A pollination method for improving the fruit setting rate of walnut hybridization

Abstract

The application discloses a pollination method for improving the fruit setting rate of walnut hybridization, and comprises the following steps: selecting female and male parents with matching flowering periods, and removing male flowers of the female parent; subsequently, a pollination shed is built by using a supporting framework and cooperating with 400-mesh nylon mesh cloth, and the whole tree of the female parent is covered to isolate external pollen; pure pollen is prepared by collecting male inflorescences of the male parent, and then performing shade drying and sieving; during the full-bloom period of the female flowers of the female parent, combined pollination is performed by using an electric pollination gun and a portable fan, the upward airflow generated by the fan is used to blow the pollen from the bottom to the top, and the pollen is uniformly deposited on the stigma. The application effectively solves the problems of the traditional bagging method, such as easy damage to the tree body, low efficiency and uneven pollination, significantly improves the pollen utilization rate and the hybridization fruit setting rate, is simple to operate, and is suitable for popularization of walnut large-scale hybridization breeding.

Description

Technical Field

[0001] This invention relates to the field of walnut hybridization technology, specifically to a pollination method for improving the fruit setting rate of walnut hybrids. Background Technology

[0002] Walnut (Juglans regia L.) is an important oilseed and economic forest tree species in China, ranking among the world's four famous dried fruits. It boasts extremely high nutritional value and health benefits, enjoys high market recognition, and is a crucial pillar industry for increasing the income and wealth of fruit farmers. However, walnuts exhibit dioecious behavior, meaning that the male and female flowers on the same walnut tree bloom at different times, and are mostly self-incompatible. This often leads to mismatched flowering periods and pollination failures, severely impacting yield. Therefore, artificial pollination is not only a key technology for high-yield walnut cultivation but also an indispensable core element in walnut hybridization breeding.

[0003] For walnut breeders, the low fruit set rate after artificial pollination has always been a prominent problem in walnut hybridization breeding. Traditional breeding methods generally use single-flower bagging and individual flower pollination, which are not only cumbersome, labor-intensive, and inefficient, but also prone to causing mechanical damage to branches. In windy weather, pollination bags increase wind resistance, further exacerbating the risk of branch bending and breakage, significantly reducing the hybridization fruit set rate and breeding effectiveness.

[0004] For example, Chinese invention patent application CN202510390117.3 discloses a pollen suspension suitable for liquid pollination of pecans and its preparation method. The pollen suspension comprises the following components by weight percentage: xanthan gum 0.01-0.06%, sucrose 5-25%, boric acid 0.01-0.03%, calcium chloride 0.01-0.03%, pecan pollen 0.85-1.5%, and the balance being water. The pollen suspension provided by this invention can provide nutrients to the pollen, ensure uniform pollen concentration in the droplets, and improve the viability of pecan pollen, reaching up to 97.89%, thereby improving the pollination effect of pecans and providing a practical basis for exploring and experimenting with drone-assisted pollination.

[0005] For example, Chinese utility model patent application CN202422788230.0 discloses a pollination device for the entire crown of thin-shelled pecans, including a pollination drone body for pollinating the entire crown of thin-shelled pecans. A liquid pollen cylinder is installed on the top of the pollination drone body, and a hydraulic pump is installed at the bottom of the liquid pollen cylinder. This utility model uses the pollination drone body as the main body of the entire device to achieve aerial operation. The hydraulic pump at the bottom of the liquid pollen cylinder can extract liquid pollen. The insertion hole at the center of the pollination drone body is connected to the delivery pipe, so that the hydraulic pump can transport liquid pollen through the delivery pipe. A detachable atomizing nozzle installed on the delivery pipe extending to the bottom can atomize and spray the liquid pollen, which is convenient for pollinating the entire crown of thin-shelled pecans. The stirring motor installed at the bottom of the outer wall of the liquid pollen cylinder and the stirring blade connected to its output shaft can stir the liquid pollen in the liquid pollen cylinder to prevent pollen sedimentation.

[0006] For example, Chinese invention patent application number CN201810014798.3 discloses a method for artificial pollination of thin-shelled pecans, including the following steps: S1, pollen collection; S2, selection of pollination time; S3, implementation of the pollination method. In this invention, the artificial pollination method involves scientific pollen collection, selection of the optimal pollination time, and preparation of a pollen solution by weight ratio of pollen:sucrose:boric acid:water = 1:50:0.2:3000. The solution is then sprayed onto the trees using a drone, which enhances pollen viability, enables rapid large-area pollination, reduces the pressure of short pollination time, and improves fruit set rate.

[0007] Existing open pollination technologies are still insufficient to meet breeding needs. Existing technologies require the preparation of special pollen suspensions, which involve many types of reagents and complex preparation processes. Existing technologies use full-crown drone pollination devices, which are expensive and also rely on liquid pollen suspensions. They are only suitable for large-scale field supplementary pollination and cannot achieve the precise and targeted pollination required for hybridization breeding. They are unable to meet the professional needs of walnut targeted hybridization breeding and have extremely limited applications. Based on the above-mentioned technical problems of existing technologies, this invention provides a pollination method to improve the fruit setting rate of walnut hybridization. Summary of the Invention

[0008] To address the aforementioned technical problems in the existing technology, this invention provides a pollination method for improving the fruit setting rate of walnut hybrids.

[0009] The present invention adopts the following technical solution:

[0010] This invention provides a pollination method for improving the fruit setting rate of walnut hybrids, comprising:

[0011] Step 1: Based on the phenological survey data, select female-progenitor female parents and male-progenitor male parents for hybridization to ensure that the male and female flowers bloom at the same time. Before or at the early stage of male flower germination of the female parent, manually remove the male flower buds or male inflorescences of the female parent to reduce nutrient consumption. Avoid damaging the bark of branches and female flower buds during the operation.

[0012] Step 2: Before the female flowers of the mother plant enter their peak flowering period, a pollination shed is built using a supporting framework and 400-mesh non-pollen-transparent nylon netting. The entire mother plant is covered inside the pollination shed to isolate it from external pollen contamination. At the same time, the opening status of the male flowers of the father plant is closely observed, and male inflorescences are collected during the peak flowering period of the male flowers of the father plant.

