CN112400484B - Cassava pollen collecting and storing method - Google Patents
Cassava pollen collecting and storing method Download PDFInfo
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- CN112400484B CN112400484B CN202011362329.4A CN202011362329A CN112400484B CN 112400484 B CN112400484 B CN 112400484B CN 202011362329 A CN202011362329 A CN 202011362329A CN 112400484 B CN112400484 B CN 112400484B
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- 240000003183 Manihot esculenta Species 0.000 title claims abstract description 82
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 title claims abstract description 80
- 238000000034 method Methods 0.000 title claims abstract description 41
- 238000001035 drying Methods 0.000 claims abstract description 50
- 238000004321 preservation Methods 0.000 claims abstract description 36
- 230000001954 sterilising effect Effects 0.000 claims abstract description 15
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 15
- 238000007710 freezing Methods 0.000 claims description 43
- 230000008014 freezing Effects 0.000 claims description 43
- 238000003860 storage Methods 0.000 claims description 33
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 28
- 239000007788 liquid Substances 0.000 claims description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 16
- 229910052757 nitrogen Inorganic materials 0.000 claims description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 claims description 8
- 238000011084 recovery Methods 0.000 claims description 7
- 238000005507 spraying Methods 0.000 claims description 7
- 238000005286 illumination Methods 0.000 claims description 6
- 230000001678 irradiating effect Effects 0.000 claims description 6
- 239000002655 kraft paper Substances 0.000 claims description 6
- 238000010257 thawing Methods 0.000 claims description 6
- 238000001514 detection method Methods 0.000 abstract description 6
- 238000003976 plant breeding Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 40
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- 244000005700 microbiome Species 0.000 description 6
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- ZOXJGFHDIHLPTG-UHFFFAOYSA-N Boron Chemical compound [B] ZOXJGFHDIHLPTG-UHFFFAOYSA-N 0.000 description 5
- 241000241413 Propolis Species 0.000 description 5
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- 239000007864 aqueous solution Substances 0.000 description 4
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- 238000004043 dyeing Methods 0.000 description 4
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- 235000010339 sodium tetraborate Nutrition 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
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- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 2
- 241000257303 Hymenoptera Species 0.000 description 2
- DKNPRRRKHAEUMW-UHFFFAOYSA-N Iodine aqueous Chemical compound [K+].I[I-]I DKNPRRRKHAEUMW-UHFFFAOYSA-N 0.000 description 2
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01D—HARVESTING; MOWING
- A01D91/00—Methods for harvesting agricultural products
- A01D91/04—Products growing above the soil
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
- A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N3/00—Preservation of plants or parts thereof, e.g. inhibiting evaporation, improvement of the appearance of leaves or protection against physical influences such as UV radiation using chemical compositions; Grafting wax
Abstract
The invention relates to the technical field of plant breeding, in particular to a cassava pollen collecting and preserving method. The method of the invention comprises the following steps: (1) collecting male flower pollen, (2) sterilizing, (3) drying pollen, (4) preserving pollen, and (5) reviving pollen; the invention firstly provides a system for cassava pollen collection, sterilization, drying, preservation, resuscitation, vitality detection and the like, and optimizes parameters in the system.
Description
[ technical field ] A method for producing a semiconductor device
The invention belongs to the technical field of plant breeding, and particularly relates to a cassava pollen collecting and storing method.
[ background of the invention ]
Cassava (the scientific name: Manihot esculenta Crantz) is a plant of the genus Manihot of the family Euphorbiaceae, is drought-resistant and barren-resistant, is widely planted in more than 100 countries or regions such as Africa, America and Asia, is one of three potato crops, is the third food crop in a hot zone, is the sixth food crop in the world, is called the king of starch, and is the grain for nearly six hundred million people in the world. Therefore, the optimization of the cassava cultivation is of great significance.
Cassava is a cross-pollinated crop with the same strain, the male flower and the female flower have the same sequence and are cross-pollinated, the common male flower on the same inflorescence blooms 1-2 weeks later than the female flower, and the cassava normally matures about 90 days after blooming and pollination. The blooming time of different varieties of cassava is inconsistent. In addition, in the breeding work, in order to better select hybrid parents according to breeding targets and select varieties with more advantages, less defects and complementary advantages and defects as the parents, the collection of cassava male flowers and the preservation of pollen vitality are necessary.
