A method for preserving sugarcane pollen

Abstract

This invention discloses a method for preserving sugarcane pollen, comprising the following steps: (1) transplanting; (2) collection; (3) drying; (4) pretreatment; (5) freezing; and (6) refrigeration. Immediately after collection, the moisture content of the sugarcane pollen is reduced to below 10% to improve its survival rate. The sugarcane pollen is then refrigerated at 8–10°C, frozen at -25–30°C, and finally preserved at ultra-low temperature. This increases the sugarcane pollen's tolerance, reduces frost damage, and avoids performance changes caused by excessive temperature differences. It also significantly reduces the respiration of the sugarcane pollen, prolongs the preservation time, and maintains stable genetic properties, resulting in good storage performance. When placed in a freezer at -65–70°C for ultra-low temperature preservation, a desiccant is added to the cryovial. Furthermore, the pollen is wrapped in white paper and oil paper. When the sugarcane pollen breaks dormancy or experiences a short power outage during ultra-low temperature preservation, pollen discomfort can be reduced, thereby improving the survival rate and fruit set rate of the sugarcane pollen.

Description

Technical Field

[0001] This invention relates to the field of pollen preservation technology, specifically to a method for preserving sugarcane pollen. Background Technology

[0002] Sugarcane, belonging to the genus *Saccharum*, is a tall, perennial, solid herbaceous plant. It has a robust and well-developed rhizome. The culm is 3-5(-6) meters tall. It is widely cultivated in the tropical southern regions of Taiwan, Fujian, Guangdong, Hainan, Guangxi, Sichuan, and Yunnan. Sugarcane thrives in fertile soil, abundant sunshine, and areas with significant temperature differences between winter and summer. A temperate and tropical crop, sugarcane is the raw material for sugar production and can also be used to extract ethanol as an energy substitute. Sugarcane is rich in sugar and water, and also contains various vitamins, fats, proteins, organic acids, calcium, iron, and other substances highly beneficial to human metabolism. It is primarily used for sugar production. The outer skin is typically purple and green, but red and brown are less common.

[0003] Pollen is a tiny mass of spores in seed plants. Mature pollen grains are actually small gametophytes that produce male gametes. Pollen is produced by the anthers in the stamen and reaches the pistil by various methods, pollinating the ovules. Pollen varies among different plant species. Based on pollen shape, size, symmetry, polarity, the number, structure, and location of germination pores, the structure of the pollen cell walls, and surface sculpting, it is often possible to identify the family and genus, and even the species. The study of pollen morphology provides a basis for classification and identification of fossil pollen in pollen analysis, and also provides valuable data for the study of plant phylogeny. Most pollen grains disperse when mature, becoming single pollen grains. However, some pollen grains are formed by two or more pollen grains adhering together, called compound pollen grains. Many pollen grains combined together, with at least two clumps within a single anther locule, are called pollen clumps. A pollen mass is a collection of pollen grains that are all bound together in one or more chambers. Germplasm resources are the genetic material that parents pass directly to their offspring through reproductive cells or somatic cells, determining their traits. They are the material basis for biological genetic variation and biodiversity, and the genetic material basis for the breeding and improvement of new varieties.

[0004] Sugarcane pollen has a relatively thin cell wall, making it highly susceptible to changes in external climate. Sugarcane pollen loses its viability within an hour of shedding, making its preservation challenging. Currently, sugarcane pollen is mostly preserved using cryopreservation methods. However, direct cryopreservation suffers from low pollen survival rates and decreased pollen viability; furthermore, breaking the dormancy of sugarcane pollen can cause discomfort, further reducing pollen survival and seed setting rates. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for preserving sugarcane pollen that offers advantages such as long preservation time, high survival rate, stable heredity, and high fruit set rate.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for preserving sugarcane pollen, comprising the following steps:

[0007] (1) Transplanting: Select sugarcane plants that have basically completed heading and are about to flower, and transplant them indoors to obtain sugarcane plants ready for harvest;

[0008] (2) Collection: Observe the cracking of the anthers of the sugarcane plant to be collected. When the anthers crack, immediately place a clean piece of white paper under the flower spike and shake the flower spike to make the pollen fall onto the white paper to collect the sugarcane pollen.

[0009] (3) Drying: Remove impurities from the sugarcane pollen to obtain clean sugarcane pollen; send the clean sugarcane pollen into a light-proof drying room and dry it until the moisture content is below 10% to obtain dried pollen;

[0010] (4) Pretreatment: Wrap the dried pollen in white paper to obtain a pollen packet; wrap the pollen packet with a layer of oil paper and tie it up to obtain an oil paper packet; insert the oil paper packet into the cryopreservation tube, seal it, and place it in a refrigerator at a temperature of 8-10℃ for 40-60 minutes. Take it out to obtain a refrigerated pollen cryopreservation tube.

[0011] (5) Freezing: Place the refrigerated pollen cryopreservation tubes in a freezer at a temperature of -25 to -30°C and freeze for 20 to 30 minutes. Remove them to obtain frozen pollen cryopreservation tubes.

[0012] (6) Refrigeration: Add desiccant to the frozen pollen cryovial, seal it, and place the frozen pollen cryovial in a freezer at a temperature of -65 to -70°C for ultra-low temperature storage.

[0013] Further, in step (1), sugarcane plants that have basically completed heading and are about to flower are selected, cut off along the middle and lower part of the sugarcane stalk, brought back indoors, and the sugarcane stalk is inserted into the nutrient solution and placed at an angle to obtain sugarcane plants to be harvested; the nutrient solution is composed of 40-45 parts magnesium sulfate, 15-20 parts ammonium sulfate, 8-10 parts sodium nitrate, 12-15 parts superphosphate, 12-15 parts superphosphate, and 100-130 parts sterile water by weight.

[0014] Furthermore, the nutrient solution in step (1) also includes 0.01 to 0.02 parts by weight of 0.01% brassinolide water-soluble powder.

