CN108849475B - Malus spectabilis pollen treatment process, Malus spectabilis pollen and application thereof - Google Patents
Malus spectabilis pollen treatment process, Malus spectabilis pollen and application thereof Download PDFInfo
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- CN108849475B CN108849475B CN201810764389.5A CN201810764389A CN108849475B CN 108849475 B CN108849475 B CN 108849475B CN 201810764389 A CN201810764389 A CN 201810764389A CN 108849475 B CN108849475 B CN 108849475B
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention provides a begonia pollen treatment process, begonia pollen and application thereof, and relates to the technical field of artificial pollination. A Chinese flowering crabapple pollen treatment process comprises the following steps: thawing the refrigerated pollen under thawing condition of 0-10 deg.C. The Chinese flowering crabapple pollen is obtained by the Chinese flowering crabapple pollen treatment process. The application of the begonia pollen in the artificial pollination of begonia. Thawing the refrigerated pollen under thawing condition of 0-10 deg.C. The obtained plum-leaf crab pollen after unfreezing can be used for artificial pollination of plum-leaf crab. The pollen thawed at the temperature has less damage to pollen vitality, so that the thawed pollen can keep better pollen vitality, the success rate of artificial pollination is favorably ensured, and the utilization rate of the pollen is also improved.
Description
Technical Field
The invention relates to the technical field of artificial pollination, and particularly relates to a begonia pollen treatment process, begonia pollen and application thereof.
Background
The ornamental value of the ornamental crabapple can be embodied differently according to various aspects such as flower color, leaf color, tree shape, fruit and the like. The tree type includes column type, drooping branch type and small arbor type; the flower type is various, and the flower type has a single valve, a half-double valve and a double valve; the leaf color has large variation range from green to purple; the flower color is white, red, pink, purple, etc.; the fruit color is red, orange red, deep red, mauve, golden yellow, orange yellow, etc. Crabapple is a species of vernonia but may exhibit different landscape during one year: in spring, the flowers are in a cluster, the fruits are tired in summer, the fruits are luxuriant in autumn, and the fruits do not fall in winter. The ornamental Chinese flowering crabapple has the characteristics of high ornamental value, rich varieties, strong adaptability of the Chinese flowering crabapple to the environment, convenience in manual management, large planting range and the like, and modern gardens have wide application to the ornamental Chinese flowering crabapple. The market demand for ornamental crabapples has also facilitated research and variety renewal for ornamental crabapples. In the production of the seeds of the Chinese flowering crabapple, artificial pollination is an important and indispensable work, so that the preservation of the Chinese flowering crabapple pollen becomes an important prerequisite for artificial pollination. How to better maintain the pollen activity of the Chinese flowering crabapple becomes an important factor for success of seed production.
Disclosure of Invention
The invention aims to provide a processing technology of Chinese flowering crabapple pollen, which can better keep the vitality of the Chinese flowering crabapple pollen and improve the utilization rate of the pollen.
The invention also aims to provide the Chinese flowering crabapple pollen which has good pollen activity and is beneficial to improving the success rate of artificial pollination.
The invention also aims to provide application of the Chinese flowering crabapple pollen, wherein the Chinese flowering crabapple pollen can be applied to artificial pollination of Chinese flowering crabapple and has high pollination success rate.
The technical problem to be solved by the invention is realized by adopting the following technical scheme.
The invention provides a processing technology of Chinese flowering crabapple pollen, which comprises the following steps:
thawing the refrigerated pollen under thawing condition of 0-10 deg.C.
The invention provides a begonia pollen which is obtained by processing the begonia pollen by the processing technology.
The invention also provides application of the begonia pollen in artificial pollination of begonia.