[ summary of the invention ]
In view of the above, the invention provides a cassava pollen collecting and preserving method, which firstly provides systems for cassava pollen collection, drying, sterilization, preservation, resuscitation, vitality detection and the like, optimizes parameters in the systems, has the characteristic of strong operability, and can maintain the vitality of the cassava pollen for a long time, thereby improving the success rate of artificial hybridization pollination of cassava.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a collection and preservation method of cassava pollen comprises the following steps:
(1) collecting male flower pollen: selecting a parent serving as a male parent, observing the flowering phase of the parent, collecting male flowers at 10:30-12:30 in the morning when the male flowers are bright in color but do not bloom, and bringing the collected male flowers back to a laboratory;
the collecting bottle is made of transparent materials and comprises a collecting chamber and a drying chamber, an interlayer is arranged between the collecting chamber and the drying chamber and is a hollow interlayer, and a breathable biological film or filter paper is arranged on the hollow interlayer; the top of the collecting chamber is provided with a first sealing cover, and the bottom of the drying chamber is provided with a second sealing cover; when the collecting bottle is used, the second sealing cover is opened, the drying agent is placed in the drying chamber, then the collecting bottle is taken to the field to collect the inflorescence of the male flowers, the first sealing cover is opened, and the male flowers are placed and then taken back to the laboratory.
(2) And (3) sterilization: uniformly spraying medical alcohol on the surface of the cassava male flowers collected in the step (1), and then placing under an ultraviolet lamp for irradiating for 15-25min to obtain sterilized cassava male flowers;
(3) drying pollen: collecting pollen of the sterilized male cassava flowers, drying the collected cassava pollen until the water content is 10-20%, classifying the collected cassava pollen according to varieties on a super-clean workbench, and shaking off the yellow pollen on the sterilized kraft paper for collection;
(4) and (3) pollen preservation: respectively filling the cassava pollen obtained in the step (3) into a freezing storage tube, and storing at low temperature;
(5) and (3) pollen recovery: taking out the preserved pollen from the freezing chamber, and thawing for 0.5h at normal temperature to finish pollen resuscitation.
In the invention, further, the specific manner of pollen collection in the step (3) is as follows: placing the sterilized male cassava flowers in a sterile room, drying at the temperature of 24-26 ℃ and under the illumination intensity of 100-.
In the invention, further, the time for collecting the male flowers in the step (1) is 10:30 in the morning.
In the present invention, the low-temperature preservation in the step (4) is specifically: storing the pollen which needs to be used within 3 months in a freezing storage tube filled with the pollen at the temperature lower than 10 ℃.
In the present invention, the low-temperature preservation in the step (4) is specifically: the pollen to be used after 3 months is stored in a freezing chamber below-20 ℃ after the freezing tube filled with the pollen is placed in liquid nitrogen for 7-9 s.
In the present invention, the medical alcohol in the step (2) is 70-75% by volume.
The application also provides a method for collecting the preserved pollen to carry out artificial pollination on cassava by using the method, which specifically comprises the following steps:
(1) and (3) detecting the activity of the pollen: detecting pollen activity by adopting a carmine acetate dyeing method or an iodine-potassium iodide dyeing method; the activity of the pollen is more than 60 percent;
(3) preparation of pollen: uniformly dispersing pollen which passes the activity detection requirement into an aqueous solution to prepare pollination liquid; the mass ratio of the aqueous solution to the pollen is 35: 1, mixing the water solution, the propolis and the borax according to a mass ratio of 25:1:0.5 to obtain the water solution;
(3) artificial hybridization and pollination: at 10-12 am of the full-bloom period of the female parent, when the female flower is about to open, the top of the female flower is lightly touched to open the petals and expose the stigma, the pollination liquid is filled into a small sprayer to spray on the stigma of the female flower, then the pollinated female flower is sleeved by a bag and fixed on a flower stem or a petiole by a clip, and pollination by other media such as wind, insects and the like is avoided.
The invention has the following beneficial effects:
1. the invention firstly provides a system for cassava pollen collection, drying, sterilization, preservation, resuscitation, activity detection and the like, and optimizes parameters in the system.