[0015] Further, in step (2), observe the cracking of the anthers of the sugarcane plant to be harvested, and set a 100-watt incandescent lamp at a position 0.5 to 0.7 meters above the top of the flower spike; when the anthers crack, immediately turn on the incandescent lamp, and at the same time use a clean white paper to place under the flower spike, shake the flower spike to make the pollen fall onto the white paper, and collect the sugarcane pollen.

[0016] Furthermore, after collecting sugarcane pollen in step (3), the sugarcane pollen is immediately passed through a 50-mesh sieve to remove impurities and obtain clean sugarcane pollen; the clean sugarcane pollen is then immediately sent to a light-proof drying room and dried until the moisture content is 7-10% to obtain dried pollen.

[0017] Further, in step (4), a layer of sterile aqueous solution of tea polyphenols with a content of 0.2 g / L is sprayed evenly on both sides of the white paper, and dried to obtain white paper containing tea polyphenols; the dried pollen is wrapped in the white paper containing tea polyphenols and wrapped into a strip to obtain a pollen packet; multiple layers of oil paper are wrapped around the outside of the pollen packet, wrapped into a strip, and tied to obtain an oil paper packet; the oil paper packet is inserted into the cryopreservation tube, sealed, and placed in a refrigerator at a temperature of 8-10℃ for 45-60 minutes, and then taken out to obtain a refrigerated pollen cryopreservation tube.

[0018] Further, in step (5), the refrigerated pollen cryopreservation tube is placed in a freezer at a temperature of -26 to -30°C and frozen for 22 to 30 minutes, then taken out to obtain the frozen pollen cryopreservation tube.

[0019] Further, in step (6), a desiccant is added to the frozen pollen cryopreservation tube, the tube is sealed, and the frozen pollen cryopreservation tube is placed in a freezer at a temperature of -65 to -70°C for ultra-low temperature storage; the desiccant is composed of 55 to 60 parts of anhydrous calcium chloride and 40 to 45 parts of starch by weight.

[0020] Further, the method for breaking dormancy of sugarcane pollen in step (6) is as follows: 1) Place the frozen pollen cryopreservation tube into a constant temperature water bath with a water bath temperature of 30-35℃ and an oscillation speed of 30-40r / min to thaw; 2) After thawing, remove the cryopreservation tube, pour out the desiccant inside the cryopreservation tube, remove the oil paper package, and rinse the oil paper package with sterile water at a temperature of 20-25℃; 3) Dry the oil paper package and take out the sugarcane pollen inside the oil paper package.

[0021] Further, the method for breaking dormancy of sugarcane pollen in step (6) is as follows: 1) Place the frozen pollen cryopreservation tube into a constant temperature water bath with a water bath temperature of 32-35℃ and an oscillation speed of 35-40r / min to thaw; 2) After thawing, remove the cryopreservation tube, pour out the desiccant inside the cryopreservation tube, remove the oil paper package, and rinse the oil paper package with sterile water at a temperature of 22-25℃; 3) Dry the oil paper package and take out the sugarcane pollen inside the oil paper package.

[0022] This invention discloses a method for preserving sugarcane pollen. After collection, the sugarcane pollen is immediately dried to reduce its moisture content to below 10%, significantly improving its survival rate. The pollen is then subjected to sequential refrigeration at 8–10°C, freezing at -25–30°C, and finally ultra-low temperature storage at -65–70°C. This step-by-step cooling increases the pollen's tolerance, reduces frost damage, and avoids performance changes caused by excessive temperature differences. It also significantly reduces pollen respiration, extending storage time while maintaining stable genetic properties, resulting in excellent storage performance. During ultra-low temperature storage at -65–70°C, a desiccant is added to the cryovials, and the pollen is wrapped in white paper and oil paper. This reduces pollen discomfort during short power outages or the breaking of dormancy during ultra-low temperature storage, thereby improving the survival and fruit set rates.

[0023] This invention discloses a method for preserving sugarcane pollen, which can significantly extend the preservation time of sugarcane pollen to over 600 days. Adding an appropriate amount of brassinolide to the nutrient solution regulates nutrient distribution in the sugarcane plant, effectively improving the activity of the collected sugarcane pollen. During pollen collection, an incandescent lamp is turned on, while the pollen is completely shaded during drying. Adjusting the light exposure at different stages of pollen development maximizes the beneficial effects of light and eliminates its adverse effects, effectively enhancing the activity of the collected sugarcane pollen. Wrapping the dried pollen in white paper containing tea polyphenols, which have strong antioxidant properties and inhibit Staphylococcus aureus, Escherichia coli, and Bacillus subtilis, effectively improves the activity of the sugarcane pollen. Detailed Implementation

[0024] The following embodiments can help those skilled in the art to more fully understand the present invention, but should not be construed as limiting the present invention in any way.

[0025] This invention discloses a method for preserving sugarcane pollen. Sugarcane plants that have essentially completed heading and are about to flower are selected. The lower part of the sugarcane stalk is cut off and brought indoors. The sugarcane stalk is then inserted into a nutrient solution and placed at an angle of 30-60 degrees. This tilting facilitates pollen collection; however, the tilt angle should not be too large, as this can lead to instability.

[0026] This invention discloses a method for preserving sugarcane pollen. The nutrient solution, by weight, consists of 40-45 parts magnesium sulfate, 15-20 parts ammonium sulfate, 8-10 parts sodium nitrate, 12-15 parts superphosphate, and 100-130 parts sterile water. Alternatively, the nutrient solution can be prepared by diluting commercially available sugarcane stalk nutrient solution, as long as it meets the normal nutrient requirements of the sugarcane stalk.

[0027] This invention discloses a method for preserving sugarcane pollen, in which an appropriate amount of brassinolide is added to the nutrient solution. Brassinolide is a broad-spectrum and highly effective plant growth regulator with the physiological functions of auxin and cytokinin. It also enhances photosynthesis, regulates nutrient distribution, promotes the transport of carbohydrates from stems and leaves to grains, improves the crop's resistance to adverse external factors, and promotes the growth of weak parts of the plant. Experiments have shown that adding an appropriate amount of brassinolide to the nutrient solution can improve the activity of the collected sugarcane pollen.

