CN108719281A - A kind of cryopreservation method of Pollen of Masson Pine - Google Patents

Abstract

The invention discloses a method for ultra-low temperature preservation of masson pine pollen, which comprises the steps of collecting flower spikes, pollen collection, pollen drying, pollen freezing, pollen thawing, pollen germination in vitro and the like. According to the characteristics of masson pine pollen, the method optimizes the ultra-low temperature pollen preservation method. The method is simple and easy to operate, which can improve the storage period and storage life of masson pine pollen, improve the utilization rate of masson pine pollen, and solve the problems of hybrid seed production such as the lack of flowering period of masson pine.

Description

A kind of cryopreservation method of masson pine pollen

technical field

The invention relates to a method for ultra-low temperature preservation of forest germplasm resources, in particular to a method for ultra-low temperature preservation of masson pine pollen.

Background technique

Pinus massoniana Lamb (Pinus massoniana Lamb) is an evergreen tall tree of the Pinaceae genus. It is the main native tree species of my country's specialty, and it is also the most widely distributed and most resourceful forest community in the southern subtropical region. It has fast growth, excellent material, strong adaptability, The characteristics of high comprehensive utilization rate occupy an extremely important position in the construction of forest resources and ecological development. Due to the large distribution of Pinus massoniana and its rich genetic variation, the preservation of its germplasm resources is of great significance.

Pollen is one of the forms of plant germplasm, which contains all gene types of the species and has extremely rich genetic information. It is an important material for germplasm preservation, exchange, artificial assisted pollination and cross breeding. Studies have shown that in the natural state, the lifespan of masson pine pollen is only one month, which brings great inconvenience to germplasm research and cross-breeding work, so it is particularly important to carry out medium and long-term preservation of pollen.

Cryopreservation is known as the most effective method for long-term preservation of germplasm resources, which can preserve shoot tips, callus, dormant buds, pollen, cells, protoplasts, etc. In the ultra-low temperature state, the metabolism of the material is almost completely stopped. As long as the method is suitable and the liquid nitrogen is sufficient, the material can be preserved indefinitely in theory. At present, many native woody plants such as grapefruit, oil palm, willow, camellia, osmanthus, etc. have successfully carried out research on the cryopreservation of pollen. Studies have shown that the cryopreservation methods and preservation parameters of different plant pollen will be different due to the different characteristics of the plant itself. Therefore, it is urgent to provide a cryopreservation method for the germplasm pollen of Pine massoniana to help prolong the life of pollen and maintain the quality of Pine massoniana. The genetic diversity of germplasm resources and artificial assisted pollination in seed gardens can solve the problem of flowering in hybrid breeding, effectively speed up breeding speed, improve breeding efficiency, and avoid the invasion of diseases and insect pests; at the same time, it is convenient for germplasm exchange, and it is a cell engineering and genetic engineering for pollen cultivation And the physiological and biochemical research of pollen provides stable test materials at any time.

Contents of the invention

In view of the above-mentioned deficiencies, the invention provides a method for ultra-low temperature preservation of pine pollen. The method is aimed at the pollen collection, drying of pollen, frozen storage of pollen, thawing of pollen, in vitro germination of pollen, etc. for the characteristics of masson pine pollen. The steps are optimized to improve the storage period and storage life of the masson pine pollen, improve the utilization rate of the masson pine pollen, and solve the problems of hybrid seed production such as the flowering period.

The present invention is realized by adopting the following technical solutions:

A method for cryopreservation of masson pine pollen, comprising the steps of:

(1) Collection of flower spikes: When the microspore leaf balls of masson pine male cones turn slightly yellow to loose powder, choose a sunny morning to collect flower spikes, cut the flower spikes with scissors, wrap them in kraft paper, and bring them back laboratory;

(2) Pollen collection: Put the flower spike sticks in a 250ml Erlenmeyer flask, place them in a constant temperature shaking incubator and shake at a constant speed for 24-48 hours; after the pollen is scattered, collect the pollen on sulfuric acid paper, and collect it every 12 hours ;