The Chinese flowering crabapple pollen processing technology, the Chinese flowering crabapple pollen and the application of the Chinese flowering crabapple pollen have the beneficial effects that:
thawing the refrigerated pollen under thawing condition of 0-10 deg.C. The obtained plum-leaf crab pollen after unfreezing can be used for artificial pollination of plum-leaf crab. The pollen thawed at the temperature has less damage to pollen vitality, so that the thawed pollen can keep better pollen vitality, the success rate of artificial pollination is favorably ensured, and the utilization rate of the pollen is also improved.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The following specifically describes the processing technology of the begonia pollen, the begonia pollen and the application thereof in the embodiment of the invention.
The embodiment provides a crabapple pollen, and the pollen treatment process mainly comprises the following steps:
collecting buds of the crabapple in the full-bloom stage, and taking out anthers by using tweezers. It should be noted that if the picking path is far, the ice box needs to be carried. The collected buds are firstly put into a sulfuric acid paper bag, and then the sulfuric acid paper bag is put into an ice box and taken back to the laboratory. The anthers were removed with tweezers in a clean environment in the laboratory. The parchment paper is firm, dense and slightly transparent, has the characteristics of strong penetration resistance to grease and water, air impermeability, high wet strength and the like, and can prevent water, moisture, oil, bacteria and disinfection. The life of pollen is short, and the pollen does not have long survival time under natural conditions after leaving the plant. The low temperature of the ice box is beneficial to protecting flower buds in the transfer process and reducing the active damage of the surrounding natural environment to pollen.
The anther is naturally dried in the shade at room temperature. And (3) placing the naturally dried anther in the shade in an incubator at 23-27 ℃ so that the anther can be dried to produce pollen. In order to improve the uniformity of artificial pollination seed production, the pollen can be firstly screened, for example, the pollen is screened by a sieve with the diameter of 10 cm and the aperture of 0.25 mm. And (3) putting the sieved pollen into a centrifugal tube (or other common experimental containers), putting the centrifugal tube into a self-sealing bag, and finally putting the self-sealing bag under corresponding storage conditions for refrigeration. The valve bag can avoid freezing on the centrifuging tube, though also being covered with the lid on the centrifuging tube, but the valve bag can avoid ice, water to enter into the centrifuging tube better, and then influences pollen vigor.
The pollen is kept at a certain water content for refrigeration, which is beneficial to keeping the pollen vitality for long-time refrigeration. Therefore, the water content of the dried pollen can be controlled to be 60-70%, then the pollen is filled into a freezing storage tube (or a centrifugal tube), the freezing storage tube is placed in liquid nitrogen for 5-10s, and then the freezing storage tube is sealed by a self-sealing bag and then transferred to a freezer at the temperature of-80 ℃ for refrigeration. The liquid nitrogen can carry out quick freezing to the pollen, and the pollen later stage of being convenient for can refrigerate longer time in the refrigerator-freezer.
In order to ensure the effectiveness of pollen selection and reduce the useless work of experimenters, a small amount of processed pollen can be taken to carry out pollen activity determination before the pollen is frozen. Pollen viability was determined by in vitro germination. If the pollen activity is higher than 60%, the pollen of the batch meets the use requirement, and the pollen can be subjected to cryopreservation.
The method for determining the pollen viability before freezing by the in vitro germination method comprises the following steps:
0.2% boric acid and 10% sucrose were added to 100ml of distilled water, and the prepared culture solution was dropped on a concave slide glass by a dropper. Uniformly dispersing pollen on the culture solution by using toothpicks, placing the glass slide in a culture dish containing wet absorbent cotton, covering, and placing in a light incubator for culture. The culture was carried out at 25 ℃ for 4 h. And (4) observing by using an optical microscope, and calculating the pollen germination rate by taking the length of the pollen tube exceeding the radius of the pollen as a reference, wherein the total number of the pollen in each visual field is not less than 30. Pollen germination rate (i.e. pollen vigor) ═ number of pollen that has germinated/total number of pollen x 100%. The pollen germination rate is higher than 60%, which indicates that the batch of pollen meets the use requirement and can be frozen for storage.
The refrigeration condition of pollen comprises storing at-20-80 deg.C for 30-250 days. Further, it may be stored at-80 ℃ for 250 days. Generally, the lower the refrigeration temperature, the longer the shelf life.