[0028] This invention discloses a method for preserving sugarcane pollen. Drying requires complete shading; the drying chamber must be completely opaque to prevent light from adversely affecting the pollen. The drying method can involve using desiccants such as silica gel or calcium chloride to absorb moisture, or direct drying with pollen drying equipment.

[0029] This invention discloses a method for preserving sugarcane pollen. Before adding desiccant to the frozen pollen cryopreservation tube, the oil paper package needs to be sealed. The sealing method is to use waterproof glue to seal it. To avoid the pollen from directly contacting the desiccant and causing adverse effects, a waterproof film layer can be wrapped around the oil paper package, and the waterproof film layer can be heat-sealed at the same time.

[0030] This invention discloses a method for preserving sugarcane pollen. Sugarcane pollen loses its activity after being dispersed for one hour, and its preservation is also quite difficult. It is difficult to treat the pollen with antioxidant components such as tea polyphenols. Therefore, this application specifically uses white paper containing tea polyphenols to wrap the dried pollen, thereby improving the activity of sugarcane pollen.

[0031] This invention discloses a method for preserving sugarcane pollen. In the following embodiments, the method for in vitro pollen culture is as follows: a solid culture method is used, the pH value of the culture medium is 6.0, the dormant sugarcane pollen is shaken onto the surface of the culture medium, and the pollen germination is observed using an optical microscope. This is repeated 3 times. The pollen germination rate = number of germinating pollen / total number of observed pollen x 100%.

[0032] This invention discloses a method for preserving sugarcane pollen, using first-year Guitang 42 sugarcane grown in Wuning District, Nanning as the research object.

[0033] Example 1

[0034] A method for preserving sugarcane pollen includes the following steps:

[0035] (1) Transplanting: Select sugarcane plants that have basically completed heading and are about to flower, cut them off from the middle and lower part of the sugarcane stalk, bring them back indoors, insert the sugarcane stalk into a nutrient solution at a depth of about 35 cm and place them at an angle of 30 degrees to obtain sugarcane plants ready for harvest; the nutrient solution is composed of 0.01 parts of 0.01% brassinolide water-soluble powder, 40 parts of magnesium sulfate, 15 parts of ammonium sulfate, 8 parts of sodium nitrate, 12 parts of superphosphate, 12 parts of superphosphate, and 100 parts of sterile water by weight;

[0036] (2) Collection: Observe the cracking of the anthers of the sugarcane plant to be collected, and set up a 100-watt incandescent lamp 0.5 meters above the top of the flower spike; when the anthers crack, immediately turn on the incandescent lamp, and at the same time place a clean white paper under the flower spike, shake the flower spike to make the pollen fall onto the white paper, and collect the sugarcane pollen.

[0037] (3) Drying: After collecting sugarcane pollen, immediately pass the sugarcane pollen through a 50-mesh sieve to remove impurities and obtain clean sugarcane pollen; immediately send the clean sugarcane pollen into a light-proof drying room and dry it until the moisture content is 7% to obtain dried pollen.

[0038] (4) Pretreatment: First, spray a layer of sterile aqueous solution of tea polyphenols with a content of 0.2 g / L evenly on both sides of white paper, dry it, and obtain white paper containing tea polyphenols; wrap the dried pollen with white paper containing tea polyphenols and wrap it into strips to obtain pollen packets; wrap one or more layers of oil paper on the outside of the pollen packets, wrap them into strips, tie them up, and obtain oil paper packets; insert the oil paper packets into cryovials, seal them, and place them in a refrigerator at a temperature of 8°C for 40 min, take them out, and obtain refrigerated pollen cryovials;

[0039] (5) Freezing: Place the refrigerated pollen cryopreservation tubes in a freezer at -25°C for 20 minutes, then remove them to obtain frozen pollen cryopreservation tubes;

[0040] (6) Cold storage: Add desiccant to the frozen pollen cryopreservation tube, seal it, and place the frozen pollen cryopreservation tube in a freezer at a temperature of -65℃ for ultra-low temperature storage; the desiccant is composed of 55 parts of anhydrous calcium chloride and 40 parts of starch by weight.

[0041] Breaking dormancy: The method for breaking dormancy of sugarcane pollen in ultra-low temperature preservation in step (6) is as follows: 1) Place the frozen pollen cryopreservation tube into a constant temperature water bath device with a water bath temperature of 30℃ and an oscillation speed of 30r / min to thaw; 2) After thawing, take out the cryopreservation tube, pour out the desiccant in the cryopreservation tube, take out the oil paper package, and rinse the oil paper package with sterile water at a temperature of 20℃; 3) Dry the oil paper package and take out the sugarcane pollen inside the oil paper package.

[0042] The in vitro germination rate of sugarcane pollen under different cryogenic storage times in this embodiment was statistically analyzed, and the results are shown in Table 1 below:

[0043] Table 1

[0044] Ultra-low temperature storage time (days) 10 100 300 600 In vitro germination rate (%) 83.5 73.5 55.5 30.5

[0045] Example 2

[0046] A method for preserving sugarcane pollen includes the following steps:

[0047] (1) Transplanting: Select sugarcane plants that have basically completed heading and are about to flower, cut them off from the middle and lower part of the sugarcane stalk, bring them back indoors, insert the sugarcane stalk into a nutrient solution at a depth of about 35 cm and place it at an angle of 60 degrees to obtain sugarcane plants ready for harvest; the nutrient solution is composed of 0.02 parts of 0.01% brassinolide water-soluble powder, 45 parts of magnesium sulfate, 20 parts of ammonium sulfate, 10 parts of sodium nitrate, 15 parts of superphosphate, 15 parts of superphosphate, and 130 parts of sterile water by weight;

[0048] (2) Collection: Observe the cracking of the anthers of the sugarcane plant to be collected, and set up a 100-watt incandescent lamp 0.7 meters above the top of the flower spike; when the anthers crack, immediately turn on the incandescent lamp, and at the same time place a clean white paper under the flower spike, shake the flower spike to make the pollen fall onto the white paper, and collect the sugarcane pollen.