[0013] Step 3: Lay the collected male inflorescences flat on sulfuric acid paper and place them in a cool and ventilated place to air dry for 1-2 days at an indoor temperature of 20-25℃ and a relative humidity of 40%-60%. After the male inflorescences naturally release pollen, use an 80-120 mesh sieve to remove impurities, collect the pure pollen, put it into a sample bag with built-in silica gel desiccant, label it, and store it in a refrigerator at 4℃ for a short period of time, not exceeding 7 days.

[0014] Step 4: When the female flower of the mother plant is in full bloom, the stigma is in an inverted "V" shape and secretes mucus on its surface, put the pollen into the pollen storage box of the pollination gun, start the pollination gun near the female flower, so that the pollen flies out from the pollen outlet at a uniform wind speed of 0.5~2.0 m / s; turn on the portable lithium battery fan at a position 10~30 cm below the pollen outlet and with a vertical height difference of 5~15 cm, with a wind speed of 1.0~3.0 m / s, and use the wind force to blow the pollen from bottom to top, so that the pollen falls evenly on the stigma of the female flower; keep the horizontal distance between the pollination gun and the female flower 20~50 cm to avoid the pollen outlet being directly facing the stigma and causing pollen to accumulate;

[0015] Step 5: Using traditional manual application of sulfuric acid paper bags and direct pollination with a pollinator as controls, the fruit set rate was investigated 20 days after flowering.

[0016] Furthermore, in step 1, the maternal parent is the female-progenitor variety "Zhipi No. 3" and the paternal parent is the male-progenitor variety "Kuixiang".

[0017] Furthermore, in step 1, the removal of male flowers from the mother plant is done before the male flower buds sprout, using a manual removal method, while protecting the integrity of the female flower buds and the bark of the branches during the operation.

[0018] Furthermore, in step 2, the pollination shed is constructed when the stigma of the female flower of the mother plant has just begun to crack and open; the supporting frame of the pollination shed is a rectangular frame constructed of lightweight metal pipes or bamboo poles, covered with 400-mesh nylon mesh, and the mesh is secured in the slots using spring clips, with the bottom edge buried in soil and compacted or pressed with heavy objects to ensure a sealed and isolated environment.

[0019] Furthermore, in step 2, the male flowers of the male parent are collected when 10% to 30% of the florets in a cluster of male flowers begin to open and release pollen.

[0020] Furthermore, in step 3, the sieving is done using a 100-mesh sieve; the air-drying time is 1.5 days; the sample bag is a self-sealing bag with a built-in color-changing silica gel desiccant, and the pollen moisture content is controlled at 8%~15%.

[0021] Furthermore, in step 4, the pollination gun is a portable electric pollination gun with a pollen storage box capacity of 100 mL; the portable fan is a handheld lithium battery rechargeable fan with a blade diameter of 15~25 cm and three adjustable wind speeds, which should be set to medium to high speed during pollination.

[0022] Furthermore, in step 4, the combined pollination operation time is from 9:00 to 11:00 am, with an air temperature of 18 to 25°C and a relative humidity of 50% to 70%. At this time, the stigma of the walnut female flower secretes abundant mucus and the pollen is most viable. A pollination gun is used for mobile spot spraying in conjunction with a fan to continuously blow pollen, so that the stigma of each female inflorescence is evenly coated with pollen, ensuring that each female flower is evenly pollinated.

[0023] Furthermore, in step 4, for female flowers in the middle and upper parts of the tree with a height exceeding 2.5 m, a pollination gun with an extension rod is used in conjunction with a portable fan to simultaneously increase the pollen dispersion height, so as to achieve uniform pollination of the entire tree from top to bottom or from bottom to top.

[0024] Furthermore, in step 5, the method for investigating the fruit set rate is to randomly select 30 to 50 pollinated female inflorescences, label them, and accurately record the number of female flowers; 20 days after flowering, count the number of fruits and calculate the fruit set rate.

[0025] Compared with the prior art, the superior effects of the present invention are as follows:

[0026] 1. The pollination method for improving the fruit setting rate of walnut hybrids described in this invention covers the entire mother tree at once by building a pollination shed, replacing the tedious work of bagging hundreds or thousands of individual flowers. This whole-tree isolation mode elevates the operation unit from individual flowers to the whole tree, greatly simplifying the hybridization process, freeing breeders from repetitive and meticulous labor, and revolutionizing pollination efficiency.

[0027] 2. The pollination method for improving the fruit setting rate of walnut hybrids described in this invention uses a pollination gun to spray pollen, while simultaneously using a fan to generate an upward airflow below the gun nozzle, so that the pollen forms a uniformly dispersed state in the pollination shed. This simulates the natural wind pollination process of walnuts, avoiding the problem of excessive pollen quantity caused by the pollination gun blowing directly onto the stigma, which would affect germination. This ensures that each female flower can receive an appropriate amount of uniform pollen, significantly improving the pollination success rate and fertilization quality.

[0028] 3. The pollination method for improving the fruit setting rate of walnut hybrids described in this invention achieves strict physical isolation by building a pollination shed, ensuring that all pollinated pollen is artificially introduced from the target male parent pollen, perfectly meeting the stringent requirements of directional hybridization breeding for pure and unmixed parents. Furthermore, through the synergistic effect of the fan and the pollination gun, it overcomes the shortcomings of low efficiency in traditional manual pollination, achieving a balance between precision and efficiency.

[0029] 4. The pollination method for improving the fruit setting rate of walnut hybrids described in this invention does not require hanging a large number of pollination bags on individual branches, fundamentally eliminating the wind resistance hazards caused by bagging. The pollination operation can be completed on the ground or with simple tools, without the need to repeatedly climb and touch the branches, thus maximizing the protection of the mother tree and ensuring the integrity and safety of the breeding materials.

[0030] 5. The pollination method for improving the fruit setting rate of walnut hybrids described in this invention utilizes simple bamboo, wood, or steel structures, nylon mesh, a handheld electric pollination gun, and a portable rechargeable fan. The materials are readily available and inexpensive. Pollen is used directly in dry powder form, eliminating the need for complex liquid preparation. The entire technical solution is simple to understand and easy to operate; ordinary technicians can master it with simple training. It is highly suitable for promotion and application in walnut breeding units and large-scale planting enterprises. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the pollination method for improving the fruit setting rate of walnut hybrids in an embodiment of the present invention;

[0032] Figure 2 This is a schematic diagram of the male flower in full bloom during the pollination method for improving the fruit setting rate of walnut hybrids in this embodiment of the invention.