The refrigeration of the begonia pollen prolongs the activity of the pollen in terms of time, and strives for a certain time period for production.
When the pollen needs artificial pollination, the refrigerated pollen is taken out for thawing.
The thawing conditions comprise thawing said pollen at 0-10 deg.C. The treatment conditions of pollen of each plant are different, and the pollen of some plants can obtain better vitality after being thawed at 35 ℃, such as corn pollen and sunflower pollen, but the inventor finds that the begonia pollen needs to be thawed at 0-10 ℃ to obtain better vitality through experiments.
The inventor finds that the pollen has the best pollen activity after being thawed in an ice-water mixture at 0 ℃. The ice-water mixture is beneficial to more stably maintaining the 0 ℃ condition in the unfreezing process and is also beneficial to maintaining the pollen vitality.
The pollen has good activity after being thawed at 0-10 ℃ for 10-15 minutes generally.
Pollen activity was best when thawed in ice water mixture at 0 ℃ for 15 minutes.
In order to ensure the success rate of artificial pollination and reduce the useless work of experimenters, a trace amount of pollen is taken firstly for activity determination after the pollen is thawed and before pollination.
Pollen viability was determined by in vitro germination. 0.2% boric acid and 10% sucrose are added into 100ml distilled water, the prepared solution is dripped on a concave glass slide by a dropper, and then pollen is uniformly dispersed on the culture solution by toothpicks. The slide glass is put in a culture dish containing the wet absorbent cotton, covered, put in a light incubator for culture and cultured for 4h at the temperature of 25 ℃. And (3) calculating the pollen germination rate (namely pollen viability) by using an optical microscope to observe, wherein the length of the pollen tube exceeding the pollen radius is taken as a reference, and the total number of the pollen in each visual field is not less than 30. Pollen germination rate ═ (number of already germinated pollen/total number of pollen) × 100%). The pollen germination rate is more than 60%, and then the artificial pollination can be continued.
The begonia pollen obtained by the pollen treatment process can be used for artificial pollination of begonia, the activity of the pollen is good, and the success rate of the artificial pollination is greatly improved.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
The embodiment provides a Chinese flowering crabapple pollen for the artificial pollination of Chinese flowering crabapple, which is obtained by the following treatment processes:
the collected and processed pollen is directly used. Putting pollen into the centrifuge tube, putting the centrifuge tube into the self-sealing bag, and refrigerating the self-sealing bag under corresponding storage conditions. The cold storage condition of pollen is to store at-80 deg.C for 180 days.
Before the pollen is refrigerated, a small amount of the treated pollen is taken for pollen viability measurement. Pollen viability was determined by in vitro germination. If the pollen activity is higher than 60%, the pollen of the batch meets the use requirement, and the pollen can be subjected to cryopreservation. If the pollen viability is lower than 60%, the pollen viability of the batch is low, and the batch is recommended to be discarded.
When the pollen needs artificial pollination, the refrigerated pollen is taken out for thawing. The unfreezing process comprises the following steps: the centrifugal tube filled with pollen is taken out, the pollen is taken out from the centrifugal tube and is put into a new centrifugal tube, the centrifugal tube is sealed, and the new centrifugal tube is convenient for unfreezing the pollen more quickly. The pollen together with the new centrifuge tube was then thawed in ice water mixture at 0 ℃ for 13 minutes.
After the pollen is unfrozen, before pollination, a trace amount of pollen is taken for activity determination. The pollen germination rate is more than 60%, and then the artificial pollination can be continued. If the pollen germination rate is less than 60%, the artificial pollination is not recommended to be continued.
Example 2
The embodiment provides a Chinese flowering crabapple pollen for the artificial pollination of Chinese flowering crabapple, which is obtained by the following treatment processes:
collecting buds of the flowering period of the crabapple. The collection distance is short, and the flower buds can be directly taken back to a laboratory for treatment. The anthers were removed with tweezers in a clean environment in the laboratory.
