A method for long-term ultra-low temperature storage of fresh dried pepper hybrid pollen

Abstract

The present invention provides a method for ultra-low temperature long-term preservation of fresh dried pepper hybrid pollen, and relates to the technical field of plant or part preservation thereof. The ultra-low temperature long-term preservation method mainly comprises collecting pollen, drying pollen, sealing with inert gas, ice water bath and low temperature cycle pretreatment, step-down precooling, and long-term freezing at minus 80°C. The present invention overcomes the shortcomings of the prior art, can effectively preserve pepper pollen at a temperature of minus 80°C for a long time, and ensures that the thawed pollen has a high activity, thereby improving the effect of pepper pollen, reducing storage costs, and facilitating promotion and use.

Description

Technical Field

[0001] The invention relates to the technical field of preservation of plants or parts thereof, and in particular to an ultra-low temperature long-term preservation method for fresh-eating dry pepper hybrid pollen. Background Art

[0002] Chili pepper is an important vegetable and condiment crop and is widely cultivated in the world. According to statistics, the global chili pepper planting area exceeded 36,800 hectares in 2021, with a total output of 36.29 million tons of green pepper and 4.84 million tons of dried chili pepper. The chili pepper planting area in my country reached 32 million mu, and the cultivated area and output accounted for 40% of the world.

[0003] Pollen is an important substance in plants and the most important substance in breeding the next generation and hybrid breeding. According to statistics, more than 90% of Chinese pepper varieties are hybrids. In the process of pepper hybrid production, the quantity and quality of male pollen are one of the important factors affecting hybrid seeds. However, in the process of pepper hybridization, the flowering period of the two parents may not meet, resulting in the failure of all female flowers to be pollinated or excess male pollen, causing unnecessary waste and insufficient number of hybrid seeds. In addition, in the process of hybrid breeding, two or more parent varieties need to be planted at the same time, which will increase the land area, water and fertilizer usage, and labor costs. If the pollen is preserved for a long time, fresh dried peppers can be hybridized and pollinated anytime and anywhere, which can effectively prevent and control the loss of the male parent of fresh dried peppers. It can reduce the land used for breeding and reduce the breeding cost, and at the same time solve the problem of excess male pollen.

[0004] At present, the ultra-low temperature preservation method for pepper pollen is mainly liquid nitrogen preservation, which is used to solve the problem that the parents of seed production do not meet the flowering period. For example, the "ultra-low temperature preservation of pepper pollen" disclosed in Patent No. CN201110237274.9 achieves long-term vitality preservation of pepper pollen through liquid nitrogen refrigeration. However, the liquid nitrogen freezing operation is very dangerous and the cost of frozen storage is also high, which is not conducive to promotion and use. Summary of the invention

[0005] In view of the shortcomings of the existing technology, the present invention provides a method for ultra-low temperature long-term storage of hybrid pollen of fresh dried peppers, which can effectively preserve pepper pollen for a long time at a temperature of minus 80°C and ensure that the pollen after thawing has high activity, thereby improving the effect of pepper pollen, reducing storage costs, and facilitating promotion and use.

[0006] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0007] A method for ultra-low temperature long-term storage of fresh dried pepper hybrid pollen, the storage method comprising the following steps:

[0008] (1) Pollen collection: collect the flowers of fresh dried pepper, roast them, press and knead them, and separate the pollen for later use;

[0009] (2) Pollen drying: The pollen separated above is placed in a desiccator filled with silica gel and dried for 6-12 hours until the moisture content of the pollen is less than 10% to obtain dry pollen;

[0010] (3) Pollen inert gas treatment: Place the above pollen in a sealed can and use inert gas to replace the air in the can for later use;

[0011] (4) Circular precooling treatment: pre-treat the sealed can in an ice water bath for 2-4 hours, and then refrigerate at 4-6°C for 2-4 hours; repeat this operation three times to obtain preliminary precooled pollen for use;

[0012] (5) Step pre-freezing treatment: After taking out the pollen package from the sealed jar, seal it with a container, freeze it at -20℃ for 4-8h, then cool it to -40℃ and continue freezing for 4-8h, and finally place it in a -80℃ environment for long-term storage.