[0033] Figure 3 This is a schematic diagram of the male flower drying and pollination method for improving the fruit setting rate of walnut hybrids in an embodiment of the present invention.

[0034] Figure 4 This is a schematic diagram of pollen collection in the pollination method for improving the fruit setting rate of walnut hybrids in an embodiment of the present invention;

[0035] Figure 5 This is a schematic diagram of the female flower's state during the greenhouse stage in the pollination method for improving the fruit setting rate of walnut hybrids in this embodiment of the invention.

[0036] Figure 6 This is a schematic diagram of the pollination shed construction in the pollination method for improving the fruit setting rate of walnut hybrids in this embodiment of the invention;

[0037] Figure 7 This is a schematic diagram of the optimal pollination state of female flowers in the pollination method for improving the fruit setting rate of walnut hybrids in an embodiment of the present invention.

[0038] Figure 8 This is a schematic diagram of a pollination gun in the pollination method for improving the fruit setting rate of walnut hybrids in this embodiment of the invention.

[0039] Figure 9 This is a schematic diagram of a portable fan used in a pollination method to improve the fruit setting rate of walnut hybrids in an embodiment of the present invention.

[0040] Figure label:

[0041] 1. 360-degree rotating head; 2. Telescopic rod; 3. Powder output adjustment button; 4. Powder storage box; 5. Gun body; 6. Electric switch; 7. Lithium battery. Detailed Implementation

[0042] To better understand the above-mentioned objectives, features and advantages of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be noted that, unless otherwise specified, the embodiments and features in the embodiments of this application can be combined with each other.

[0043] Example

[0044] In this invention, walnuts are selected as the hybridization breeding target. The female parent is the protist variety "Zhipi No. 3," which has female flowers that bloom first and produces excellent fruit quality, but with a relatively strong astringent taste. The male parent is the promassioideous variety "Kuixiang," which has male flowers that bloom first, produces abundant and vigorous pollen, produces excellent fruit quality, and has a mild astringent taste. Its flowering period coincides well with that of "Zhipi No. 3," and their pollen compatibility is high. The purpose of using these two varieties for hybridization is to construct a hybrid offspring population with different astringent tastes.

[0045] The experimental site was located at the Science and Technology Innovation Base of Changli Fruit Tree Research Institute of Hebei Academy of Agricultural and Forestry Sciences and the National Bohai Rim Region Characteristic Fruit Tree Germplasm Resource Nursery (Changli) Walnut Breeding Base. The trees were 8-12 years old, 3.5-5.0 m tall, with a crown width of 3.0-4.5 m, vigorous growth, and free from serious pests and diseases. The experimental site had an average annual temperature of 11.1℃, an annual precipitation of 550-650 mm, and a frost-free period of 175-185 days. It belonged to the warm temperate semi-humid continental monsoon climate zone. The soil was loamy brown soil with a pH of 7.2-7.8 and an organic matter content of 1.2%-1.8%.

[0046] The pollination greenhouse support frame is made of galvanized steel pipe or bamboo with a diameter of 2.5~3.5 cm; the nylon mesh is 400 mesh with an aperture of approximately 38 μm and a width of 2~4 m, used as the covering material for the pollination greenhouse to isolate foreign pollen; the portable pollination gun is a handheld electric pollination gun with four 100 mL pollen storage boxes; the portable lithium battery fan is handheld with a blade diameter of 15~25 cm, three adjustable speeds, to assist in the upward dispersal of pollen; the pollination gun with an extension rod is 1.2 m long; 80 g / m² of neutral, acid-free tracing paper is used as a mat for drying male inflorescences in the shade; standard sieves, with mesh sizes of 80, 100, and 120, are used for sieving pollen to remove impurities; self-sealing bags are 10 cm × 15 cm, containing color-changing silica gel desiccant for short-term pollen preservation; a portable thermometer and hygrometer, with an accuracy of ±0.5℃ / ±3%RH, monitors environmental conditions; the anemometer has a range of 0~10 m / s and an accuracy of ±0.1 m / s. m / s, used to measure the wind speed of the pollination gun and fan; vernier caliper with an accuracy of 0.02mm, used to measure the morphology of the stigma; stereomicroscope with an accuracy of 40×~160×, used to observe the pollination state of the stigma; digital camera with macro lens, used to record the phenological period and pollination process.

[0047] like Figures 1 to 9 As shown, the pollination method for improving the fruit setting rate of walnut hybrids includes:

[0048] Step 1: Based on the phenological survey data, select female-progenitor female parents and male-progenitor male parents for hybridization to ensure that the male and female flowers bloom at the same time. Before or at the early stage of male flower germination of the female parent, manually remove the male flower buds or male inflorescences of the female parent to reduce nutrient consumption. Avoid damaging the bark of branches and female flower buds during the operation.

[0049] In the year preceding hybridization and in the spring of that year, systematic phenological observations were conducted on all candidate parents within the breeding base. Observations began from bud break, with fixed-point and fixed-time observations conducted daily from 9:00 AM to 11:00 AM, recording the following key phenological stages:

[0050] Male flower phenology: male inflorescence budding stage → inflorescence elongation stage → inflorescence separation stage → early pollen shedding stage, 10% of florets shedding pollen → peak pollen shedding stage, more than 50% of florets shedding pollen → pollen shedding stage;

[0051] Female flower phenology: female flower bud sprouting period → inflorescence elongation period → stigma exposure period → stigma cracking period, stigma begins to open → stigma full bloom period, "inverted V" shape, surface secretes mucus → stigma withering period;

[0052] Through two consecutive years of observation, the overlapping flowering periods of "Zhipi No. 3" and "Kuixiang" were determined. The observation results showed that the stigma of the female flower of "Zhipi No. 3" split open from April 18 to 20, and the stigma was in full bloom from April 21 to 25; the initial pollen shedding of the male flower of "Kuixiang" was from April 18 to 20, and the peak pollen shedding period was from April 20 to 23. The overlapping period of the flowering periods of the two was from April 21 to 23, which fully met the requirements for hybridization pollination.