The anther is naturally dried in the shade at room temperature. And (3) placing the naturally dried anther in the shade in an incubator at 26 ℃ so that the anther can dry out pollen. The pollen is first sieved. And (3) putting the sieved pollen into a centrifuge tube, putting the centrifuge tube into a self-sealing bag, and finally placing the self-sealing bag under corresponding storage conditions for refrigeration. The cold storage condition of pollen is to store at-80 deg.C for 250 days.
Before the pollen is refrigerated, a small amount of the treated pollen is taken for pollen viability measurement. Pollen viability was determined by in vitro germination. If the pollen activity is higher than 60%, the pollen of the batch meets the use requirement, and the pollen can be subjected to cryopreservation. If the pollen viability is lower than 60%, the pollen viability of the batch is low, and the batch is recommended to be discarded.
When the pollen needs artificial pollination, the refrigerated pollen is taken out for thawing. The unfreezing process comprises the following steps: the centrifugal tube filled with pollen is taken out, the pollen is taken out from the centrifugal tube and is put into a new centrifugal tube, the centrifugal tube is sealed, and the new centrifugal tube is convenient for unfreezing the pollen more quickly. Then putting the pollen and a new centrifugal tube into an ice-water mixture at 0 ℃ for unfreezing for 15 minutes.
After the pollen is unfrozen, before pollination, a trace amount of pollen is taken for activity determination. The pollen germination rate is more than 60%, and then the artificial pollination can be continued. If the pollen germination rate is less than 60%, the artificial pollination is not recommended to be continued.
Example 3
The embodiment provides a Chinese flowering crabapple pollen for the artificial pollination of Chinese flowering crabapple, which is obtained by the following treatment processes:
collecting buds of the flowering period of the crabapple. The collection distance is short, and the flower buds can be directly taken back to a laboratory for treatment. The anthers were removed with tweezers in a clean environment in the laboratory.
The anther is naturally dried in the shade at room temperature. And (3) placing the naturally dried anther in the shade in an incubator at 26 ℃ so that the anther can dry out pollen. The pollen is first sieved. And (3) putting the sieved pollen into a centrifuge tube, putting the centrifuge tube into a self-sealing bag, and finally placing the self-sealing bag under corresponding storage conditions for refrigeration. The cold storage condition of pollen is to store at-80 deg.C for 250 days.
Before the pollen is refrigerated, a small amount of the treated pollen is taken for pollen viability measurement. Pollen viability was determined by in vitro germination. If the pollen activity is higher than 60%, the pollen of the batch meets the use requirement, and the pollen can be subjected to cryopreservation. If the pollen viability is lower than 60%, the pollen viability of the batch is low, and the batch is recommended to be discarded.
When the pollen needs artificial pollination, the refrigerated pollen is taken out for thawing. The unfreezing process comprises the following steps: the centrifugal tube filled with pollen is taken out, the pollen is taken out from the centrifugal tube and is put into a new centrifugal tube, the centrifugal tube is sealed, and the new centrifugal tube is convenient for unfreezing the pollen more quickly. Then putting the pollen and a new centrifugal tube into water with the temperature of 10 ℃ for unfreezing for 15 minutes.
After the pollen is unfrozen, before pollination, a trace amount of pollen is taken for activity determination. The pollen germination rate is more than 60%, and then the artificial pollination can be continued. If the pollen germination rate is less than 60%, the artificial pollination is not recommended to be continued.
Example 4
The embodiment provides a Chinese flowering crabapple pollen for the artificial pollination of Chinese flowering crabapple, which is obtained by the following treatment processes:
collecting buds of the flowering period of the crabapple. The collection distance is short, and the flower buds can be directly taken back to a laboratory for treatment. The anthers were removed with tweezers in a clean environment in the laboratory.
The anther is naturally dried in the shade at room temperature. And (3) placing the naturally dried anther in the shade in an incubator at 26 ℃ so that the anther can dry out pollen. The pollen is first sieved. And (3) putting the sieved pollen into a centrifuge tube, putting the centrifuge tube into a self-sealing bag, and finally placing the self-sealing bag under corresponding storage conditions for refrigeration. The cold storage condition of pollen is preservation at-35 deg.C for 100 days.