[0013] Preferably, the fresh dried pepper flowers collected in step (1) are unopened flowers.

[0014] Preferably, the baking method in step (1) is to remove the petals and sepals and then use a 50-100W incandescent lamp to bake for 30-60 minutes.

[0015] Preferably, the inert gas in step (3) is nitrogen.

[0016] Preferably, the container is sealed in step (5) by placing the pollen and silica gel particles in a 2 mL cryotube and tightening the cap, then placing the 2 mL cryotube in a 10 mL cryotube, injecting glycerol into the 10 mL cryotube, and then tightening the cap to seal the 10 mL cryotube.

[0017] Preferably, the pollen is wrapped in two layers of sulfur paper and then in one layer of tin foil, and the amount of silica gel particles added is 1 / 10-2 / 10 of the weight of the pollen.

[0018] Preferably, the pollen after long-term storage includes the following thawing steps before use:

[0019] S1. Pre-thawing treatment: Take out the frozen pollen and the sealed container, put the container into a refrigerator at -20℃ for 10-12h, and then put it in an ice water bath for 4-6h;

[0020] S2. Open the pre-thawed container, take out the pollen inside, rinse with cold water to thaw and rehydrate, and then wait for use.

[0021] Preferably, the temperature of the cold water during the cold water flushing in S2 is 4-15° C., and the flushing time is 0.5-1 h.

[0022] The present invention provides a method for ultra-low temperature long-term storage of fresh dried pepper hybrid pollen, which has the following advantages over the prior art:

[0023] (1) The present invention pre-dries pepper pollen, seals it with inert gas, and then performs ice water bath and 2-4°C cyclic low-temperature pretreatment, which can effectively put the initially dried pollen into a dormant state, inhibit respiration, reduce the reduction in activity during subsequent long-term storage, and ensure the early retention of pollen activity. After pretreatment, the pollen is subjected to step-by-step low-temperature treatment at -20°C and -40°C, and then to low-temperature refrigeration at -80°C, which effectively prevents frostbite of the pollen, ensures a good appearance structure and subsequent germination activity of the pollen, and enables the pollen to be stored at -80°C for one year while still having a high activity, thereby achieving long-term storage of pepper pollen at a relatively low cost. The storage condition at -80°C is safer than liquid nitrogen storage, and can effectively reduce storage costs.

[0024] (2) The present invention collects unopened fresh dried pepper flowers, uses a 50-100W incandescent lamp to bake the powder in a short time, and dries the flowers in a constant temperature box at 25°C-30°C with a silica gel desiccant, thereby controlling the water content of the pollen to less than 10%, thereby protecting the pollen to a great extent. If the water content in the pollen is too high, the pollen will crystallize internally in an ultra-low temperature environment, piercing the pollen cell wall, causing the fresh dried pepper pollen to lose vitality. The fresh dried pepper pollen can be kept active for a long time through subsequent ultra-low temperature storage, and normal hybrid seeds can be obtained. The problem of the parents' flowering period not meeting, as well as the problem of less pollen excess and waste of human and material resources can be solved to a great extent, while ensuring the stable quality of fresh dried pepper hybrid seeds. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is a schematic diagram of the growth and development process of fresh dried pepper flowers;

[0026] Figure 2 This is a schematic diagram of the microscopic structure of pollen in the control group in the embodiment of the present invention;

[0027] Figure 3 This is a schematic diagram of the pollen microstructure of Experimental Group 1 in the embodiment of the present invention;

[0028] Figure 4 This is a schematic diagram of the microscopic structure of pollen in experimental group 2 in the embodiment of the present invention;

[0029] Figure 5 This is a schematic diagram of the microstructure of pollen in experimental group 3 in the embodiment of the present invention;

[0030] Figure 6This is a schematic diagram of the pollen microstructure of Experimental Group 7 in the embodiment of the present invention;

[0031] Figure 7 This is a schematic diagram of the appearance of seeds harvested from the pollen of the control group and the experimental groups 1-7 after the hybridization experiment in the embodiment of the present invention;