[0053] Parental selection principles: Select female-progenitor varieties as the female parent and male-progenitor varieties as the male parent to ensure that the flowering periods of male and female flowers coincide; at the same time, consider the genetic distance, combining ability and breeding objectives between the parents;

[0054] The timing for thinning male flower buds is chosen before or at the early stage of male flower bud emergence, from late March to early April, when the local temperature is stable at 8-12℃. At this time, the morphological differences between male and female flower buds are obvious: male flower buds are conical, with very small scales that cannot cover the bud body, and small flowers can be seen protruding inside, growing on two-year-old branches; female flower buds are nearly spherical, plump and smooth, with scales tightly covering the entire bud body, growing at the tip of the current year's branches.

[0055] Method for removing male hormones:

[0056] Tools required: Wear gardening gloves and prepare pruning shears or work by hand;

[0057] Thinning extent: The entire mother tree is thinned of male flowers, leaving no male flowers;

[0058] Key points of operation: Pinch the base of the male flower bud with your thumb and forefinger and break it off to the side and downward, avoiding tearing the bark of the branch;

[0059] Special care should be taken to protect the female flower buds, which are usually located at the top of the branches and have a clear morphological difference from the male flower buds;

[0060] After thinning out the male flowers, inspect the branches to ensure that no male flowers are missed and that no female flower buds are damaged.

[0061] Verification of the effect of male flower thinning: Ten standard branches were randomly selected, and the number of male flower buds before and after male flower thinning was counted. The results showed that the number of male flower buds on a single main branch decreased from an average of 18.5 to 0, and the integrity rate of female flower buds was 100%. After male flower thinning, the tree's nutrients were concentrated to supply the development of female flowers, laying the foundation for improving the fruit setting rate in the future.

[0062] Step 2: Before the female flowers of the mother plant enter their peak flowering period, a pollination shed is built using a supporting framework and 400-mesh non-pollen-transparent nylon netting. The entire mother plant is covered inside the pollination shed to isolate it from external pollen contamination. At the same time, the opening status of the male flowers of the father plant is closely observed, and male inflorescences are collected during the peak flowering period of the male flowers of the father plant.

[0063] The timing of pollination shed construction is crucial to ensuring effective isolation and normal development of female flowers. Based on phenological observations, the shed should be constructed when the stigmas of the female flowers of the parent plant "Zhipi No. 3" are just beginning to crack and open (20% to 30% of the female flower stigmas show tiny cracks and have not yet fully opened). At this time, the female flowers have not yet entered the pollination period, thus avoiding contamination by foreign pollen before pollination. The stigmas are just beginning to develop, and slight touches during the construction of the pollination shed will not damage the mature stigmas. Sufficient time (usually 1 to 2 days) should be allowed to complete the construction and sealing inspection of the shed.

[0064] Risks of building pollination sheds too early: If the shed is built before the female flowers have emerged, poor ventilation inside may lead to delayed development of the female flowers; Risks of building sheds too late: If the stigma has fully bloomed, building the pollination shed at this time may cause damage to the stigma due to vibration during the operation, and there may already be foreign pollen contamination.

[0065] The pollination greenhouse adopts a rectangular frame structure, and the specific design parameters are as follows:

[0066] The height of the canopy should be 1 to 1.5 meters above the tree height to ensure sufficient space between the top of the tree canopy and the canopy roof, preventing branches from touching the netting.

[0067] The top surface of the canopy is 0.8 to 1.2 m longer than the canopy width, completely covering the canopy projection area, with the edges extending outwards to prevent lateral pollen from drifting in;

[0068] For the supporting frame, use galvanized steel pipes with a diameter of 2.5~3.5 cm or bamboo with a diameter of 3~4 cm. Galvanized steel pipes are durable but more expensive, while bamboo is more economical but needs to be replaced every year.

[0069] The spacing between the frames is 3-4 m longitudinally and 2-3 m laterally to ensure that the mesh does not sag after being stretched.

[0070] The mesh is made of 400-mesh nylon mesh with a pore size of about 38 μm. Walnut pollen particles are about 40~44 μm in size, and the 400-mesh mesh can effectively block them.

[0071] The mesh fabric is 2-4 m wide and is spliced ​​according to the perimeter of the shed. The splicing is done with nylon thread or special mesh clips to ensure no gaps. Special spring clips are used to secure the fabric into the specially designed grooves of the shed to ensure that the fabric is tightly held in place.

[0072] The bottom is fixed, and the edge of the net extends outward by 30-50 cm. It is buried in soil for 15-20 cm or weighed down with bricks and stones to prevent the ground airflow from blowing foreign pollen into it from the bottom.

[0073] Steps for building a rectangular shed:

[0074] Positioning and layout: Using the tree trunk as the center, draw lines according to the tree projection and the spacing between trees to determine the boundary of the greenhouse;

[0075] Embedding posts: Embedding posts every 3-4 m along the line, with a soil depth of 40-60 cm, and the above-ground height is determined according to the shed design;

[0076] The structure is a rectangular shed with a row spacing of 3*5m to ensure that the trees are completely covered.

[0077] Longitudinal connection: Longitudinal tie rods are installed along the roof and both sides to enhance overall stability;

[0078] Covering with mesh: Cover the skeleton with 400 mesh nylon mesh, unfold it from the top to both sides, tighten it and fix it in the skeleton groove with snap rings;

[0079] Bottom sealing: Lay the mesh fabric flat on the ground with the edges facing outwards, cover it with soil and compact it or press it with bricks and stones to ensure there are no gaps;

[0080] Inspection and acceptance: Observe from the outside of the shed to check for any damage to the mesh, gaps at the joints, and whether the bottom is sealed; shine a flashlight from inside the shed to check for light leaks and repair them.

[0081] After the pollination greenhouse was built, a portable thermometer and hygrometer were hung inside to monitor the environment regularly. The monitoring results showed that the daytime temperature inside the greenhouse was 3-5°C higher than the outside temperature, while the nighttime temperature was basically the same as the outside temperature. The relative humidity inside the greenhouse was 10-15% higher than the outside temperature. The wind speed inside the greenhouse was more than 80% lower than the outside temperature. This microenvironment is conducive to keeping the stigma of the female flower moist and prolonging the pollination period.