(3) Pollen drying: the collected pollen is dried in a desiccator equipped with silica gel until its water content is 9-11%;

(4) Pollen Freezing: Pollen freezing method is one of the following methods:

① Wrap the dried pollen in aluminum foil and put it into a 2ml cryovial, then put it directly into liquid nitrogen for storage;

②Wrap the dried pollen in aluminum foil and put it into a 2ml cryovial, store it at 4°C for one week, and then put it into liquid nitrogen for storage;

③Wrap the dried pollen in aluminum foil and put it into a 2ml cryovial, store it at 4°C for one week, then at -20°C for one week, then put it into liquid nitrogen for storage;

(5) Pollen thawing: Take out the freezing tube from liquid nitrogen to thaw;

(6) Pollen germination in vitro: First, add 3 drops of semi-solid medium for pollen germination in vitro on the glass slide at equal distances, 50ul per drop, then dip a small amount of pollen with a straw and gently spray it on the semi-solid medium , put the glass slide into a petri dish with wet filter paper, seal the petri dish with parafilm, and incubate in the dark at 28°C for 36h.

Further optimization, the method of pollen collection in the above step (2), specifically put the flower spike sticks into a dried 250ml Erlenmeyer flask, place them in a constant temperature shaking incubator, shake at a constant speed of 120rpm for 24-48h, and keep 27 hours during the period. ℃ constant temperature and 2000lux light; after the pollen disperses, tilt the conical flask 45°-90° downward, pat the bottom of the conical flask with the palm of your hand, collect the pollen on the sulfuric acid paper, and collect it every 12 hours.

Further optimization, the method of thawing in the above step (5) can be to take the cryovial out of the liquid nitrogen, open the aluminum foil wrapped with pollen to expose it to the air, and thaw at room temperature for 30 minutes; or take the cryovial out of the liquid nitrogen, After thawing in a water bath at 38°C for 1.5 minutes, open the aluminum foil wrapped with pollen to expose it to the air, and thaw at room temperature for 30 minutes; or take the cryovial out of the liquid nitrogen and thaw in running water for 30 minutes.

Further optimization, the semi-solid medium in the above step (6) is composed of sucrose 50-200 mg/ml, boric acid 0.1-10 mg/ml, complex amino acid chelated calcium 0.3-3 mg/ml, PEG6000 10-150 mg/ml , 3-6 mg/ml of agar is dissolved in distilled water, and the volume is adjusted to 100ml, and it is prepared by conventional high-pressure sterilization; the compound amino acid chelated calcium is produced by chemical synthesis of various amino acids and inorganic calcium salts. An organic calcium substance.

Compared with the prior art, this technical solution has the following beneficial effects:

1. In the pollen collection step, the present invention combines constant temperature, light, and shaking culture, which greatly improves the pollen loosening efficiency of flower ears and shortens the pollen collection time; at the same time, 2000lux light and 27°C constant temperature culture can ensure that the pollen is dry and not easy to mold. block; in addition, using the characteristics of the wide bottom and narrow top of the Erlenmeyer bottle, the pollen can be easily poured out by using tweezers or a small medicine spoon to stop the bottle mouth with a 45°-90° downward slope, and the flower spikes can be easily poured out. The stick stays in the bottle.

2. In the pollen drying step of the present invention, the water content of the pollen is strictly controlled to prevent the water in the pollen cells of the masson pine pollen from being crystallized during freezing to damage the cells and improve the survival rate of the pollen.

3. In the pollen freezing step of the present invention, the dried pollen is first packaged with aluminum foil paper, which can effectively isolate the pollen and avoid pollution, while ensuring homogeneous cooling of the pollen and reducing the formation of heterogeneous ice crystals; Freezing treatment is to drop the temperature in different temperature stages and then drop into liquid nitrogen for preservation, which can effectively freeze the pine pollen and improve the pollen survival rate; as can be seen from the experimental data of the present invention, the pine pollen is 9-11 % water content does not need to be refrigerated, it can be directly put into liquid nitrogen for storage, and a good survival rate of pollen can also be obtained.