Before the pollen is refrigerated, a small amount of the treated pollen is taken for pollen viability measurement. Pollen viability was determined by in vitro germination. If the pollen activity is higher than 60%, the pollen of the batch meets the use requirement, and the pollen can be subjected to cryopreservation. If the pollen viability is lower than 60%, the pollen viability of the batch is low, and the batch is recommended to be discarded.
When the pollen needs artificial pollination, the refrigerated pollen is taken out for thawing. The unfreezing process comprises the following steps: the centrifugal tube filled with pollen is taken out, the pollen is taken out from the centrifugal tube and is put into a new centrifugal tube, the centrifugal tube is sealed, and the new centrifugal tube is convenient for unfreezing the pollen more quickly. Then putting the pollen and a new centrifugal tube into water with the temperature of 0 ℃ for unfreezing for 10 minutes.
After the pollen is unfrozen, before pollination, a trace amount of pollen is taken for activity determination. The pollen germination rate is more than 60%, and then the artificial pollination can be continued. If the pollen germination rate is less than 60%, the artificial pollination is not recommended to be continued.
Example 5
The embodiment provides a Chinese flowering crabapple pollen for the artificial pollination of Chinese flowering crabapple, which is obtained by the following treatment processes:
collecting buds of the flowering period of the crabapple. The collected buds are firstly put into a sulfuric acid paper bag, and then the sulfuric acid paper bag is put into an ice box and taken back to the laboratory. The anthers were removed with tweezers in a clean environment in the laboratory.
The anther is naturally dried in the shade at room temperature. And (3) placing the naturally dried anther in the shade in an incubator at 25 ℃ so that the anther can dry out pollen. The pollen is first sieved. And (3) putting the sieved pollen into a centrifuge tube, putting the centrifuge tube into a self-sealing bag, and finally placing the self-sealing bag under corresponding storage conditions for refrigeration. The cold storage condition of pollen is to store at-80 deg.C for 250 days.
Before the pollen is refrigerated, a small amount of the treated pollen is taken for pollen viability measurement. Pollen viability was determined by in vitro germination. If the pollen activity is higher than 60%, the pollen of the batch meets the use requirement, and the pollen can be subjected to cryopreservation. If the pollen viability is lower than 60%, the pollen viability of the batch is low, and the batch is recommended to be discarded.
When the pollen needs artificial pollination, the refrigerated pollen is taken out for thawing. The unfreezing process comprises the following steps: the centrifugal tube filled with pollen is taken out, the pollen is taken out from the centrifugal tube and is put into a new centrifugal tube, the centrifugal tube is sealed, and the new centrifugal tube is convenient for unfreezing the pollen more quickly. Then putting the pollen and a new centrifugal tube into water with the temperature of 8 ℃ for unfreezing for 13 minutes.
After the pollen is unfrozen, before pollination, a trace amount of pollen is taken for activity determination. The pollen germination rate is more than 60%, and then the artificial pollination can be continued. If the pollen germination rate is less than 60%, the artificial pollination is not recommended to be continued.
Example 6
The embodiment provides a Chinese flowering crabapple pollen for the artificial pollination of Chinese flowering crabapple, which is obtained by the following treatment processes:
collecting buds of the flowering period of the crabapple. The collected buds are firstly put into a sulfuric acid paper bag, and then the sulfuric acid paper bag is put into an ice box and taken back to the laboratory. The anthers were removed with tweezers in a clean environment in the laboratory.
The anther is naturally dried in the shade at room temperature. And (3) placing the naturally dried anther in the shade in an incubator at 23 ℃ so that the anther can dry out pollen. Controlling the water content of the pollen to be 70%, sieving the pollen, then filling the pollen into a freezing storage tube, placing the freezing storage tube in liquid nitrogen for 10s, sealing the freezing storage tube by a self-sealing bag, and then transferring the freezing storage tube to a freezer at the temperature of minus 80 ℃ for refrigeration for 200 days.