[0082] While the pollination shed is being built, the opening status of the male flowers of the male parent "Kuixiang" is closely observed. The timing for collection is when 10% to 30% of the florets in a cluster of male flowers begin to open and release pollen, that is, from the initial stage to the peak stage of pollen release.

[0083] Select robust male inflorescences from the upper middle part of the male parent tree, facing south or southeast, where the inflorescences are well-developed and have a large amount of pollen; cut them from the base of the inflorescence with pruning shears and place them in breathable collection bags (gauze bags or paper bags); the amount of each bag should not exceed 2 / 3 of the bag's capacity to prevent compression and heat generation; after collection, immediately bring them back to the laboratory for processing, avoiding direct sunlight and high temperatures;

[0084] Step 3: Lay the collected male inflorescences flat on sulfuric acid paper and place them in a cool and ventilated place to air dry for 1-2 days at an indoor temperature of 16-25℃ and a relative humidity of 40%-60%. After the male inflorescences naturally release pollen, use an 80-120 mesh sieve to remove impurities, collect the pure pollen, put it into a sample bag with built-in silica gel desiccant, label it, and store it in a refrigerator at 4℃ for a short period of time, not exceeding 7 days.

[0085] The collected male inflorescences were evenly spread on sulfuric acid paper in a single layer, ensuring good ventilation and controlled air-drying conditions.

[0086] The temperature should be between 16 and 25°C; avoid temperatures exceeding 30°C as this can cause pollen inactivation.

[0087] The relative humidity should be between 40% and 60%. If the humidity is too high, the plant is prone to mold growth and pollen will not be easily released.

[0088] Natural ventilation or a light breeze (0.5~1.0 m / s) should be used to avoid strong winds that could cause pollen loss.

[0089] Keep in a cool, dark place to avoid ultraviolet rays damaging the pollen.

[0090] The air-drying time is usually 1 to 2 days. In this embodiment, the air-drying time is 1.5 days. During the air-drying process, the inflorescence is turned over 1 to 2 times a day to ensure even drying. When the inflorescence turns from yellowish-green to yellowish-brown and the anthers naturally dehisce, and yellow pollen can be seen being released, it can be collected.

[0091] After the male inflorescences have been dried in the shade, the pollen collected on the sulfuric acid paper is placed on a 100-mesh sieve and gently rubbed and shaken to allow the pollen to fall through the sieve into a clean sulfuric acid paper or glass dish below. The material on the sieve is anther shells, filaments and other impurities, which should be discarded. The collected pollen is then passed through a 120-mesh fine sieve to remove small impurities and obtain pure pollen.

[0092] Under a stereomicroscope, pure pollen appears as spherical or ellipsoidal pollen with a diameter of 30-35 μm, a reticulated surface, and a bright yellow color. If the pollen is found to be shriveled, discolored, or moldy, it should be discarded.

[0093] Pack the pure pollen into resealable bags, with each bag containing 15-20 mL. Place a small packet of color-changing silica gel desiccant (2-3 g) inside the bag and seal it tightly. The label should include the variety name, collection date, pollen batch, and storage conditions.

[0094] For short-term storage, store in a refrigerator at 4°C for no more than 7 days. During storage, check the color of the silica gel regularly. If it changes from blue to pink, it indicates that it has lost its moisture-absorbing properties and needs to be replaced or pollen should be used immediately.

[0095] The drying and weighing method was adopted. 1.000 g of pollen was dried at 60℃ to constant weight and the moisture content was calculated. In this embodiment, the moisture content of pollen was controlled at 8%~15%, with an average of 11.2%. This moisture content range can maintain the viability of pollen and prevent it from becoming moldy.

[0096] The in vitro germination method was adopted. An agar medium containing 10% sucrose and 0.01% boric acid was prepared. After inoculation with pollen, the medium was incubated at 25°C for 4-6 hours, and the pollen germination rate was observed under a microscope.

[0097] Step 4: When the female flower of the mother plant is in full bloom, the stigma is in an inverted "V" shape and secretes mucus on its surface, put the pollen into the pollen storage box of the pollination gun. Press the electric button near the female flower to start the pollination gun, so that the pollen flies out from the pollen outlet at a uniform wind speed of 0.5~2.0 m / s. Turn on the portable lithium battery fan at a position 10~30 cm below the pollen outlet and with a vertical height difference of 5~15 cm, with a wind speed of 1.0~3.0 m / s, and use the wind force to blow the pollen from bottom to top, so that the pollen falls evenly on the stigma. Keep the horizontal distance between the pollination gun and the female flower 20~50 cm to avoid the pollen outlet being directly facing the stigma and causing pollen to accumulate.

[0098] Before pollination, observe the condition of the female flowers of the mother plant daily. The best time for pollination is when the following conditions are met simultaneously: the stigma is completely split open, forming a typical "inverted V" or "human" shape; the surface of the stigma is moist and secretes mucus; the temperature is 18~25℃, the relative humidity is 50%~70%, and there is no continuous rainfall; the stigma secretes mucus vigorously between 9:00 and 11:00 am, at which time the condition is optimal.

[0099] Use a portable electric pollination gun with a 100 mL capacity for the pollen storage box 4. Before use, clean the pollen storage box and the pollen outlet pipe to ensure there is no residual pollen or foreign matter. Pour the stored pollen into the pollen storage box 4, filling it to no more than 2 / 3 of its capacity (about 60 mL). Gently tap the box to compact it and avoid clumping. Tighten the pollen storage box 4 onto the pollination gun and check for a seal.

[0100] Use a handheld lithium battery rechargeable fan with a blade diameter of 15~25 cm and three adjustable wind speeds. Fully charge the fan before pollination to ensure continuous operation for at least 2 hours. Set the wind speed to medium to high and measure the wind speed at 15 cm below the pollen outlet (horizontal distance) to confirm that it reaches 1.0~3.0 m / s.

[0101] The pollination operation is carried out by two people: one holds the pollination gun and the other holds the fan;

[0102] The pollinator stands on the outer edge of the tree canopy, with the horizontal distance between the pollination gun and the target female flower maintained at 30-40 cm (20-50 cm is acceptable); the fan operator is positioned below the pollination gun, with the horizontal distance between the fan outlet and the pollination gun outlet at 15-20 cm (10-30 cm is acceptable), and the vertical height difference at 8-12 cm (5-15 cm is acceptable).