4. The present invention provides three thawing methods in the pollen thawing step, especially the staged thawing method that first thaws in a water bath and then thaws at room temperature. The first stage can quickly pass through the dangerous temperature zone of refreezing (-10°C to -50°C °C) to avoid secondary freezing in the cells during thawing; the second stage absorbs moisture in the air at room temperature for rehydration, avoiding the osmotic impact caused by water absorption during thawing or pollen germination that damages the cell membrane system and improves the survival rate of pollen.

5. The present invention has developed a special in vitro germination semi-solid medium after frozen pollen, sucrose 50-200 mg/ml, boric acid 0.1-10 mg/ml, compound amino acid chelated calcium 0.3-3 mg/ml, PEG6000 10 -150 mg/ml, agar 3-6 mg/ml is dissolved in distilled water, and the volume is adjusted to 100ml, and obtained through conventional high-pressure sterilization, the medium meets the nutrients required for the germination of Pinus massoniana pollen, and the culture medium used The addition of complex amino acid chelated calcium components in the base not only provides calcium for the germination of Pinus massoniana pollen, but also promotes the germination of Pinus massoniana pollen; PEG6000 and boric acid also effectively promote the germination efficiency of pollen.

6. In the in vitro culture stage of the present invention, 3 drops of semi-solid medium are added equidistantly on the glass slide, without a cover glass, because the semi-solid medium is thick and difficult to flow, so it is easy to observe, and 3 drops can be made on one glass slide. Repeat, save on slides and coverslips while still allowing for better air permeability and germination rates.

7. The germination rate of the pollen preserved by this method and the collected fresh pollen under the same conditions are not much different, and the pollen stored at ultra-low temperature for 1, 2, 3, and 4 months were respectively taken for in vitro germination detection and analyzed. There was no obvious decrease in the germination rate, so it can be seen that this method is suitable for the germplasm characteristics of Pine masson, can effectively improve the storage period and storage life of Pine masson pollen, and improve the utilization rate of Pine masson pollen, and the method is simple and easy to operate.

Detailed ways

The present invention is further illustrated by the following examples, but not as a limitation of the present invention. For the specific experimental conditions and methods not indicated in the following examples, the technical means used are generally conventional means well known to those skilled in the art.

Example 1:

A method for cryopreservation of masson pine pollen, comprising the steps of:

(1) Collection of flower spikes: From March 1 to 5, 2018, the pollen of Pinus massoniana in Nanning, Guangxi was collected. When the microspore leaf balls of the male cones of Masson pine were slightly yellowed to loose powder, the flower spikes were collected in the morning on a sunny day. , use scissors to cut the spikes, wrap them in kraft paper, and bring them back to the laboratory;

(2) Pollen collection: Put the flower spike sticks in a dried 250ml Erlenmeyer flask, place them in a constant temperature shaking incubator, and shake at a constant speed of 120rpm for 48h, during which time keep a constant temperature of 27°C and a light of 2000lux; after the pollen disperses , tilt the Erlenmeyer flask 45° downward, pat the bottom of the Erlenmeyer flask with the palm of your hand, collect the pollen on the sulfuric acid paper, and collect once every 12 hours;

(3) Pollen drying: the collected pollen is dried in a desiccator equipped with silica gel until its water content is 9%;

(4) Pollen cryopreservation: Wrap the dried pollen in aluminum foil and put it into a 2ml cryovial, and put it directly into liquid nitrogen for storage;

(5) Pollen thawing: Take the cryovial out of the liquid nitrogen, open the aluminum foil wrapped with pollen to expose it to the air, and thaw at room temperature for 30 minutes;

(6) Pollen germination in vitro: First, add 3 drops of semi-solid medium for pollen germination in vitro on the glass slide at equal distances, 50ul per drop, then dip a small amount of pollen with a straw and gently spray it on the semi-solid medium , put the glass slide into a petri dish with wet filter paper, seal the petri dish with parafilm, and incubate in the dark at 28°C for 36h.

The semi-solid medium described in the above step (6) is prepared by dissolving in distilled water according to the amount of sucrose, agar, boric acid, complex amino acid chelated calcium and PEG6000 in Table 1, and distilled to 100ml, and prepared by conventional autoclaving ; The compound amino acid chelated calcium is a product produced by Chengdu Chelated Biotechnology Co., Ltd.