[0032] Figure 8 This is a schematic diagram of the germination of seeds harvested 3d and 5d after the hybridization experiment of the control group and the experimental groups 1-7 pollen in the embodiment of the present invention;

[0033] Fig. 9 The present invention is a flow chart of the method for ultra-low temperature long-term storage of fresh dried pepper hybrid pollen. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. Embodiment 1:

[0035] Experiment on long-term ultra-low temperature storage of fresh dried pepper hybrid pollen:

[0036] 1. Sample acquisition of the experimental group:

[0037] (1) The fresh dried pepper varieties grown at the planting base of the Institute of Ecological Agriculture in the Hot Zone of Yuanmou County, Chuxiong Yi Autonomous Prefecture, Yunnan Province (25°69′N, 101°87′E, 1120.90m above sea level) were used as samples. The female parent was numbered 7A and the male parent was numbered 7B-1.

[0038] (2) Collect the male pepper flowers that have not fully opened in November 2022; Figure 1 As shown, the flowers of fresh dried peppers were not open during the 5th collection period;

[0039] 2. Sample pre-drying treatment:

[0040] Remove the petals and sepals, bake them under a 100W incandescent lamp for 60 minutes, and gently rub them to separate the pollen (take a portion of the pollen as a control group and do not do the following treatment);

[0041] The collected pollen was placed in a thermostatic box filled with silica gel and dried for 12 hours (25°C) until the moisture content of the pollen was less than 10%, and the dried pollen was used for later use;

[0042] 3. Low temperature frozen storage:

[0043] The dried pollen was divided into 7 parts, which were designated as experimental groups 1-7, and each group was frozen as follows:

[0044] 3.1 Experimental Group 1:

[0045] (1) Place the pollen in a sealed jar and use nitrogen to replace the air in the jar for later use;

[0046] (2) Pre-treat the sealed can in an ice water bath for 3 h, then place it in a refrigerator at 4°C for 3 h, and repeat the ice water bath and 4°C refrigeration process 3 times to complete the initial refrigeration;

[0047] (3) Select a 2 mL small cryotube and a 10 mL large cryotube for later use;

[0048] (4) Take out the pollen from the sealed jar, wrap it with 2 layers of sulfur paper, then wrap it with tin foil, place it in a small cryotube, and put silica gel particles 1 / 10 of the weight of the pollen in the small cryotube, tighten the tube cover, place the small cryotube in the large cryotube, fill it with glycerol, and tighten the tube cover to seal it;

[0049] (5) Freeze the sealed large cryovial at -20°C for 6 hours, then cool it to -40°C and continue freezing for 6 hours, and then place it in a -80°C environment for long-term storage.

[0050] 3.2 Experimental Group 2:

[0051] (1) Place the pollen in a sealed jar and use nitrogen to replace the air in the jar for later use;

[0052] (2) Pre-treat the sealed can in an ice water bath for 3 h, and then place it in a refrigerator at 4°C for 3 h to complete the initial refrigeration;

[0053] (3) Select a 2 mL small cryotube and a 10 mL large cryotube for later use;

[0054] (4) Take out the pollen from the sealed jar, wrap it with 2 layers of sulfur paper, then wrap it with tin foil, place it in a small cryotube, and put silica gel particles 1 / 10 of the weight of the pollen in the small cryotube, tighten the tube cover, place the small cryotube in the large cryotube, fill it with glycerol, and tighten the tube cover to seal it;

[0055] (5) Freeze the sealed large cryovial at -20°C for 6 hours, then cool it to -40°C and continue freezing for 6 hours, and then place it in a -80°C environment for long-term storage.

[0056] 3.3 Experimental Group 3:

[0057] (1) Place the pollen in a sealed jar, pre-treat it in an ice water bath for 3 hours, then place it in a refrigerator at 4°C for 3 hours, and repeat the ice water bath and 4°C refrigeration process 3 times to complete the initial refrigeration;

[0058] (2) Select a 2 mL small cryotube and a 10 mL large cryotube for later use;

[0059] (3) Take out the pollen from the sealed jar, wrap it with 2 layers of sulfur paper, then wrap it with tin foil, place it in a small cryotube, and put silica gel particles 1 / 10 of the weight of the pollen in the small cryotube, tighten the tube cover, place the small cryotube in the large cryotube, fill it with glycerin, and tighten the tube cover to seal it;

[0060] (4) Freeze the sealed large cryovial at -20°C for 6 hours, then cool it to -40°C and continue freezing for 6 hours, and then place it in a -80°C environment for long-term storage.