2. According to the method, the optimal collection time of the inflorescence of the male flowers is determined, the male flowers are filled into the collection bottle and are taken back, the collection bottle comprises a collection chamber and a drying chamber, a drying agent is placed in the drying chamber, the drying chamber and the collection chamber are separated by a partition plate, the drying agent can dry the male cassava flowers in the collection chamber through the partition plate, and the phenomenon that the pollen activity preservation is influenced due to too large humidity in the collection chamber caused by water drops carried by the drying agent is avoided; the drying chamber is separated from the collecting chamber, so that the phenomenon that drying is uneven and the like due to direct contact of the drying agent and the flowers is avoided. The method is characterized in that the male flower inflorescence is brought back and sprayed on the surface of the male flower by medical alcohol, then the male flower is irradiated under an ultraviolet lamp for sterilization treatment, because the collection of the male cassava flower needs to be carried out in the flowering phase, insects such as bees and the like are easy to be incurred in the flowering phase, at the moment, collected pollen has more or less harmful microorganisms, and the microorganisms influence the activity of the pollen in the pollen preservation process. For solving above-mentioned problem, this application is gathering the male flower when the male flower shows that the colour is comparatively bright-coloured and not open, insects such as honeybee of adopting honey this moment are less, it uses medical alcohol and ultraviolet lamp to shine to disinfect to coordinate again, can collect pollen, kill the microorganism before the preservation, pollen can be preserved for a long time and not metamorphism after the disinfection, after that through carrying out drying process with the male flower in aseptic chamber, can not destroy the activity of pollen simultaneously by rapid draing, can observe the condition of bursting of anther simultaneously, be convenient for collect.
3. The pollen preservation method comprises the steps of preserving within 3 months and preserving after 3 months, and the activity of the pollen with the foreign bacteria removed is greatly improved due to the sterilization treatment, and the preservation of the activity is facilitated in the storage process, so that the pollen used within 3 months only needs to be preserved in an environment at the temperature lower than 10 ℃, does not need to be preserved in a freezing chamber with low temperature, and saves the cost and resources; secondly, pollen used after 3 months is firstly treated by liquid nitrogen and then stored in a freezing chamber, the liquid nitrogen can enable the pollen to be frozen quickly in a short time, the occurrence of the freezing injury phenomenon is reduced, the liquid nitrogen treatment time is not too long or too short, and the treatment time is preferably 8 s.
4. The invention also provides a pollination method of cassava, the collected and stored pollen is used for pollination treatment, in the pollination process, firstly, the pollen is subjected to activity detection, the quality of the pollen is ensured, the pollination success rate is improved, secondly, the pollen is dispersed into suspension, pollination is carried out by spraying a stigma, the suspension also comprises propolis and borax, the propolis can attract bees and is also beneficial to pollen germination, a pollen tube is formed, the pollinated flower can quickly complete the fertilization process, and in addition, the propolis has certain viscosity to promote the contact of the pollen and the surface of a female flower; borax provides boron element, boron is a special essential element of higher plants, the amount of boron in flowers in each organ of the plants is the highest, and stigma and ovary are the highest in the flowers; boron has important influence on the reproductive process of plants and is closely related to pollen formation, pollen tube germination and fertilization; boron can promote pollen germination and pollen container growth, and is beneficial to completing the fertilization process; this send and to overlap the bag processing to the style after the pollination, can effectually avoid the influence of above-mentioned condition to the pollination, can also play after the cover bag and moisturize and keep style pollen concentration in certain space, promote the pollination fertile fruit.
[ description of the drawings ]
FIG. 1 is a schematic view of the structure of a collection bottle of the present invention.
In the figure, 1 is a collection chamber, 2 is a drying chamber, 3 is an interlayer, 4 is a first sealing cover, and 5 is a second sealing cover.
The following detailed description will further illustrate the invention in conjunction with the above-described figures.
[ detailed description ] embodiments
The present invention will be further described with reference to examples and tests.