Table 1 pollen germination semi-solid medium formula table

Detection of pollen survival rate: According to the method in this example, the pollen of Pinus massoniana was preserved, and after the pollen of Pinus massoniana was cultured in vitro for 12 hours, transparent pollen tubes could be seen growing from the germination groove between the two air sacs of the germinated pollen grains. In the control group, the collected pollen was directly germinated in vitro using the same medium. After 24h, the length of pollen tube was longer than the diameter of pollen grain, and the length of some pollen tube was more than twice the diameter of pollen grain at 36h. In this experiment, the length of the pollen tube was greater than the diameter of the pollen grain after 36 hours. The pollen was observed with a 10×10 magnification epifluorescence microscope. Three eyes were collected and about 100 pollen were counted. The average germination rate of Pinus massoniana pollen is shown in Table 2.

Table 2 The average germination rate of masson pine pollen treated by the method of this embodiment

Example 2:

A method for cryopreservation of masson pine pollen, comprising the steps of:

(1) Collection of flower spikes: From March 1 to 5, 2018, the pollen of Pinus massoniana in Nanning, Guangxi was collected. When the microspore leaf balls of the male cones of Masson pine were slightly yellowed to loose powder, the flower spikes were collected in the morning on a sunny day. , use scissors to cut the spikes, wrap them in kraft paper, and bring them back to the laboratory;

(2) Pollen collection: Put the flower spike sticks into a dried 250ml Erlenmeyer flask, place them in a constant temperature shaking incubator, and shake at a constant speed of 120rpm for 36h, during which time keep a constant temperature of 27°C and a light of 2000lux; after the pollen disperses , tilt the Erlenmeyer flask 60° downward, pat the bottom of the Erlenmeyer flask with the palm of your hand, collect the pollen on the sulfuric acid paper, and collect once every 12 hours;

(3) Pollen drying: the collected pollen is dried in a desiccator equipped with silica gel until its water content is 9%;

(4) Freeze storage of pollen: Wrap the dried pollen in aluminum foil and put it into a 2ml cryovial, store it at 4°C for one week, and then put it into liquid nitrogen for storage;

(5) Pollen thawing: Take the cryovial out of the liquid nitrogen, open the aluminum foil wrapped with pollen to expose it to the air, and thaw at room temperature for 30 minutes;

(6) Pollen germination in vitro: First, add 3 drops of semi-solid medium for pollen germination in vitro on the glass slide at equal distances, 50ul per drop, then dip a small amount of pollen with a straw and gently spray it on the semi-solid medium , put the glass slide into a petri dish with wet filter paper, seal the petri dish with parafilm, and incubate in the dark at 28°C for 36h.

The semi-solid medium described in the above step (6) is prepared by dissolving in distilled water according to the amount of sucrose, agar, boric acid, compound amino acid chelated calcium and PEG6000 in Table 3, and distilled to 100ml, and prepared by conventional autoclaving ; The compound amino acid chelated calcium is a product produced by Chengdu Chelated Biotechnology Co., Ltd.

Table 3 pollen germination semi-solid medium formula table

Detection of pollen survival rate: According to the method in this example, the pollen of Pinus massoniana was preserved, and after the pollen of Pinus massoniana was cultured in vitro for 12 hours, transparent pollen tubes could be seen growing from the germination groove between the two air sacs of the germinated pollen grains. In the control group, the collected pollen was directly germinated in vitro using the same medium. After 24h, the length of pollen tube was longer than the diameter of pollen grain, and the length of some pollen tube was more than twice the diameter of pollen grain at 36h. In this experiment, after 36 hours, the length of the pollen tube was greater than the diameter of the pollen grain. The pollen was observed by a 10×10 magnification epifluorescence microscope. Three eyes were collected and about 100 pollen were counted. The average germination rate of Pinus massoniana pollen is shown in Table 4.