[0049] (3) Drying: After collecting sugarcane pollen, immediately pass the sugarcane pollen through a 50-mesh sieve to remove impurities and obtain clean sugarcane pollen; immediately send the clean sugarcane pollen into a light-proof drying room and dry it until the moisture content is 10% to obtain dried pollen.

[0050] (4) Pretreatment: First, spray a layer of sterile aqueous solution of tea polyphenols with a content of 0.2 g / L evenly on both sides of white paper, and dry it to obtain white paper containing tea polyphenols; wrap the dried pollen in the white paper containing tea polyphenols and wrap it into a strip to obtain a pollen bag; wrap one or more layers of oil paper on the outside of the pollen bag, wrap it into a strip, and tie it to obtain an oil paper bag; insert the oil paper bag into the cryovial, seal it, and place it in a refrigerator at a temperature of 10℃ for 60 min, take it out, and obtain a refrigerated pollen cryovial;

[0051] (5) Freezing: Place the refrigerated pollen cryopreservation tubes in a freezer at -30℃ for 30 minutes, then remove them to obtain frozen pollen cryopreservation tubes;

[0052] (6) Refrigeration: Add desiccant to the frozen pollen cryopreservation tube, seal it, and place the frozen pollen cryopreservation tube in a freezer at a temperature of -70°C for ultra-low temperature storage; the desiccant is composed of 60 parts of anhydrous calcium chloride and 45 parts of starch by weight.

[0053] Breaking dormancy: The method for breaking dormancy of sugarcane pollen in ultra-low temperature preservation in step (6) is as follows: 1) Place the frozen pollen cryopreservation tube into a constant temperature water bath device with a water bath temperature of 35℃ and an oscillation speed of 40r / min to thaw; 2) After thawing, take out the cryopreservation tube, pour out the desiccant in the cryopreservation tube, take out the oil paper package, and rinse the oil paper package with sterile water at a temperature of 25℃; 3) Dry the oil paper package and take out the sugarcane pollen inside the oil paper package.

[0054] The in vitro germination rate of sugarcane pollen under different cryogenic storage times in this embodiment was statistically analyzed, and the results are shown in Table 2 below:

[0055] Table 2

[0056] Ultra-low temperature storage time (days) 10 100 300 600 In vitro germination rate (%) 80.5 71.5 52.5 28.5

[0057] Example 3

[0058] A method for preserving sugarcane pollen includes the following steps:

[0059] (1) Transplanting: Select sugarcane plants that have basically completed heading and are about to flower, cut them off from the middle and lower part of the sugarcane stalk, bring them back indoors, insert the sugarcane stalk into a nutrient solution at a depth of about 35 cm and place them at an angle of 45 degrees to obtain sugarcane plants ready for harvest; the nutrient solution is composed of 0.015 parts of 0.01% brassinolide water-soluble powder, 42 parts of magnesium sulfate, 17 parts of ammonium sulfate, 9 parts of sodium nitrate, 13 parts of superphosphate, 14 parts of superphosphate, and 120 parts of sterile water by weight;

[0060] (2) Collection: Observe the cracking of the anthers of the sugarcane plant to be collected, and set up a 100-watt incandescent lamp 0.6 meters above the top of the flower spike; when the anthers crack, immediately turn on the incandescent lamp, and at the same time place a clean white paper under the flower spike, shake the flower spike to make the pollen fall onto the white paper, and collect the sugarcane pollen.

[0061] (3) Drying: After collecting sugarcane pollen, immediately pass the sugarcane pollen through a 50-mesh sieve to remove impurities and obtain clean sugarcane pollen; immediately send the clean sugarcane pollen into a light-proof drying room and dry it until the moisture content is 9% to obtain dried pollen.

[0062] (4) Pretreatment: First, spray a layer of sterile aqueous solution of tea polyphenol with a content of 0.2 g / L evenly on both sides of white paper, dry it, and obtain white paper containing tea polyphenol; wrap the dried pollen with white paper containing tea polyphenol and wrap it into a strip to obtain pollen bag; wrap one or more layers of oil paper on the outside of the pollen bag, wrap it into a strip, tie it well, and obtain oil paper bag; insert the oil paper bag into the cryopreservation tube, seal it, and place it in a cold storage room at a temperature of 9℃ for 45 min, take it out, and obtain refrigerated pollen cryopreservation tube;

[0063] (5) Freezing: Place the refrigerated pollen cryopreservation tubes in a freezer at -26℃ for 22 minutes, then remove them to obtain frozen pollen cryopreservation tubes;

[0064] (6) Refrigeration: Add desiccant to the frozen pollen cryopreservation tube, seal it, and place the frozen pollen cryopreservation tube in a freezer at a temperature of -68°C for ultra-low temperature storage; the desiccant is composed of 58 parts of anhydrous calcium chloride and 43 parts of starch by weight.

[0065] Breaking dormancy: The method for breaking dormancy of sugarcane pollen in step (6) is as follows: 1) Place the frozen pollen cryopreservation tube into a constant temperature water bath with a water bath temperature of 32℃ and an oscillation speed of 35r / min to thaw; 2) After thawing, take out the cryopreservation tube, pour out the desiccant in the cryopreservation tube, take out the oil paper package, and rinse the oil paper package with sterile water at a temperature of 22℃; 3) Dry the oil paper package and take out the sugarcane pollen inside the oil paper package.

[0066] The in vitro germination rate of sugarcane pollen under different cryogenic storage times in this embodiment was statistically analyzed, and the results are shown in Table 3 below:

[0067] Table 3

[0068] Ultra-low temperature storage time (days) 10 100 300 600 In vitro germination rate (%) 85.5 78.5 57.5 38.5

[0069] Example 4

[0070] A method for preserving sugarcane pollen includes the following steps:

[0071] (1) Transplanting: Select sugarcane plants that have basically completed heading and are about to flower, cut them off from the middle and lower part of the sugarcane stalk, bring them back indoors, insert the sugarcane stalk into a nutrient solution at a depth of about 35 cm and place it at an angle of 45 degrees to obtain sugarcane plants ready for harvest; the nutrient solution is composed of 42 parts magnesium sulfate, 17 parts ammonium sulfate, 9 parts sodium nitrate, 13 parts superphosphate, 14 parts superphosphate and 120 parts sterile water by weight.