Before the pollen is refrigerated, a small amount of sieved pollen is taken for pollen viability measurement. Pollen viability was determined by in vitro germination. If the pollen activity is higher than 60%, the pollen of the batch meets the use requirement, and the pollen can be subjected to cryopreservation. If the pollen viability is lower than 60%, the pollen viability of the batch is low, and the batch is recommended to be discarded.
When the pollen needs artificial pollination, the refrigerated pollen is taken out for thawing. The unfreezing process comprises the following steps: the centrifugal tube filled with pollen is taken out, the pollen is taken out from the centrifugal tube and is put into a new centrifugal tube, the centrifugal tube is sealed, and the new centrifugal tube is convenient for unfreezing the pollen more quickly. Then putting the pollen and a new centrifugal tube into water with the temperature of 9 ℃ for unfreezing for 14 minutes.
After the pollen is unfrozen, before pollination, a trace amount of pollen is taken for activity determination. The pollen germination rate is more than 60%, and then the artificial pollination can be continued. If the pollen germination rate is less than 60%, the artificial pollination is not recommended to be continued.
Example 7
The embodiment provides a Chinese flowering crabapple pollen for the artificial pollination of Chinese flowering crabapple, which is obtained by the following treatment processes:
collecting buds of the flowering period of the crabapple. The collected buds are firstly put into a sulfuric acid paper bag, and then the sulfuric acid paper bag is put into an ice box and taken back to the laboratory. The anthers were removed with tweezers in a clean environment in the laboratory.
The anther is naturally dried in the shade at room temperature. Placing the naturally dried anther in the shade in an incubator at 27 ℃ to dry the anther out pollen. Controlling the water content of the pollen to be 60%, sieving the pollen, then filling the pollen into a freezing storage tube, placing the freezing storage tube in liquid nitrogen for 5s, sealing the freezing storage tube by a self-sealing bag, and then transferring the freezing storage tube to a freezer at the temperature of-20 ℃ for refrigeration for 30 days.
Before the pollen is refrigerated, a small amount of sieved pollen is taken for pollen viability measurement. Pollen viability was determined by in vitro germination. If the pollen activity is higher than 60%, the pollen of the batch meets the use requirement, and the pollen can be subjected to cryopreservation. If the pollen viability is lower than 60%, the pollen viability of the batch is low, and the batch is recommended to be discarded.
When the pollen needs artificial pollination, the refrigerated pollen is taken out for thawing. The unfreezing process comprises the following steps: the centrifugal tube filled with pollen is taken out, the pollen is taken out from the centrifugal tube and is put into a new centrifugal tube, the centrifugal tube is sealed, and the new centrifugal tube is convenient for unfreezing the pollen more quickly. Then putting the pollen and a new centrifugal tube into water with the temperature of 10 ℃ for unfreezing for 20 minutes.
After the pollen is unfrozen, before pollination, a trace amount of pollen is taken for activity determination. The pollen germination rate is more than 60%, and then the artificial pollination can be continued. If the pollen germination rate is less than 60%, the artificial pollination is not recommended to be continued.
Example 8
The embodiment provides a Chinese flowering crabapple pollen for the artificial pollination of Chinese flowering crabapple, which is obtained by the following treatment processes:
collecting buds of the flowering period of the crabapple. The collected buds are firstly put into a sulfuric acid paper bag, and then the sulfuric acid paper bag is put into an ice box and taken back to the laboratory. The anthers were removed with tweezers in a clean environment in the laboratory.
The anther is naturally dried in the shade at room temperature. And (3) placing the naturally dried anther in the shade in an incubator at 24 ℃ so that the anther can dry out pollen. Controlling the water content of the pollen to be 65%, sieving the pollen, then filling the pollen into a freezing storage tube, placing the freezing storage tube in liquid nitrogen for 8s, sealing the freezing storage tube by a self-sealing bag, and then transferring the freezing storage tube to a freezer at the temperature of-30 ℃ for refrigeration for 60 days.