Table 4 The average germination rate of masson pine pollen treated by the method of this embodiment

Example 3:

A method for cryopreservation of masson pine pollen, comprising the steps of:

(1) Collection of flower spikes: From March 1 to 5, 2018, the pollen of Pinus massoniana in Nanning, Guangxi was collected. When the microspore leaf balls of the male cones of Masson pine were slightly yellowed to loose powder, the flower spikes were collected in the morning on a sunny day. , use scissors to cut the spikes, wrap them in kraft paper, and bring them back to the laboratory;

(2) Pollen collection: Put the flower spike sticks into a dried 250ml Erlenmeyer flask, place them in a constant temperature shaking incubator, and shake at a constant speed of 120rpm for 36h, during which time keep a constant temperature of 27°C and a light of 2000lux; after the pollen disperses , tilt the Erlenmeyer flask 60° downward, pat the bottom of the Erlenmeyer flask with the palm of your hand, collect the pollen on the sulfuric acid paper, and collect once every 12 hours;

(3) Pollen drying: the collected pollen is dried in a desiccator equipped with silica gel until its water content is 10%;

(4) Freezing storage of pollen: Wrap the dried pollen in aluminum foil and put it into a 2ml cryovial, store it at 4°C for one week, then at -20°C for one week, and then put it into liquid nitrogen for storage;

(5) Pollen thawing: Take the cryovial out of the liquid nitrogen, open the aluminum foil wrapped with pollen to expose it to the air, and thaw at room temperature for 30 minutes;

(6) Pollen germination in vitro: First, add 3 drops of semi-solid medium for pollen germination in vitro on the glass slide at equal distances, 50ul per drop, then dip a small amount of pollen with a straw and gently spray it on the semi-solid medium , put the glass slide into a petri dish with wet filter paper, seal the petri dish with parafilm, and incubate in the dark at 28°C for 36h.

The semi-solid medium described in the above step (6) is prepared by dissolving in distilled water according to the amount of sucrose, agar, boric acid, compound amino acid chelated calcium and PEG6000 in Table 5, and distilled to 100ml, and prepared by conventional autoclaving ; The compound amino acid chelated calcium is a product produced by Chengdu Chelated Biotechnology Co., Ltd.

Table 5 pollen germination semi-solid medium formula table

Detection of pollen survival rate: According to the method in this example, the pollen of Pinus massoniana was preserved, and after the pollen of Pinus massoniana was cultured in vitro for 12 hours, transparent pollen tubes could be seen growing from the germination groove between the two air sacs of the germinated pollen grains. In the control group, the collected pollen was directly germinated in vitro using the same medium. After 24h, the length of pollen tube was longer than the diameter of pollen grain, and the length of some pollen tube was more than twice the diameter of pollen grain at 36h. In this experiment, the length of the pollen tube was greater than the diameter of the pollen grain after 36 hours. The pollen was observed by a 10×10 magnification epifluorescence microscope, and 3 eyes were taken, and about 100 pollen were counted. The average germination rate of Pinus massoniana pollen is shown in Table 6.

Table 6 The average germination rate of masson pine pollen treated by the method of this embodiment

Example 4:

A method for cryopreservation of masson pine pollen, comprising the steps of:

(1) Collection of flower spikes: From March 1 to 5, 2018, the pollen of Pinus massoniana in Nanning, Guangxi was collected. When the microspore leaf balls of the male cones of Masson pine were slightly yellowed to loose powder, the flower spikes were collected in the morning on a sunny day. , use scissors to cut the spikes, wrap them in kraft paper, and bring them back to the laboratory;

(2) Pollen collection: Put the flower spike sticks into a dried 250ml Erlenmeyer flask, place them in a constant temperature shaking incubator, and shake at a constant speed of 120rpm for 36h, during which time keep a constant temperature of 27°C and a light of 2000lux; after the pollen disperses , tilt the Erlenmeyer flask 45° downward, pat the bottom of the Erlenmeyer flask with the palm of your hand, collect the pollen on the sulfuric acid paper, and collect once every 12 hours;

(3) Pollen drying: the collected pollen is dried in a desiccator equipped with silica gel until its water content is 11%;