[0072] (2) Collection: Observe the cracking of the anthers of the sugarcane plant to be collected, and set up a 100-watt incandescent lamp 0.6 meters above the top of the flower spike; when the anthers crack, immediately turn on the incandescent lamp, and at the same time place a clean white paper under the flower spike, shake the flower spike to make the pollen fall onto the white paper, and collect the sugarcane pollen.

[0073] (3) Drying: After collecting sugarcane pollen, immediately pass the sugarcane pollen through a 50-mesh sieve to remove impurities and obtain clean sugarcane pollen; immediately send the clean sugarcane pollen into a light-proof drying room and dry it until the moisture content is 9% to obtain dried pollen.

[0074] (4) Pretreatment: First, spray a layer of sterile aqueous solution of tea polyphenol with a content of 0.2 g / L evenly on both sides of white paper, dry it, and obtain white paper containing tea polyphenol; wrap the dried pollen with white paper containing tea polyphenol and wrap it into a strip to obtain pollen bag; wrap one or more layers of oil paper on the outside of the pollen bag, wrap it into a strip, tie it well, and obtain oil paper bag; insert the oil paper bag into the cryopreservation tube, seal it, and place it in a cold storage room at a temperature of 9℃ for 45 min, take it out, and obtain refrigerated pollen cryopreservation tube;

[0075] (5) Freezing: Place the refrigerated pollen cryopreservation tubes in a freezer at -26℃ for 22 minutes, then remove them to obtain frozen pollen cryopreservation tubes;

[0076] (6) Refrigeration: Add desiccant to the frozen pollen cryopreservation tube, seal it, and place the frozen pollen cryopreservation tube in a freezer at a temperature of -68°C for ultra-low temperature storage; the desiccant is composed of 58 parts of anhydrous calcium chloride and 43 parts of starch by weight.

[0077] Breaking dormancy: The method for breaking dormancy of sugarcane pollen in step (6) is as follows: 1) Place the frozen pollen cryopreservation tube into a constant temperature water bath with a water bath temperature of 32℃ and an oscillation speed of 35r / min to thaw; 2) After thawing, take out the cryopreservation tube, pour out the desiccant in the cryopreservation tube, take out the oil paper package, and rinse the oil paper package with sterile water at a temperature of 22℃; 3) Dry the oil paper package and take out the sugarcane pollen inside the oil paper package.

[0078] The in vitro germination rate of sugarcane pollen under different cryogenic storage times in this embodiment was statistically analyzed, and the results are shown in Table 4 below:

[0079] Table 4

[0080] Ultra-low temperature storage time (days) 10 100 300 600 In vitro germination rate (%) 75.5 62.5 43.5 19.5

[0081] Example 5

[0082] A method for preserving sugarcane pollen includes the following steps:

[0083] (1) Transplanting: Select sugarcane plants that have basically completed heading and are about to flower, cut them off from the middle and lower part of the sugarcane stalk, bring them back indoors, insert the sugarcane stalk into a nutrient solution at a depth of about 35 cm and place them at an angle of 45 degrees to obtain sugarcane plants ready for harvest; the nutrient solution is composed of 0.015 parts of 0.01% brassinolide water-soluble powder, 42 parts of magnesium sulfate, 17 parts of ammonium sulfate, 9 parts of sodium nitrate, 13 parts of superphosphate, 14 parts of superphosphate, and 120 parts of sterile water by weight;

[0084] (2) Collection: Observe the cracking of the anthers of the sugarcane plant to be collected. When the anthers crack, immediately turn on the incandescent light and place a clean white paper under the flower spike. Shake the flower spike to make the pollen fall onto the white paper and collect the sugarcane pollen.

[0085] (3) Drying: After collecting sugarcane pollen, immediately pass the sugarcane pollen through a 50-mesh sieve to remove impurities and obtain clean sugarcane pollen; immediately send the clean sugarcane pollen into a light-proof drying room and dry it until the moisture content is 9% to obtain dried pollen.

[0086] (4) Pretreatment: First, spray a layer of sterile aqueous solution of tea polyphenol with a content of 0.2 g / L evenly on both sides of white paper, dry it, and obtain white paper containing tea polyphenol; wrap the dried pollen with white paper containing tea polyphenol and wrap it into a strip to obtain pollen bag; wrap one or more layers of oil paper on the outside of the pollen bag, wrap it into a strip, tie it well, and obtain oil paper bag; insert the oil paper bag into the cryopreservation tube, seal it, and place it in a cold storage room at a temperature of 9℃ for 45 min, take it out, and obtain refrigerated pollen cryopreservation tube;

[0087] (5) Freezing: Place the refrigerated pollen cryopreservation tubes in a freezer at -26℃ for 22 minutes, then remove them to obtain frozen pollen cryopreservation tubes;

[0088] (6) Refrigeration: Add desiccant to the frozen pollen cryopreservation tube, seal it, and place the frozen pollen cryopreservation tube in a freezer at a temperature of -68°C for ultra-low temperature storage; the desiccant is composed of 58 parts of anhydrous calcium chloride and 43 parts of starch by weight.

[0089] Breaking dormancy: The method for breaking dormancy of sugarcane pollen in step (6) is as follows: 1) Place the frozen pollen cryopreservation tube into a constant temperature water bath with a water bath temperature of 32℃ and an oscillation speed of 35r / min to thaw; 2) After thawing, take out the cryopreservation tube, pour out the desiccant in the cryopreservation tube, take out the oil paper package, and rinse the oil paper package with sterile water at a temperature of 22℃; 3) Dry the oil paper package and take out the sugarcane pollen inside the oil paper package.