Before the pollen is refrigerated, a small amount of sieved pollen is taken for pollen viability measurement. Pollen viability was determined by in vitro germination. If the pollen activity is higher than 60%, the pollen of the batch meets the use requirement, and the pollen can be subjected to cryopreservation. If the pollen viability is lower than 60%, the pollen viability of the batch is low, and the batch is recommended to be discarded.
When the pollen needs artificial pollination, the refrigerated pollen is taken out for thawing. The unfreezing process comprises the following steps: the centrifugal tube filled with pollen is taken out, the pollen is taken out from the centrifugal tube and is put into a new centrifugal tube, the centrifugal tube is sealed, and the new centrifugal tube is convenient for unfreezing the pollen more quickly. Then putting the pollen and a new centrifugal tube into water with the temperature of 0 ℃ for unfreezing for 15 minutes.
After the pollen is unfrozen, before pollination, a trace amount of pollen is taken for activity determination. The pollen germination rate is more than 60%, and then the artificial pollination can be continued. If the pollen germination rate is less than 60%, the artificial pollination is not recommended to be continued.
Comparative example 1
The refrigerated pollen provided in example 2 was thawed by the following thawing steps:
the centrifugal tube filled with pollen is taken out, the pollen is taken out from the centrifugal tube and is put into a new centrifugal tube, the centrifugal tube is sealed, and the new centrifugal tube is convenient for unfreezing the pollen more quickly. Then putting the pollen and a new centrifugal tube into a 35 ℃ water bath kettle for unfreezing for 15 minutes.
Comparative example 2
The refrigerated pollen provided in example 2 was thawed by the following thawing steps:
the centrifugal tube filled with pollen is taken out, the pollen is taken out from the centrifugal tube and is put into a new centrifugal tube, the centrifugal tube is sealed, and the new centrifugal tube is convenient for unfreezing the pollen more quickly. The pollen together with the new centrifuge tube was placed in a room and thawed at room temperature for 15 minutes.
Comparative example 3
The refrigerated pollen provided in example 2 was thawed by the following thawing steps:
the centrifugal tube filled with pollen is taken out, the pollen is taken out from the centrifugal tube and is put into a new centrifugal tube, the centrifugal tube is sealed, and the new centrifugal tube is convenient for unfreezing the pollen more quickly. Then placing the pollen and a new centrifugal tube in a refrigerator at the temperature of-20 ℃ for 12 hours. The tube was then thawed in a 0 ℃ ice water mixture for 15 minutes.
Test example 1
The thawed pollen provided in example 2 and comparative examples 1-3 was subjected to viability assay, respectively. Pollen activity is measured by an in vitro germination method.
In 100ml of distilled water, 0.2% boric acid and 10% sucrose were added to prepare a medium.
Dropping the prepared culture medium on a concave glass slide by using a dropper, and then uniformly dispersing the pollen on the culture medium (the surface of the culture medium has a layer of yellowish color) by using a pin. The slide glass is put in a culture dish containing the wet absorbent cotton, covered, put in a light incubator for culture and cultured for 4h at the temperature of 25 ℃. And (4) observing by using an optical microscope, and calculating the pollen germination rate by taking the length of the pollen tube exceeding the radius of the pollen as a reference, wherein the total number of the pollen in each visual field is not less than 30. Pollen germination rate (i.e. pollen vigor) ═ number of pollen that has germinated/total number of pollen x 100%. Each group of pollen was tested for 50 treatments. The test results are shown in table 1. During testing, three Chinese flowering crabapple varieties of 'Jinfengshou', 'Ziraindrop' and 'plateau red' are respectively selected.