[0061] 3.4 Experimental Group 4:

[0062] (1) Select a 2 mL small cryotube and a 10 mL large cryotube for later use;

[0063] (2) Wrap the dried fresh pepper pollen with two layers of sulfur paper, then wrap it with tin foil, place it in a small cryovial, and put silica gel particles 1 / 10 of the pollen weight in the small cryovial, tighten the tube cap, place the small cryovial in a large cryovial, fill it with glycerol, and tighten the tube cap to seal it;

[0064] (3) Freeze the large cryovial at -20°C for 6 hours, then cool it to -40°C for another 6 hours, and then store it at -80°C.

[0065] 3.5 Experimental Group 5:

[0066] (1) Place the pollen in a sealed jar and use nitrogen to replace the air in the jar for later use;

[0067] (2) Pre-treat the sealed can in an ice water bath for 3 h, then place it in a refrigerator at 4°C for 3 h, and repeat the ice water bath and 4°C refrigeration process 3 times to complete the initial refrigeration;

[0068] (3) Select a 2 mL small cryotube and a 10 mL large cryotube for later use;

[0069] (4) Take out the pollen from the sealed jar, wrap it with 2 layers of sulfur paper, then wrap it with tin foil, place it in a small cryotube, and put silica gel particles 1 / 10 of the weight of the pollen in the small cryotube, tighten the tube cover, place the small cryotube in the large cryotube, fill it with glycerol, and tighten the tube cover to seal it;

[0070] (5) Freeze the sealed large cryovial at -20°C for 12 hours and then store it at -80°C.

[0071] 3.6 Experimental Group 6:

[0072] (1) Place the pollen in a sealed jar and use nitrogen to replace the air in the jar for later use;

[0073] (2) Pre-treat the sealed can in an ice water bath for 3 h, then place it in a refrigerator at 4°C for 3 h, and repeat the ice water bath and 4°C refrigeration process 3 times to complete the initial refrigeration;

[0074] (3) Select a 2 mL small cryotube and a 10 mL large cryotube for later use;

[0075] (4) Take out the pollen from the sealed jar, wrap it with 2 layers of sulfur paper, then wrap it with tin foil, place it in a small cryotube, and put silica gel particles 1 / 10 of the weight of the pollen in the small cryotube, tighten the tube cover, place the small cryotube in the large cryotube, fill it with glycerol, and tighten the tube cover to seal it;

[0076] (5) Place the sealed large cryovial at -40°C for 12 hours, then store at -80°C.

[0077] 3.7 Experimental Group 7:

[0078] (1) Select a 2 mL small cryotube and a 10 mL large cryotube for later use;

[0079] (2) Wrap the dried fresh pepper pollen with two layers of sulfur paper, then wrap it with tin foil, place it in a small cryovial, and put silica gel particles 1 / 10 of the pollen weight in the small cryovial, tighten the tube cap, place the small cryovial in a large cryovial, fill it with glycerol, and tighten the tube cap to seal it;

[0080] (3) Freeze the large cryovial at -20°C for 6 hours, then cool to -40°C for another 6 hours, and then store in liquid nitrogen.

[0081] 4. Thawing and recovery:

[0082] (1) Take out the frozen pollen and cryotubes, place the cryotubes in a -20°C refrigerator for 12 hours, and then place them in an ice water bath for another 5 hours;

[0083] (2) Open the pre-thawed cryotube, take out the pollen inside, rinse and thaw it with cold water at 4-15°C and rehydrate it for 40 minutes to obtain thawed and revived pollen.

[0084] 5. Testing:

[0085] 5.1. The pollens of experimental groups 1, 2, 3 and 7 and the untreated control group were thawed and observed under a microscope. Figure 2-6 As shown, Figure 2 The pollen of the control group, Figure 3 The pollen of experimental group 1, Figure 4 The pollen of experimental group 2, Figure 5 The pollen of experimental group 3, Figure 6 This is the pollen of experimental group 7. As can be seen from the figure, the pollen structure of experimental group 1 is closest to that of the control group.