(4) Pollen cryopreservation: Wrap the dried pollen in aluminum foil and put it into a 2ml cryovial, and put it directly into liquid nitrogen for storage;

(5) Pollen thawing: thawing in the following three ways:

① Take the cryovial out of the liquid nitrogen, open the aluminum foil wrapped with pollen to expose it to the air, and thaw at room temperature for 30 minutes;

② Take the cryovial out of the liquid nitrogen, thaw in a water bath at 38°C for 1.5 minutes, open the aluminum foil wrapped with pollen to expose it to the air, and thaw at room temperature for 30 minutes;

③ Take the cryovial out of the liquid nitrogen and thaw it in running water for 30 minutes.

(6) Pollen germination in vitro: First, add 3 drops of semi-solid medium for pollen germination in vitro on the glass slide at equal distances, 50ul per drop, then dip a small amount of pollen with a straw and gently spray it on the semi-solid medium , put the glass slide into a petri dish with wet filter paper, seal the petri dish with parafilm, and incubate in the dark at 28°C for 36h.

The semi-solid medium in the above step (6) is the M2 semi-solid medium in Example 1.

Detection of pollen survival rate: According to the method in this example, the pollen of Pinus massoniana was preserved, and after the pollen of Pinus massoniana was cultured in vitro for 12 hours, transparent pollen tubes could be seen growing from the germination groove between the two air sacs of the germinated pollen grains. In the control group, the collected pollen was directly germinated in vitro using the same medium. After 24h, the length of pollen tube was longer than the diameter of pollen grain, and the length of some pollen tube was more than twice the diameter of pollen grain at 36h. In this experiment, after 36 hours, the length of the pollen tube is greater than the diameter of the pollen grains, which is regarded as survival. The pollen is observed by a 10×10 times epifluorescence microscope, and 3 eyes are taken, and about 100 pollen grains are counted. Rates are shown in Table 7.

Table 7 The average germination rate of masson pine pollen after different thawing methods

Embodiment 6:

A method for cryopreservation of masson pine pollen, comprising the steps of:

(1) Collection of flower spikes: From March 1 to 5, 2018, the pollen of Pinus massoniana in Nanning, Guangxi was collected. When the microspore leaf balls of the male cones of Masson pine were slightly yellowed to loose powder, the flower spikes were collected in the morning on a sunny day. , use scissors to cut the spikes, wrap them in kraft paper, and bring them back to the laboratory;

(2) Pollen collection: Put the flower spike sticks into a dried 250ml Erlenmeyer flask, place them in a constant temperature shaking incubator, and shake them at a constant speed of 120rpm for 48 hours. During this period, keep a constant temperature of 27°C and light; Tilt the Erlenmeyer flask 45° downward, pat the bottom of the Erlenmeyer flask with your palm, collect pollen on sulfuric acid paper, and collect once every 12 hours;

(3) Pollen drying: the collected pollen is dried in a desiccator equipped with silica gel until its water content is 10%;

(4) Pollen cryopreservation: Wrap the dried pollen in aluminum foil and put it into a 2ml cryovial, then put it directly into liquid nitrogen for storage;

(5) Pollen thawing: take out the frozen tubes of pollen that have been stored in liquid nitrogen for 30 days, 60 days, 90 days, and 120 days respectively, and thaw them in a water bath at 38°C for 1.5 minutes, then open the aluminum foil wrapped with pollen and use them. When exposed to the air, thaw at room temperature for 30 minutes;

(6) Pollen germination in vitro: First, add 3 drops of semi-solid medium for pollen germination in vitro on the glass slide at equal distances, 50ul per drop, then dip a small amount of pollen with a straw and gently spray it on the semi-solid medium , put the glass slide into a petri dish with wet filter paper, seal the petri dish with parafilm, and incubate in the dark at 28°C for 36h.

The semi-solid medium in the above step (6) is the M2 semi-solid medium in Example 1.