TABLE 1 pollen viability after thawing
As can be seen from the comparison of the results in table 1, the vigor of the pollen after thawing by the thawing method provided in example 2 is significantly higher than the pollen vigor of the three comparative examples. As can be seen from table 1, the pollen viability of the "jinfeng" crabapple pollen thawed by the thawing method provided in example 2 is significantly higher than the pollen viability of the three comparative examples; the vitality of the pollen of the crabapple thawed by the thawing method provided by the embodiment 2 is obviously higher than the vitality of the pollen of the three comparative examples; the vigor of the pollen of the plateau red begonia thawed by the thawing method provided by the embodiment 2 is obviously higher than that of the three comparative examples.
Test example 2
The refrigerated pollen provided in example 2 was taken and the thawing procedure in example 2 was used, but minute amounts of pollen were taken at different thawing time periods for viability assays, respectively. After thawing for 5 minutes, 10 minutes, 15 minutes, 20 minutes and 25 minutes, a trace amount of pollen was taken out and subjected to activity measurement. Each group was determined to be 20 treatments. The measurement results are shown in Table 2. Pollen viability assay was performed according to the method of test example 1. Pollen varieties are all 'purple raindrops'.
TABLE 2 pollen viability at different thawing time
| Time of thawing | Vitality of pollen |
| 5 minutes | 80.16％ |
| 10 minutes | 96.75％ |
| 15 minutes | 97.90％ |
| 20 minutes | 79.56％ |
| 25 minutes | 67.89％ |
From the results shown in Table 2, it is clear that the thawing time also has a certain influence on the pollen viability. The pollen viability is highest when the thawing time is 10-15 minutes, and the pollen viability is reduced when the thawing time is too short or too long.
The embodiments described above are some, but not all embodiments of the invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the invention as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Claims (7)
1. The Chinese flowering crabapple pollen treatment process is characterized by comprising the following steps:
and taking out the refrigerated pollen for thawing, wherein the thawing condition comprises that the pollen is thawed in an ice-water mixture at the temperature of 0 ℃ for 10-15 minutes.
2. The begonia pollen processing process of claim 1, wherein the thawing conditions comprise thawing the pollen in an ice water mixture at 0 ℃ for 15 minutes.
3. The process for treating begonia pollen according to claim 1, wherein the cold storage condition before thawing the pollen comprises storing the pollen at-20 to-80 ℃ for 30 to 250 days.
4. The process of claim 3, wherein the cold storage conditions before thawing of the pollen comprises storage at-80 ℃ for 250 days.
5. The begonia pollen processing process according to any one of claims 1-4, wherein the collection of the pollen before cold storage comprises the following steps:
collecting buds of the flowering phase of the crabapple, taking out anthers by using tweezers, putting the naturally dried anthers in an incubator at 23-27 ℃ to dry the anthers to generate pollen, and refrigerating the pollen.
6. The begonia pollen processing technology according to claim 5, wherein the water content of the obtained pollen after drying the anther is 60-70%, then the pollen is loaded into a freezing storage tube, the freezing storage tube is placed in liquid nitrogen for 5-10s, and then the freezing storage tube is transferred to a freezer at-80 ℃ for refrigeration.
7. Use of the plum-leaf crab pollen treatment process according to any one of claims 1 to 6 in artificial pollination of plum-leaf crab.
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Application publication date: 20181123 Assignee: Jiangsu Qinghao Landscape Horticulture Co.,Ltd. Assignor: NANJING FORESTRY University Contract record no.: X2021320000137 Denomination of invention: A processing technology of Begonia pollen and Begonia pollen and its application Granted publication date: 20210907 License type: Common License Record date: 20211209 Application publication date: 20181123 Assignee: Nanjing Qingzhu Orchard Horticulture Co.,Ltd. Assignor: NANJING FORESTRY University Contract record no.: X2021320000140 Denomination of invention: A processing technology of Begonia pollen and Begonia pollen and its application Granted publication date: 20210907 License type: Common License Record date: 20211209 Application publication date: 20181123 Assignee: Nanjing Mingke Intelligent Information Technology Co.,Ltd. Assignor: NANJING FORESTRY University Contract record no.: X2021320000138 Denomination of invention: A processing technology of Begonia pollen and Begonia pollen and its application Granted publication date: 20210907 License type: Common License Record date: 20211209 |
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