in cryovials, refrigerated at 4 ~ 5℃ for 16 ~ 36 hours, then frozen at -20 ~ -30℃ for 36 ~ 60 hours, and finally stored at -196℃ in an ultra-low temperature environment. (3) Resurgence of male pollen viability Before pollinating the female parent plant, *Coptis chinensis*, place the male parent pollen preserved in step (2) in an environment of 4-5℃ for 2-3 hours to allow the pollen to recover its viability; (4) Male flowers of the mother plant were removed and bagged for isolation. Healthy, disease-free *Cinnamomum camphora* plants were selected as the mother plants. Flowers in the large bud stage that were about to open the next day were selected, and all stamens were completely removed by manual emasculation. After emasculation, the plants were immediately bagged and sealed to prevent contamination by exogenous pollen. (5) Pretreatment of the stigma of the mother plant When the flowers of the mother plant are half-open, the stigma is fully exposed and free of secretions, spray the stigma evenly with a mixed aqueous solution of 5-6% sucrose and 1-2% boric acid. (6) Pollen mixing and blending Collect pollen from healthy *Phellodendron chinense* buds of the same variety, and inactivate it with dry heat at 121℃ for 15-20 minutes to destroy its reproductive activity and obtain inactivated homologous pollen from *Phellodendron chinense*. Mix the inactivated homologous pollen from *Phellodendron chinense* with heterologous pollen from *Phellodendron chinense* after resuscitation in step (3) at a mass ratio of 3:7-5:5 to obtain composite pollen. (7) Artificial pollination During the peak pollination window after flowering, the compound pollen from step (6) was applied to the stigma of the pretreated *Phellodendron chinense* parent plant in step (5). After pollination, an isolation bag was immediately placed on top. (8) Post-pollination management After pollination, maintain an air humidity of 60% to 70% in the field and avoid strong sunlight exposure and heavy rain. Remove the isolation bag 7 to 10 days after pollination, and cultivate the hybrid germplasm according to conventional water and fertilizer management and pest and disease control until the fruit matures.

2. The method for germplasm creation through interspecific distant hybridization of *Phellodendron amurense* according to claim 1, characterized in that, In step (1), the pollen of the male parent is collected on a sunny, windless morning between 9:00 and 11:

00. The drying chamber is controlled at a temperature of 20-25°C and a relative humidity of 30-40% for 2-3 hours.

3. The method for germplasm creation through interspecific distant hybridization of *Phellodendron amurense* according to claim 1, characterized in that, The three-step gradient cooling of the male parent pollen in step (2) involves placing the pollen from step (1) into cryopreservation tubes, refrigerating it at 4 ℃ for 24 h, then freezing it at -20 ℃ for 48 h, and finally storing it in a liquid nitrogen ultra-low temperature environment at -196 ℃.

4. The method for germplasm creation through interspecific distant hybridization of *Phellodendron amurense* according to claim 1, characterized in that, In step (3), the preserved paternal pollen is placed in an environment of 4 ℃ for 2 h to revive.

5. The method for germplasm creation through interspecific distant hybridization of *Phellodendron amurense* according to claim 1, characterized in that, In step (4), the manual removal of male reproductive organs is performed gently to avoid damaging the stigma; the bagging is done using a breathable sulfuric acid paper bag.

6. The method for germplasm creation through interspecific distant hybridization of *Phellodendron amurense* according to claim 1, characterized in that, In step (4), the manual removal of male reproductive organs is performed gently to avoid damaging the stigma; the bagging is done using a breathable sulfuric acid paper bag.

7. The method for germplasm creation through interspecific distant hybridization of *Phellodendron amurense* according to claim 1, characterized in that, In step (6), the inactivated homologous pollen of *Cinnamomum camphora* and the revived heterologous pollen of *Cinnamomum camphora* are mixed uniformly at a mass ratio of 3:

7.

8. The method for germplasm creation through interspecific distant hybridization of *Phellodendron amurense* according to claim 1, characterized in that, In step (6), the peak pollination window period is 10 to 14 days after flowering of the *Phellodendron amurense* parent plant.

Images