[0103] First, turn on the fan. After the airflow stabilizes, turn on the pollination gun to spray pollen. The direction of the fan airflow and the direction of the pollen sprayed by the pollination gun should form an upward angle (about 30°~60°). Use the wind power to lift and diffuse the pollen airflow from bottom to top.

[0104] The pollination gun uses a "moving spot spraying" method, which means spraying briefly (1-2 seconds) at a cluster of female flowers and then moving to the next cluster to avoid pollen accumulation caused by spraying in a fixed position for a long time; the fan blows continuously and follows the movement of the pollination gun to maintain a stable relative position.

[0105] Move the pollen in the order of "from top to bottom, from inside to outside" or "from bottom to top, spiral upward" to ensure that all parts of the canopy are evenly pollinated. For the hidden parts inside the canopy, adjust the angle appropriately and use the airflow of the fan to deliver the pollen.

[0106] The amount of pollen per cluster of female flowers should be such that the stigma surface is evenly covered with a thin layer of pollen, avoiding excessive accumulation, as excessive pollen will inhibit germination and waste pollen resources;

[0107] After pollination is completed, the pollination shed should be kept open for 3-5 days until the stigma turns brown and withers and loses its pollination ability. During this period, the environment inside the shed should be monitored. If the temperature is too high (>30℃), the top part of the netting can be lifted to ventilate and cool down. If the humidity is too high, quicklime can be hung inside the shed to absorb moisture.

[0108] Observe the stigma on the second day after pollination. If there are fresh open female flowers, a second pollination can be performed.

[0109] Step 5: Using traditional manual application of sulfuric acid paper bags and direct pollination with a pollinator as controls, the fruit set rate was investigated 20 days after flowering.

[0110] Comparison between traditional manual application of sulfuric acid paper bags and direct pollination with a pollinator gun:

[0111] Numbered CK, the traditional bagging and direct pollination with a pollinator gun were used, repeated 3 times, with 50 female flowers per repeat;

[0112] Numbered T1, pollination was carried out using a pollination greenhouse and direct pollination gun (without a fan), repeated 3 times, with 50 female flowers per repeat;

[0113] Number T2, pollination shed with pollination gun and fan, repeated 3 times, each repeat with 50 female flowers;

[0114] Control treatment (CK) procedure: Before the stigma of the female flower splits open, cover it with a sulfuric acid paper bag (30 cm × 40 cm) and tie it to the branch to seal the opening; when the female flower is in full bloom, cut a small opening in one corner of the sulfuric acid paper bag with scissors, avoiding the leaves, and spray pollen directly onto the stigma with the pollination gun nozzle. After pollination, fold the small opening twice and staple it closed. Remove the bag after the stigma withers.

[0115] On the day of pollination, 30-50 pollinated female flowers were randomly selected from each treatment and labeled with waterproof plastic tags. The treatment number, pollination date and number of female flowers were recorded. The tags were fixed to the branches at the base of the inflorescence with thin wire.

[0116] Twenty days after flowering (approximately 25-30 days after pollination), the young fruit is about 1.0-1.5 cm in diameter, making it easy to identify, and no natural fruit drop has occurred.

[0117] Survey content: count the number of fruits per flower cluster; calculate the fruit set rate: fruit set rate (%) = (total number of fruits / total number of marked female flowers) × 100%; observe the development status of young fruits and record the condition of deformed and diseased fruits;

[0118] The isolation effect of the pollination greenhouse is significant: the fruit set rate of the pollination greenhouse and direct pollination in T1 was 45.33%, which was 15.33 percentage points higher than the fruit set rate of 30.00% in CK. This indicates that the isolation of the pollination greenhouse avoids competition from foreign pollen, and the microenvironment inside the greenhouse is conducive to keeping the stigma in a receptive state.

[0119] Fan assistance further improves fruit set rate: T2 fruit set rate is 65.45%, which is 20.12 percentage points higher than T1 and 35.45 percentage points higher than CK. The airflow assistance of the fan makes the pollen disperse evenly, avoiding pollen accumulation caused by direct pollination with pollination gun, and significantly improving pollination uniformity and success rate.

[0120] like Figure 8 As shown, the 360-degree rotating head 1 enables all-around, blind-spot-free pollination, adapting to the distribution of female flowers at different angles and heights on walnut trees. It can precisely target the stigma, avoiding pollen waste. In densely planted orchards, it can flexibly bypass foliage obstacles, ensuring uniform pollen coverage and improving pollination success rate. The extension rod adjusts the working distance and height, solving the problem of pollinating female flowers at the top of tall walnut trees. It allows access to high flowers without the need for a ladder, and when shortened, it facilitates close-range, precise operation of lower flowers, reducing operator fatigue from prolonged arm lifting, and is easy to store, carry, and transport. The pollen release adjustment button 3 precisely controls the amount of pollen released, adjusting according to the pollen requirements of different varieties and flowering stages to avoid excessive pollen waste. Walnut pollen is expensive; to prevent insufficient pollination and low fruit set due to insufficient pollen output, the system adapts to different weather conditions (adjusting the size when windy and the size when there is no wind) to ensure that each flower receives an appropriate amount of pollen and improve the uniformity of fruit set; the pollen storage box 4 is used for temporary storage and supply of pollen, keeping the pollen dry and preventing it from becoming inactive due to moisture. The transparent or semi-transparent design makes it easy to observe the remaining amount, and the good sealing reduces the loss of pollen during operation. The capacity is moderate, and a single filling can complete the pollination operation of a certain area. It is easy to disassemble and clean, and prevents cross-contamination of pollen from different varieties; the gun body 5 is the main support and structural integration of the whole machine; the electric switch 6 is used for starting and stopping the pollination operation; and the lithium battery 7 is the power source of the whole machine.

[0121] Furthermore, in step 1, the maternal parent is the female-progenitor variety "Zhipi No. 3" and the paternal parent is the male-progenitor variety "Kuixiang".

[0122] Furthermore, in step 1, the removal of male flowers from the mother plant is done before the male flower buds sprout, and is done manually by breaking them off. During the operation, care should be taken to protect the integrity of the female flower buds and the bark of the branches.