Detection of pollen survival rate: According to the method in this example, the pollen of Pinus massoniana was preserved, and after the pollen of Pinus massoniana was cultured in vitro for 12 hours, transparent pollen tubes could be seen growing from the germination groove between the two air sacs of the germinated pollen grains. In the control group, the collected pollen was directly germinated in vitro using the same medium. After 24h, the length of pollen tube was longer than the diameter of pollen grain, and the length of some pollen tube was more than twice the diameter of pollen grain at 36h. In this experiment, after 36 hours, the length of the pollen tube was greater than the diameter of the pollen grain. The pollen was observed by a 10×10 magnification epifluorescence microscope. Three eyes were collected and about 100 pollen were counted. The average germination rate of Pinus massoniana pollen is shown in Table 8.

The average germination rate of masson pine pollen after the method of table 8 is processed

In addition, it should be understood that although this specification is described according to implementation modes, not each implementation mode only includes an independent technical solution, and this description in the specification is only for clarity, and those skilled in the art should take the specification as a whole , the technical solutions in the various embodiments can also be properly combined to form other implementations that can be understood by those skilled in the art.

Claims (4)

1. a method for cryopreservation of masson pine pollen, characterized in that: it may further comprise the steps: (1) Collection of flower spikes: When the microspore leaf balls of masson pine male cones turn slightly yellow to loose powder, choose a sunny morning to collect flower spikes, cut the flower spikes with scissors, wrap them in kraft paper, and bring them back laboratory; (2) Pollen collection: Put the flower spike sticks in a 250ml Erlenmeyer flask, place them in a constant temperature shaking incubator and shake at a constant speed for 24-48 hours; after the pollen is scattered, collect the pollen on sulfuric acid paper, and collect it every 12 hours ; (3) Pollen drying: the collected pollen is dried in a desiccator equipped with silica gel until its water content is 9-11%; (4) Pollen Freezing: Pollen freezing method is one of the following methods: Wrap the dried pollen in aluminum foil and put it into a 2ml cryovial, then put it directly into liquid nitrogen for storage; Wrap the dried pollen in aluminum foil and put it into a 2ml cryovial, store it at 4°C for one week, and then put it into liquid nitrogen for storage; Wrap the dried pollen in aluminum foil and put it into a 2ml cryovial, store it at 4°C for one week, then at -20°C for one week, and put it into liquid nitrogen for storage; (5) Pollen thawing: Take out the freezing tube from liquid nitrogen to thaw; (6) Pollen germination in vitro: First, add 3 drops of semi-solid medium for pollen germination in vitro on the glass slide at equal distances, 50ul per drop, then dip a small amount of pollen with a straw and gently spray it on the semi-solid medium , put the glass slide into a petri dish with wet filter paper, seal the petri dish with parafilm, and incubate in the dark at 28°C for 36h. 2. A method for cryopreservation of masson pine pollen according to claim 1, characterized in that: the method of collecting pollen in said step (2) is to put flower spike sticks into a dried 250ml Erlenmeyer flask , cultured in a constant temperature shaking incubator, shaking at a constant speed of 120rpm for 24-48h, maintaining a constant temperature of 27°C and a light of 2000lux during the period; At the bottom of the bottle, the pollen was collected on sulfuric acid paper and collected every 12 hours. 3. The ultra-low temperature preservation method for masson pine pollen according to claim 1, characterized in that: in the step (5), the method of thawing is to take out the freezing tube from liquid nitrogen, and open the aluminum foil wrapped with pollen Expose the paper to the air, and thaw at room temperature for 30 minutes; or take the cryovial out of the liquid nitrogen, thaw in a 38°C water bath for 1.5 minutes, open the aluminum foil wrapped with pollen, expose it to the air, and thaw at room temperature for 30 minutes; or Take the cryovial out of the liquid nitrogen and thaw in running water for 30 min. 4. The cryopreservation method of masson pine pollen according to claim 1, characterized in that: the formula of the semi-solid medium in the step (6) is 50-200 mg/ml sucrose, 0.1-10 boric acid mg/ml, compound amino acid chelated calcium 0.3-3 mg/ml, PEG6000 10-150 mg/ml, agar 3-6 mg/ml dissolved in distilled water, and dilute to 100ml, prepared by conventional autoclaving.