Example 1:
the embodiment provides a cassava pollen collecting and storing method, which comprises the following steps:
(1) collecting male flower pollen: selecting a parent serving as a male parent, observing the flowering phase of the parent, collecting male flowers at 10:30 a.m. when the male flowers are bright in color but do not bloom, and bringing the collected male flowers back to a laboratory; the collecting bottle is made of transparent materials and comprises a collecting chamber 1 and a drying chamber 2, an interlayer 3 is arranged between the collecting chamber 1 and the drying chamber 2 and is a hollow interlayer, and a breathable biological film or filter paper is arranged on the hollow interlayer; the top of the collecting chamber is provided with a first sealing cover 4, and the bottom of the drying chamber is provided with a second sealing cover 5; when the device is used, the second sealing cover 5 is opened, the drying agent is placed in the drying chamber 2, then the collecting bottle is taken to the field to collect the inflorescence of the male flowers, the first sealing cover 4 is opened, and the male flowers are placed and then taken back to the laboratory;
(2) and (3) sterilization: uniformly spraying medical alcohol on the surface of the cassava male flowers collected in the step (1), and then placing under an ultraviolet lamp for irradiating for 15min to obtain sterilized cassava male flowers;
(3) drying pollen: collecting pollen of the sterilized male cassava flowers, drying the collected cassava pollen until the water content is 10%, classifying the collected cassava pollen according to varieties on an ultra-clean workbench, and shaking off yellow pollen on the sterilized kraft paper for collection; the specific way of pollen collection is as follows: placing the sterilized male cassava flowers in a sterile room, drying at 24 ℃ under the condition of illumination intensity of 100W, shaking the male cassava flowers to collect scattered pollen after anthers burst, and finishing the operation of collecting the pollen; the medical alcohol is 70% medical alcohol by volume;
(4) and (3) pollen preservation: filling the pollen obtained in the step (3) into a freezing storage tube, and storing at low temperature; wherein, the pollen which needs to be used within 3 months is preserved in a freezing storage tube filled with the pollen at the temperature lower than 10 ℃; placing the pollen in a freezing storage tube filled with pollen in liquid nitrogen for 7s after 3 months, and then transferring the pollen into a freezing chamber below-20 ℃ for storage;
(5) and (3) pollen recovery: taking out the preserved pollen from the freezing chamber, and thawing for 0.5h at normal temperature to finish pollen resuscitation.
Example 2:
the embodiment provides a cassava pollen collecting and storing method, which comprises the following steps:
(1) collecting male flower pollen: selecting a parent serving as a male parent, observing the flowering phase of the parent, collecting male flowers at 11:30 am when the male flowers are bright in color but do not bloom, and bringing the collected male flowers back to a laboratory; the collecting bottle is made of transparent materials and comprises a collecting chamber 1 and a drying chamber 2, an interlayer 3 is arranged between the collecting chamber 1 and the drying chamber 2 and is a hollow interlayer, and a breathable biological film or filter paper is arranged on the hollow interlayer; the top of the collecting chamber is provided with a first sealing cover 4, and the bottom of the drying chamber is provided with a second sealing cover 5; when the device is used, the second sealing cover 5 is opened, the drying agent is placed in the drying chamber 2, then the collecting bottle is taken to the field to collect the inflorescence of the male flowers, the first sealing cover 4 is opened, and the male flowers are placed and then taken back to the laboratory;
(2) and (3) sterilization: uniformly spraying medical alcohol on the surface of the cassava male flowers collected in the step (1), and then placing under an ultraviolet lamp for irradiating for 20min to obtain sterilized cassava male flowers;
(3) drying pollen: collecting pollen of the sterilized male cassava flowers, drying the collected cassava pollen until the water content is 15%, classifying the collected cassava pollen according to varieties on an ultra-clean workbench, and shaking off yellow pollen on the sterilized kraft paper for collection; the specific way of pollen collection is as follows: placing the sterilized male cassava flowers in a sterile room, drying at the temperature of 25 ℃ and under the illumination intensity of 125W, shaking the male cassava flowers to collect scattered pollen after anthers burst, and finishing the operation of collecting the pollen; the medical alcohol is 72% medical alcohol by volume;
(4) and (3) pollen preservation: filling the pollen obtained in the step (3) into a freezing storage tube, and storing at low temperature; wherein, the pollen which needs to be used within 3 months is preserved in a freezing storage tube filled with the pollen at the temperature lower than 10 ℃; placing the pollen in a freezing storage tube filled with pollen in liquid nitrogen for 8s after 3 months, and then transferring the pollen into a freezing chamber below-20 ℃ for storage;
(5) and (3) pollen recovery: taking out the preserved pollen from the freezing chamber, and thawing for 0.5h at normal temperature to finish pollen resuscitation.