[0090] The in vitro germination rate of sugarcane pollen under different cryogenic storage times in this embodiment was statistically analyzed, and the results are shown in Table 5 below:

[0091] Table 5

[0092] Ultra-low temperature storage time (days) 10 100 300 600 In vitro germination rate (%) 77.5 61.5 42.5 18.5

[0093] Example 6

[0094] A method for preserving sugarcane pollen includes the following steps:

[0095] (1) Transplanting: Select sugarcane plants that have basically completed heading and are about to flower, cut them off from the middle and lower part of the sugarcane stalk, bring them back indoors, insert the sugarcane stalk into a nutrient solution at a depth of about 35 cm and place them at an angle of 45 degrees to obtain sugarcane plants ready for harvest; the nutrient solution is composed of 0.015 parts of 0.01% brassinolide water-soluble powder, 42 parts of magnesium sulfate, 17 parts of ammonium sulfate, 9 parts of sodium nitrate, 13 parts of superphosphate, 14 parts of superphosphate, and 120 parts of sterile water by weight;

[0096] (2) Collection: Observe the cracking of the anthers of the sugarcane plant to be collected, and set up a 100-watt incandescent lamp 0.6 meters above the top of the flower spike; when the anthers crack, immediately turn on the incandescent lamp, and at the same time place a clean white paper under the flower spike, shake the flower spike to make the pollen fall onto the white paper, and collect the sugarcane pollen.

[0097] (3) Drying: After collecting sugarcane pollen, immediately pass the sugarcane pollen through a 50-mesh sieve to remove impurities and obtain clean sugarcane pollen; immediately send the clean sugarcane pollen into a light-proof drying room and dry it until the moisture content is 9% to obtain dried pollen.

[0098] (4) Pretreatment: Wrap the dried pollen in white paper and roll it into a strip to obtain a pollen packet; wrap one or more layers of oil paper on the outside of the pollen packet, roll it into a strip, tie it up, and obtain an oil paper packet; insert the oil paper packet into the cryopreservation tube, seal it, and place it in a refrigerator at 9°C for 45 minutes. Take it out to obtain a refrigerated pollen cryopreservation tube.

[0099] (5) Freezing: Place the refrigerated pollen cryopreservation tubes in a freezer at -26℃ for 22 minutes, then remove them to obtain frozen pollen cryopreservation tubes;

[0100] (6) Refrigeration: Add desiccant to the frozen pollen cryopreservation tube, seal it, and place the frozen pollen cryopreservation tube in a freezer at a temperature of -68°C for ultra-low temperature storage; the desiccant is composed of 58 parts of anhydrous calcium chloride and 43 parts of starch by weight.

[0101] Breaking dormancy: The method for breaking dormancy of sugarcane pollen in step (6) is as follows: 1) Place the frozen pollen cryopreservation tube into a constant temperature water bath with a water bath temperature of 32℃ and an oscillation speed of 35r / min to thaw; 2) After thawing, take out the cryopreservation tube, pour out the desiccant in the cryopreservation tube, take out the oil paper package, and rinse the oil paper package with sterile water at a temperature of 22℃; 3) Dry the oil paper package and take out the sugarcane pollen inside the oil paper package.

[0102] The in vitro germination rate of sugarcane pollen under different cryogenic storage times in this embodiment was statistically analyzed, and the results are shown in Table 6 below:

[0103] Table 6

[0104] Ultra-low temperature storage time (days) 10 100 300 600 In vitro germination rate (%) 70.5 58.5 40.5 15.5

[0105] Comparative Example 1

[0106] A method for preserving sugarcane pollen includes the following steps:

[0107] (1) Transplanting: Select sugarcane plants that have basically completed heading and are about to flower, cut them off from the middle and lower part of the sugarcane stalk, bring them back indoors, insert the sugarcane stalk into a nutrient solution at a depth of about 35 cm and place them at an angle of 45 degrees to obtain sugarcane plants ready for harvest; the nutrient solution is composed of 0.015 parts of 0.01% brassinolide water-soluble powder, 42 parts of magnesium sulfate, 17 parts of ammonium sulfate, 9 parts of sodium nitrate, 13 parts of superphosphate, 14 parts of superphosphate, and 120 parts of sterile water by weight;

[0108] (2) Collection: Observe the cracking of the anthers of the sugarcane plant to be collected, and set up a 100-watt incandescent lamp 0.6 meters above the top of the flower spike; when the anthers crack, immediately turn on the incandescent lamp, and at the same time place a clean white paper under the flower spike, shake the flower spike to make the pollen fall onto the white paper, and collect the sugarcane pollen.

[0109] (3) Drying: After collecting sugarcane pollen, immediately pass the sugarcane pollen through a 50-mesh sieve to remove impurities and obtain clean sugarcane pollen; immediately send the clean sugarcane pollen into a light-proof drying room and dry it until the moisture content is 9% to obtain dried pollen.

[0110] (4) Refrigeration: Add dried pollen to the cryovial, seal it, and place the cryovial in a freezer at a temperature of -68°C for ultra-low temperature storage.

[0111] Breaking dormancy: The method for breaking dormancy of sugarcane pollen in step (6) is as follows: 1) Place the frozen pollen cryopreservation tube into a constant temperature water bath with a water bath temperature of 32℃ and an oscillation speed of 35r / min to thaw; 2) After thawing, take out the cryopreservation tube, pour out the desiccant in the cryopreservation tube, take out the oil paper package, and rinse the oil paper package with sterile water at a temperature of 22℃; 3) Dry the oil paper package and take out the sugarcane pollen inside the oil paper package.