[0123] Furthermore, in step 2, the pollination shed is constructed when the stigma of the female flower of the mother plant has just begun to crack and open; the supporting frame of the pollination shed is a rectangular frame constructed of lightweight metal pipes or bamboo poles, covered with 400-mesh nylon mesh, and the edges of the mesh are buried in soil and compacted or pressed with heavy objects to ensure sealing and isolation.

[0124] Furthermore, in step 2, the male flowers of the male parent are collected when 10% to 30% of the florets in a cluster of male flowers begin to open and release pollen.

[0125] Furthermore, in step 3, the sieving is done using a 100-mesh sieve; the air-drying time is 1.5 days; the sample bag is a self-sealing bag with a built-in color-changing silica gel desiccant, and the pollen moisture content is controlled at 8%~15%.

[0126] Furthermore, in step 4, the pollination gun is a portable electric pollination gun with a pollen storage box capacity of 100 mL; the portable fan is a handheld lithium battery rechargeable fan with a blade diameter of 15~25 cm and three adjustable wind speeds, which should be set to medium to high speed during pollination.

[0127] Furthermore, in step 4, the combined pollination operation time is from 9:00 to 11:00 AM, with an air temperature of 18 to 25°C and a relative humidity of 50% to 70%, when the stigma secretion is vigorous and the pollination activity is at its highest; a moving pollination gun is used in conjunction with a fan to continuously blow pollen to ensure uniform pollination in all directions.

[0128] Furthermore, in step 4, for female flowers in the middle and upper parts of the tree with a height exceeding 2.5 m, a pollination gun with an extension rod is used in conjunction with a portable fan to simultaneously increase the pollen dispersion height, so as to achieve uniform pollination of the entire tree from top to bottom or from bottom to top.

[0129] Furthermore, in step 5, the method for investigating the fruit set rate is to randomly select 30 to 50 pollinated female flowers, label them, and accurately record the number of female flowers; count the number of fruits 20 days after flowering, and calculate the fruit set rate.

[0130] Example 2: Screening of different pollination greenhouse mesh sizes

[0131] To verify the isolation effect of 400-mesh nylon mesh, a comparative experiment was conducted with different mesh sizes:

[0132] Number M1, mesh specification 200 mesh, pore size 75μm, walnut pollen particle size 40~44 μm, pore size > pollen particle size, pollen may be lost;

[0133] Product number M2, mesh specification 300 mesh, pore size 50μm, walnut pollen grain size 40~44 μm, pore size slightly larger than pollen grain size, posing a risk;

[0134] Numbered M3, with a mesh size of 400 mesh and an aperture of 38μm, the walnut pollen grain size is 40~44 μm. The aperture is smaller than the pollen grain size, effectively blocking it.

[0135] Number M4, mesh specification 500 mesh, pore size 25μm, walnut pollen grain size 40~44 μm, pore size is much smaller than pollen grain size, excessive blocking;

[0136] Number CK, no header, naturally authorized for comparison;

[0137] Method for testing the isolation effect: Before formal pollination, spray pollen outside the greenhouse with a pollination gun, place a glass slide in the corresponding position inside the greenhouse, count the number of pollen on the glass slide under a microscope, and see if any pollen falls onto the glass slide.

[0138] Both 400-mesh and 500-mesh netting achieve an isolation efficiency of over 99%, but 500-mesh netting has poor light and air permeability, resulting in a greenhouse temperature 2-3°C higher than 400-mesh netting, which has a slight adverse effect on female flower development. 400-mesh netting effectively isolates pollen while maintaining light and air permeability, making it the best choice. 200-mesh and 300-mesh netting pose a risk of pollen leakage, and the purity of hybridization cannot be guaranteed.

[0139] Example 3: Optimization of pollination gun and fan parameters

[0140] To determine the optimal operating parameters for the pollination gun and fan, an orthogonal experimental design was used, with the factors and levels shown below:

[0141] A: Pollination gun wind speed: 0.5m / s for horizontal level 1, 1.2m / s for horizontal level 2, and 2.0m / s for horizontal level 3;

[0142] B: Fan speed: 1.0m / s for horizontal 1, 2.0m / s for horizontal 2, and 3.0m / s for horizontal 3;

[0143] C: Horizontal distance between pollination gun and fan: 10cm for horizontal 1, 20cm for horizontal 2, and 30cm for horizontal 3;

[0144] D: Vertical height difference between pollination gun and fan: 5cm for horizontal 1, 10cm for horizontal 2, and 15cm for horizontal 3;

[0145] The experimental results are shown in Table 1 below:

[0146] Table 1

[0147]

[0148] Range R-values: R_B(8.9) > R_A(5.2) > R_D(3.8) > R_C(2.1);

[0149] The optimal combination is 5, which is: pollination gun wind speed 1.2 m / s, fan wind speed 2.0 m / s, horizontal distance 30 cm, and vertical height difference 5 cm.

[0150] The optimal combination was validated three times, and the average fruit set rate was 65.7% ± 2.8%, which is close to the optimal experiment number 5 (64.2%) in the orthogonal table, confirming that the parameter optimization results are reliable.

[0151] Example 4: Effects of different tassel thinning periods on fruit set rate

[0152] To verify the technical effectiveness of the male mascarpone removal pretreatment, a comparison was set up at different male mascarpone removal stages, as shown in Table 2 below:

[0153] Table 2

[0154]

[0155] According to Table 2, male flower bud thinning can significantly improve the fruit set rate, and the earlier the thinning is done, the better the effect. The fruit set rate is highest (66.2%) when male flower buds are thinned before bud break (S1), which is 17.7 percentage points higher than that when no male flower buds are thinned (S0). The effect of male flower bud thinning in the early stage of pollen shedding (S4) is not obvious because nutrient consumption has already occurred. This invention prefers to perform male flower bud thinning from before the male flower buds sprout to the early stage of sprouting.

[0156] Example 5: The effect of pollen shelf life on viability

[0157] To determine the optimal short-term preservation period for pollen, a preservation time gradient experiment was conducted, as shown in Table 3 below:

[0158] Table 3

[0159]

[0160] According to Table 3, within 7 days of storage, the pollen germination rate remains above 70% and the fruit setting rate remains above 60%, meeting production needs; after 10 days of storage, pollen viability decreases significantly and the fruit setting rate decreases significantly; this invention limits short-term storage to no more than 7 days to ensure pollen viability.