Example 3:
the embodiment provides a cassava pollen collecting and storing method, which comprises the following steps:
(1) collecting male flower pollen: selecting a parent serving as a male parent, observing the flowering phase of the parent, collecting male flowers at 12:30 a.m. when the male flowers are bright in color but do not bloom, and bringing the collected male flowers back to a laboratory; the collecting bottle is made of transparent materials and comprises a collecting chamber 1 and a drying chamber 2, an interlayer 3 is arranged between the collecting chamber 1 and the drying chamber 2 and is a hollow interlayer, and a breathable biological film or filter paper is arranged on the hollow interlayer; the top of the collecting chamber is provided with a first sealing cover 4, and the bottom of the drying chamber is provided with a second sealing cover 5; when the device is used, the second sealing cover 5 is opened, the drying agent is placed in the drying chamber 2, then the collecting bottle is taken to the field to collect the inflorescence of the male flowers, the first sealing cover 4 is opened, and the male flowers are placed and then taken back to the laboratory;
(2) and (3) sterilization: uniformly spraying medical alcohol on the surface of the cassava male flowers collected in the step (1), and then placing under an ultraviolet lamp for irradiating for 25min to obtain sterilized cassava male flowers;
(3) drying pollen: collecting pollen of the sterilized male cassava flowers, drying the collected cassava pollen until the water content is 20%, classifying the collected cassava pollen according to varieties on an ultra-clean workbench, and shaking off the yellow pollen on the sterilized kraft paper for collection; the specific way of pollen collection is as follows: placing the sterilized male cassava flowers in a sterile room, drying at 26 ℃ under the condition of illumination intensity of 150W, shaking the male cassava flowers to collect scattered pollen after anthers burst, and finishing the operation of collecting the pollen; the medical alcohol is 75% by volume;
(4) and (3) pollen preservation: filling the pollen obtained in the step (3) into a freezing storage tube, and storing at low temperature; wherein, the pollen which needs to be used within 3 months is preserved in a freezing storage tube filled with the pollen at the temperature lower than 10 ℃; placing the pollen in a freezing storage tube filled with pollen in liquid nitrogen for 9s after 3 months, and then transferring the pollen into a freezing chamber below-20 ℃ for storage;
(5) and (3) pollen recovery: taking out the preserved pollen from the freezing chamber, and thawing for 0.5h at normal temperature to finish pollen resuscitation.
Example 4
The embodiment provides a cassava pollination method, which comprises the following steps:
(1) collecting male flower pollen: selecting a parent serving as a male parent, observing the flowering phase of the parent, collecting male flowers at 11:30 am when the male flowers are bright in color but do not bloom, and bringing the collected male flowers back to a laboratory; the collecting bottle is made of transparent materials and comprises a collecting chamber 1 and a drying chamber 2, an interlayer 3 is arranged between the collecting chamber 1 and the drying chamber 2 and is a hollow interlayer, and a breathable biological film or filter paper is arranged on the hollow interlayer; the top of the collecting chamber is provided with a first sealing cover 4, and the bottom of the drying chamber is provided with a second sealing cover 5; when the device is used, the second sealing cover 5 is opened, the drying agent is placed in the drying chamber 2, then the collecting bottle is taken to the field to collect the inflorescence of the male flowers, the first sealing cover 4 is opened, and the male flowers are placed and then taken back to the laboratory;
(2) and (3) sterilization: uniformly spraying medical alcohol on the surface of the cassava male flowers collected in the step (1), and then placing under an ultraviolet lamp for irradiating for 20min to obtain sterilized cassava male flowers;
(3) drying pollen: collecting pollen of the sterilized male cassava flowers, drying the collected cassava pollen until the water content is 15%, classifying the collected cassava pollen according to varieties on an ultra-clean workbench, and shaking off yellow pollen on the sterilized kraft paper for collection; the specific way of pollen collection is as follows: placing sterilized male cassava flowers in a sterile room, drying for 17h under the conditions that the temperature is 25 ℃ and the illumination intensity is 125W, shaking the male cassava flowers to collect scattered pollen after anthers burst, and finishing the operation of collecting pollen; the medical alcohol is 80% by volume;
(4) and (3) pollen preservation: filling the pollen obtained in the step (3) into a freezing storage tube, and storing at low temperature; wherein, the pollen which needs to be used within 3 months is preserved in a freezing storage tube filled with the pollen at the temperature lower than 10 ℃; placing the pollen in a freezing storage tube filled with pollen in liquid nitrogen for 8s after 3 months, and then transferring the pollen into a freezing chamber below-20 ℃ for storage;
(5) and (3) pollen recovery: taking out the preserved pollen from the freezing chamber, and thawing for 0.5h at normal temperature to finish pollen resuscitation;
(6) and (3) detecting the activity of the pollen: detecting pollen activity by adopting a carmine acetate dyeing method or an iodine-potassium iodide dyeing method; the activity of the pollen is more than 60 percent;
(7) preparation of pollen: uniformly dispersing pollen which passes the activity detection requirement into an aqueous solution to prepare pollination liquid; the mass ratio of the aqueous solution to the pollen is 35: 1, mixing the water solution, the propolis and the borax according to a mass ratio of 25:1:0.5 to obtain the water solution;
(8) artificial hybridization and pollination: at 10-12 am of the full-bloom period of the female parent, when the female flower is about to open, the top of the female flower is lightly touched to open the petals and expose the stigma, the pollination liquid is filled into a small sprayer to spray on the stigma of the female flower, then the pollinated female flower is sleeved by a bag and fixed on a flower stem or a petiole by a clip, and pollination by other media such as wind, insects and the like is avoided.