[0112] The in vitro germination rate of sugarcane pollen under different cryogenic storage times in this embodiment was statistically analyzed, and the results are shown in Table 7 below:

[0113] Table 7

[0114] Ultra-low temperature storage time (days) 10 100 300 600 In vitro germination rate (%) 60.5 25.5 -- --

[0115] Comparative Example 2

[0116] A method for preserving sugarcane pollen includes the following steps:

[0117] (1) Transplanting: Select sugarcane plants that have basically completed heading and are about to flower, cut them off from the middle and lower part of the sugarcane stalk, bring them back indoors, insert the sugarcane stalk into a nutrient solution at a depth of about 35 cm and place them at an angle of 45 degrees to obtain sugarcane plants ready for harvest; the nutrient solution is composed of 0.015 parts of 0.01% brassinolide water-soluble powder, 42 parts of magnesium sulfate, 17 parts of ammonium sulfate, 9 parts of sodium nitrate, 13 parts of superphosphate, 14 parts of superphosphate, and 120 parts of sterile water by weight;

[0118] (2) Collection: Observe the cracking of the anthers of the sugarcane plant to be collected, and set up a 100-watt incandescent lamp 0.6 meters above the top of the flower spike; when the anthers crack, immediately turn on the incandescent lamp, and at the same time place a clean white paper under the flower spike, shake the flower spike to make the pollen fall onto the white paper, and collect the sugarcane pollen.

[0119] (3) Drying: After collecting sugarcane pollen, immediately pass the sugarcane pollen through a 50-mesh sieve to remove impurities and obtain clean sugarcane pollen; immediately send the clean sugarcane pollen into a light-proof drying room and dry it until the moisture content is 9% to obtain dried pollen.

[0120] (4) Pretreatment: Add dried pollen to the cryovial, seal it, and place it in a refrigerator at 9°C for 45 minutes. Take it out to obtain the refrigerated pollen cryovial.

[0121] (5) Freezing: Place the refrigerated pollen cryopreservation tubes in a freezer at -26℃ for 22 minutes, then remove them to obtain frozen pollen cryopreservation tubes;

[0122] (6) Refrigeration: Add desiccant to the frozen pollen cryopreservation tube, seal it, and place the frozen pollen cryopreservation tube in a freezer at a temperature of -68°C for ultra-low temperature storage; the desiccant is composed of 58 parts of anhydrous calcium chloride and 43 parts of starch by weight.

[0123] Breaking dormancy: The method for breaking dormancy of sugarcane pollen in step (6) is as follows: 1) Place the frozen pollen cryopreservation tube into a constant temperature water bath with a water bath temperature of 32℃ and an oscillation speed of 35r / min to thaw; 2) After thawing, take out the cryopreservation tube, pour out the desiccant in the cryopreservation tube, take out the oil paper package, and rinse the oil paper package with sterile water at a temperature of 22℃; 3) Dry the oil paper package and take out the sugarcane pollen inside the oil paper package.

[0124] The in vitro germination rate of sugarcane pollen under different cryogenic storage times in this embodiment was statistically analyzed, and the results are shown in Table 8 below:

[0125] Table 8

[0126] Ultra-low temperature storage time (days) 10 100 300 600 In vitro germination rate (%) 63.5 46.5 25.5 --

[0127] Comparative Example 3

[0128] A method for preserving sugarcane pollen includes the following steps:

[0129] (1) Transplanting: Select sugarcane plants that have basically completed heading and are about to flower, cut them off from the middle and lower part of the sugarcane stalk, bring them back indoors, insert the sugarcane stalk into a nutrient solution at a depth of about 35 cm and place them at an angle of 45 degrees to obtain sugarcane plants ready for harvest; the nutrient solution is composed of 0.015 parts of 0.01% brassinolide water-soluble powder, 42 parts of magnesium sulfate, 17 parts of ammonium sulfate, 9 parts of sodium nitrate, 13 parts of superphosphate, 14 parts of superphosphate, and 120 parts of sterile water by weight;

[0130] (2) Collection: Observe the cracking of the anthers of the sugarcane plant to be collected, and set up a 100-watt incandescent lamp 0.6 meters above the top of the flower spike; when the anthers crack, immediately turn on the incandescent lamp, and at the same time place a clean white paper under the flower spike, shake the flower spike to make the pollen fall onto the white paper, and collect the sugarcane pollen.

[0131] (3) Drying: After collecting sugarcane pollen, immediately pass the sugarcane pollen through a 50-mesh sieve to remove impurities and obtain clean sugarcane pollen; immediately send the clean sugarcane pollen into a light-proof drying room and dry it until the moisture content is 9% to obtain dried pollen.

[0132] (4) Pretreatment: First, spray a layer of sterile aqueous solution of tea polyphenol with a content of 0.2 g / L evenly on both sides of white paper, dry it, and obtain white paper containing tea polyphenol; wrap the dried pollen with white paper containing tea polyphenol and wrap it into a strip to obtain pollen bag; wrap one or more layers of oil paper on the outside of the pollen bag, wrap it into a strip, tie it well, and obtain oil paper bag; insert the oil paper bag into the cryopreservation tube, seal it, and place it in a cold storage room at a temperature of 9℃ for 45 min, take it out, and obtain refrigerated pollen cryopreservation tube;

[0133] (5) Freezing: Place the refrigerated pollen cryopreservation tubes in a freezer at -26℃ for 22 minutes, then remove them to obtain frozen pollen cryopreservation tubes;

[0134] (6) Refrigeration: Place the frozen pollen cryopreservation tubes in a freezer at a temperature of -68℃ for ultra-low temperature storage.

[0135] Breaking dormancy: The method for breaking dormancy of sugarcane pollen in step (6) is as follows: 1) Place the frozen pollen cryopreservation tube into a constant temperature water bath with a water bath temperature of 32℃ and an oscillation speed of 35r / min to thaw; 2) After thawing, take out the cryopreservation tube, pour out the desiccant in the cryopreservation tube, take out the oil paper package, and rinse the oil paper package with sterile water at a temperature of 22℃; 3) Dry the oil paper package and take out the sugarcane pollen inside the oil paper package.

[0136] The in vitro germination rate of sugarcane pollen under different cryogenic storage times in this embodiment was statistically analyzed, and the results are shown in Table 9 below:

[0137] Table 9

[0138] Ultra-low temperature storage time (days) 10 100 300 600 In vitro germination rate (%) 65.5 40.5 22.0 --

[0139] Pollination and hybridization experiments were conducted using sugarcane pollen stored at different cryogenic times as described in Example 3. The pollination and seed set results are shown in Table 10 below.