[0161] Example 6: A comparison between the combination of pollination greenhouse, pollination gun, and fan and a single technology is shown in Table 4 below:

[0162] Table 4

[0163]

[0164] According to Table 4, the pollination greenhouse increased the fruit set rate by 12.6 percentage points and eliminated branch damage; the pollination gun replaced the brush, increasing the operation efficiency by 3.3 times; on the basis of using the pollination gun, the addition of the fan further increased the fruit set rate by 20.2 percentage points. The fan not only improved the uniformity of pollen dispersal, but also optimized the pollination microenvironment of the stigma by simulating natural wind pollination through airflow; the combination of the three produced a synergistic effect, which is not a simple superposition.

[0165] This invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the invention as claimed. The scope of protection of this invention is defined by the appended claims.

Claims

1. A pollination method for improving the fruit setting rate of walnut hybrids, characterized in that, include: Step 1: Based on the phenological survey data, select female-progenitor female parents and male-progenitor male parents for hybridization to ensure that the male and female flowers bloom at the same time. Before or at the early stage of male flower germination of the female parent, manually remove the male flower buds or male inflorescences of the female parent to reduce nutrient consumption. Avoid damaging the bark of branches and female flower buds during the operation. Step 2: Before the female flowers of the mother plant enter their peak flowering period, a pollination shed is built using a supporting framework and 400-mesh non-pollen-transparent nylon netting. The entire mother plant is covered inside the pollination shed to isolate it from external pollen contamination. At the same time, the opening status of the male flowers of the father plant is closely observed, and male inflorescences are collected during the peak flowering period of the male flowers of the father plant. Step 3: Lay the collected male inflorescences flat on sulfuric acid paper and place them in a cool and ventilated place to air dry for 1-2 days at an indoor temperature of 20-25℃ and a relative humidity of 40%-60%. After the male inflorescences naturally release pollen, use an 80-120 mesh sieve to remove impurities, collect the pure pollen, put it into a sample bag with built-in silica gel desiccant, label it, and store it in a refrigerator at 4℃ for a short period of time, not exceeding 7 days. Step 4: When the female flower of the mother plant is in full bloom, the stigma is in an inverted "V" shape and secretes mucus on its surface, put the pollen into the pollen storage box of the pollination gun, start the pollination gun near the female flower, so that the pollen flies out from the pollen outlet at a uniform wind speed of 0.5~2.0 m / s; turn on the portable lithium battery fan at a position 10~30 cm below the pollen outlet of the pollination gun and with a vertical height difference of 5~15 cm, with a wind speed of 1.0~3.0 m / s, and use the wind force to blow the pollen from bottom to top, so that the pollen falls evenly on the stigma of the female flower; keep the horizontal distance between the pollination gun and the female flower 20~50 cm to avoid the pollen outlet being directly facing the stigma and causing pollen to accumulate; Step 5: Using traditional manual application of sulfuric acid paper bags and direct pollination with a pollinator as controls, the fruit set rate was investigated 20 days after flowering.

2. The pollination method for improving the fruit setting rate of walnut hybrids according to claim 1, characterized in that, In step 1, the female parent is the progenitor variety "Zhipi No. 3" and the male parent is the progenitor variety "Kuixiang".

3. The pollination method for improving the fruit setting rate of walnut hybrids according to claim 1, characterized in that, In step 1, the removal of male flowers from the mother plant is done before or after the male flower buds sprout, and is done manually by breaking them off. During the operation, care should be taken to protect the integrity of the female flower buds and the bark of the branches.

4. The pollination method for improving the fruit setting rate of walnut hybrids according to claim 1, characterized in that, In step 2, the pollination shed is built when the stigma of the female flower of the mother plant has just begun to crack and open; the supporting frame of the pollination shed is a rectangular frame built of lightweight metal pipes or bamboo poles, covered with 400-mesh nylon mesh, and the mesh is secured in the slots using spring clips. The bottom edge is buried in soil and compacted or pressed with heavy objects to ensure a sealed and isolated environment.

5. The pollination method for improving the fruit setting rate of walnut hybrids according to claim 1, characterized in that, In step 2, the male flowers of the male parent are collected when 10% to 30% of the florets in a cluster of male flowers begin to open and release pollen.

6. The pollination method for improving the fruit setting rate of walnut hybrids according to claim 1, characterized in that, In step 3, the sieving is done using a 100-mesh sieve; the air-drying time is 1.5 days; the sample bag is a self-sealing bag with a built-in color-changing silica gel desiccant, and the pollen moisture content is controlled at 8%~15%.

7. The pollination method for improving the fruit setting rate of walnut hybrids according to claim 1, characterized in that, In step 4, the pollination gun is a portable electric pollination gun with a pollen storage box capacity of 100 mL; the portable fan is a handheld lithium battery rechargeable fan with a blade diameter of 15~25 cm and three adjustable wind speeds, which should be set to medium to high speed during pollination.

8. The pollination method for improving the fruit setting rate of walnut hybrids according to claim 1, characterized in that, In step 4, the combined pollination operation time is from 9:00 to 11:00 am, with an air temperature of 18 to 25°C and a relative humidity of 50% to 70%. At this time, the stigma of the walnut female flower secretes abundant mucus and the pollen is most viable. A pollination gun is used to spray the pollen in a mobile manner, combined with a fan to continuously blow the pollen, so that the stigma of each female inflorescence is evenly coated with pollen, ensuring that each female flower is evenly pollinated.

9. The pollination method for improving the fruit setting rate of walnut hybrids according to claim 1, characterized in that, In step 4, for female flowers in the middle and upper parts of the tree with a height of more than 2.5 m, a pollination gun with an extension rod is used in conjunction with a portable fan to simultaneously increase the height of pollen dispersal, so as to achieve uniform pollination of the entire tree from top to bottom or from bottom to top.

10. The pollination method for improving the fruit setting rate of walnut hybrids according to claim 1, characterized in that, In step 5, the method for investigating the fruit set rate is to randomly select 30 to 50 pollinated female inflorescences, label them, and accurately record the number of female flowers; count the number of fruits 20 days after flowering, and calculate the fruit set rate.

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