[0086] 5.2. The pollen of the experimental group 1-7 stored for one year was thawed and revived according to the above method and the fresh pollen of the control group was used to conduct a cross-pollination experiment on the female parent 7A. The cross-pollination rate, number of seeds per fruit, thousand-grain weight, germination rate and germination index of each group were statistically tested. The specific results are shown in Table 1 below:

[0087] Table 1

[0088]

[0089] And the seeds collected after the pollen hybridization of each group are as follows Figure 7 As shown, the seeds after hybridization of pollen from experimental group 1 were most similar to those from the control group.

[0090] The seeds harvested after pollen hybridization in each group were germinated. The growth morphology at 3d and 5d was as follows: Figure 8 As shown, experimental group 1, experimental group 2, experimental group 5 and the control group all had good germination morphology. Overall, the freezing preservation effect of experimental group 1 was the best.

[0091] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for long-term ultra-low temperature storage of fresh dried pepper hybrid pollen, characterized in that: The storage method comprises the following steps: (1) Pollen collection: collect the flowers of fresh dried pepper, roast them, press and knead them, and separate the pollen for later use; (2) Pollen drying: The pollen separated above is placed in a desiccator filled with silica gel and dried for 6-12 hours until the moisture content of the pollen is less than 10% to obtain dry pollen; (3) Pollen inert gas treatment: Place the above pollen in a sealed can and use inert gas to replace the air in the can for later use; (4) Circular precooling treatment: pre-treat the sealed can in an ice water bath for 2-4 hours, and then refrigerate at 4-6°C for 2-4 hours; repeat this operation three times to obtain preliminary precooled pollen for use; (5) Step pre-freezing treatment: After taking out the pollen package from the sealed jar, seal it with a container, freeze it at -20℃ for 4-8h, then cool it to -40℃ and continue freezing for 4-8h, and finally place it in a -80℃ environment for long-term storage.

2. A method for ultra-low temperature long-term storage of fresh dried pepper hybrid pollen according to claim 1, characterized in that: The fresh dried pepper flowers collected in step (1) are unopened flowers.

3. A method for ultra-low temperature long-term storage of fresh dried pepper hybrid pollen according to claim 1, characterized in that: The baking method in step (1) is to remove the petals and sepals and then use a 50-100W incandescent lamp to bake for 30-60 minutes.

4. The method for ultra-low temperature long-term storage of fresh dried pepper hybrid pollen according to claim 1, characterized in that: The inert gas in step (3) is nitrogen.

5. The method for ultra-low temperature long-term storage of fresh dried pepper hybrid pollen according to claim 1, characterized in that: The container sealing method in step (5) is to place the pollen and silica gel particles in a 2 mL cryotube and tighten the tube cap, then place the 2 mL cryotube in a 10 mL cryotube, inject glycerol into the 10 mL cryotube, and then tighten the 10 mL cryotube with the tube cap.

6. The method for ultra-low temperature long-term storage of fresh dried pepper hybrid pollen according to claim 5, characterized in that: The pollen is wrapped in two layers of sulfur paper and then in one layer of tin foil, and the amount of silica gel particles added is 1 / 10-2 / 10 of the weight of the pollen.

7. The method for ultra-low temperature long-term storage of fresh dried pepper hybrid pollen according to claim 1, characterized in that: The pollen after long-term storage comprises the following thawing steps before use: S1. Pre-thawing treatment: Take out the frozen pollen and the sealed container, put the container into a refrigerator at -20℃ for 10-12h, and then put it in an ice water bath for 4-6h; S2. Open the pre-thawed container, take out the pollen inside, rinse with cold water to thaw and rehydrate, and then wait for use.

8. The method for ultra-low temperature long-term storage of fresh dried pepper hybrid pollen according to claim 7, characterized in that: The temperature of the cold water during the cold water flushing in S2 is 4-15° C., and the flushing time is 0.5-1 h.

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