[0140] Table 10

[0141] Ultra-low temperature storage time (days) 10 100 300 600 Number of plants that germinate after self-pollination (plants) 535 341 147 78 Number of plants germinating after hybridization with Guitang 92-66 (in plants) 867 562 389 178 Number of plants germinating after hybridization with YueTang 83-257 (in plants) 1036 857 676 452 Number of plants germinating from cross with Taiwan Sugar 146 754 513 336 190

[0142] As can be seen from the results of in vitro germination rate and pollination and fruit set of sugarcane pollen in Examples 1-6 and Comparative Examples 1-3, the sugarcane pollen preservation method of the present invention involves drying the sugarcane pollen immediately after collection to reduce its moisture content to below 10%, which greatly improves the survival rate of sugarcane pollen. The sugarcane pollen is then subjected to sequential refrigeration at 8-10℃, freezing at -25-30℃, and finally ultra-low temperature storage in a freezer at -65-70℃. This step-by-step cooling increases the tolerance of the sugarcane pollen, reduces the frost damage rate, and avoids performance changes caused by excessive temperature differences. It also significantly reduces the respiration of the sugarcane pollen, prolongs the storage time, and maintains stable genetic properties, resulting in good storage performance. When storing in the freezer at -65-70℃, a desiccant is added to the cryovial, and the pollen is wrapped in white paper and oil paper. When sugarcane pollen is released from dormancy or briefly interrupted during ultra-low temperature preservation, pollen discomfort can be reduced, thereby improving the survival rate and seed setting rate of sugarcane pollen. The sugarcane pollen preservation method of this invention can greatly extend the preservation time of sugarcane pollen, up to 600 days or more. Adding an appropriate amount of brassinolide to the nutrient solution regulates the nutrient distribution of the sugarcane plant, effectively improving the activity of the collected sugarcane pollen. Turning on incandescent lamps during pollen collection and completely blocking out light during pollen drying, adjusting the light exposure at different stages of pollen development, maximizing the beneficial effects of light and eliminating its adverse effects, effectively improves the activity of the collected sugarcane pollen. Wrapping the dried pollen in white paper containing tea polyphenols, which have strong antioxidant properties and inhibit Staphylococcus aureus, Escherichia coli, and Bacillus subtilis, effectively enhances the activity of sugarcane pollen.

[0143] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preserving sugarcane pollen, characterized in that, Includes the following steps: (1) Transplanting: Select sugarcane plants that have basically completed heading and are about to flower, and transplant them indoors to obtain sugarcane plants ready for harvest; (2) Collection: Observe the cracking of the anthers of the sugarcane plant to be collected, and set up a 100-watt incandescent lamp 0.5 to 0.7 meters above the top of the flower spike; when the anthers crack, immediately turn on the incandescent lamp, and at the same time place a clean white paper under the flower spike, shake the flower spike to make the pollen fall onto the white paper, and collect the sugarcane pollen. (3) Drying: Immediately pass the sugarcane pollen through a 50-mesh sieve to remove impurities and obtain clean sugarcane pollen; immediately send the clean sugarcane pollen into a light-proof drying room and dry it until the moisture content is 7-10% to obtain dried pollen. (4) Pretreatment: First, spray a layer of sterile aqueous solution of tea polyphenols with a content of 0.2 g / L evenly on both sides of white paper, and dry it to obtain white paper containing tea polyphenols; wrap the dried pollen with white paper containing tea polyphenols and wrap it into a strip to obtain a pollen bag; wrap multiple layers of oil paper on the outside of the pollen bag, wrap it into a strip, and tie it to obtain an oil paper bag; insert the oil paper bag into the cryovial, seal it, and place it in a cold storage room at a temperature of 8-10℃ for 45-60 min, take it out, and obtain a cold storage pollen cryovial; (5) Freezing: Place the refrigerated pollen cryopreservation tubes in a freezer at a temperature of -25 to -30°C and freeze for 20 to 30 minutes. Remove them to obtain frozen pollen cryopreservation tubes. (6) Refrigeration: Add desiccant to the frozen pollen cryovial, seal it, and place the frozen pollen cryovial in a freezer at a temperature of -65 to -70°C for ultra-low temperature storage; The method for breaking dormancy of sugarcane pollen in step (6) is as follows: 1) Place the frozen pollen cryopreservation tube into a constant temperature water bath with a water bath temperature of 30-35℃ and an oscillation speed of 30-40r / min to thaw; 2) After thawing, remove the cryopreservation tube, pour out the desiccant inside the cryopreservation tube, remove the oil paper package, and rinse the oil paper package with sterile water at a temperature of 20-25℃; 3) Dry the oil paper package and take out the sugarcane pollen inside the oil paper package.

2. The method for preserving sugarcane pollen according to claim 1, characterized in that, In step (5), the refrigerated pollen cryopreservation tubes are placed in a freezer at a temperature of -26 to -30°C and frozen for 22 to 30 minutes. They are then removed to obtain frozen pollen cryopreservation tubes.

3. The method for preserving sugarcane pollen according to claim 1, characterized in that, In step (6), a desiccant is added to the frozen pollen cryopreservation tube, the tube is sealed, and the frozen pollen cryopreservation tube is placed in a freezer at a temperature of -65 to -70°C for ultra-low temperature storage; the desiccant is composed of 55 to 60 parts of anhydrous calcium chloride and 40 to 45 parts of starch by weight.

4. The method for preserving sugarcane pollen according to claim 1, characterized in that, The method for breaking dormancy of sugarcane pollen in step (6) is as follows: 1) Place the frozen pollen cryopreservation tube into a constant temperature water bath with a water bath temperature of 32-35℃ and an oscillation speed of 35-40r / min to thaw; 2) After thawing, remove the cryopreservation tube, pour out the desiccant inside the cryopreservation tube, remove the oil paper package, and rinse the oil paper package with sterile water at a temperature of 22-25℃; 3) Dry the oil paper package and take out the sugarcane pollen inside the oil paper package.