Effect verification
To further illustrate the utility value of the present application, applicants conducted the following tests:
test one: comparing pollination success rates before and after preservation of pollen in each group
A first group: pollen according to example 2 of the present application;
second group: collecting male flower pollen at 10:30 of the day of male flower blooming, directly collecting male flower pollen in a test field, and carrying out other modes strictly according to the example 2;
third group: the sterilization step was eliminated and the other procedure was carried out exactly as in example 2;
and a fourth group: the steps of pollen preservation are modified as follows: subpackaging cassava pollen into a freezing storage tube, and storing the freezing storage tube filled with the cassava pollen in an environment with the temperature lower than 10 ℃; otherwise exactly as in example 2;
and a fifth group: the steps of pollen preservation are modified as follows: subpackaging sterilized cassava pollen into a freezing tube, placing the freezing tube filled with the pollen in liquid nitrogen for 8s, and then transferring the freezing tube into a freezing chamber below-20 ℃ for storage; otherwise exactly as in example 2;
a sixth group: the collecting bottle is changed into a common collecting bag or a common collecting bottle, and the drying chamber is not arranged, and only the collecting chamber is arranged; otherwise exactly as in example 2;
the above groups were stored as follows: after the pollen in the step (2) is dried, the pollination operation is carried out according to the steps (5) to (7) in the embodiment 4, and after the pollen is stored, the pollination operation is carried out according to the steps (5) to (7) in the embodiment 4; each group of test fields is one mu of land, and the average pollination success rate of each group is calculated; the recorded data are shown in table 1:
and (3) successful pollination:
TABLE 1 pollination success Rate before and after pollen preservation
According to the results in table 1, the success rate of pollination of cassava pollen stored by the method is not reduced, but slightly improved, because the pollen passes through a dormancy stage after being subjected to cryopreservation and activity recovery after returning to the temperature by a scientific and reasonable storage method, the success rate of actual pollination is improved, and because the cassava flower is sterilized, microorganisms carried by the cassava flower are killed, so that the success rate of pollination of the pollen is higher. The third group of microorganisms affects the activity of pollen during pollen preservation, so the pollination success rate after preservation is not much different and is lower than that before preservation. The fourth group can be stored only for 3-4 months as it is stored at 10 deg.C, and thus the activity is reduced after 8 months of storage, resulting in a low success rate of authorization at this time. The pollination survival rate was also high in the fifth group, but the preservation cost was much higher than that described in example 2 of the present application.
And (2) test II: comparing pollen Activity of groups
The method comprises the following steps of (1) dividing the pollen into the following groups, and respectively measuring the vitality of the pollen;
the following is the same way except for the different steps or parameters highlighted, i.e. the collection and preservation method described in example 2.
1. The effect of different collection times on pollen activity was compared as shown in table 2:
TABLE 2 Effect of different collection times on pollen Activity
As can be seen from Table 2, the pollen vitality of the cassava pollen collected at different times in one day is different, and the vitality value of the cassava pollen at the ratio of 11:30 is the highest value, which is the best collection time.
2. Comparing the effect of different treatment modes on the activity of the pollen preserved at ultralow temperature for more than 3 months, the data are recorded as shown in table 3:
table 3 compares the effect of different treatments on pollen activity
According to the table 3, the activity of the pollen can be maintained by performing liquid nitrogen treatment on the pollen before refrigeration in the mode of the application, the pollen is frozen and loses activity due to direct refrigeration without adopting liquid nitrogen treatment, the liquid nitrogen treatment time is not too short or too long, and the applicant determines that the optimal liquid nitrogen treatment time is 8s through multiple experiments.
3. Comparing the influence of different preservation modes on the pollen activity, the activity of the pollen after 3 months of preservation is detected and classified into the following groups, and the recorded data are shown in table 4:
a first group: pollen Collection and preservation method described in example 2
Second group: the sterilization step was eliminated and the other procedure was carried out exactly as in example 2;
third group: the pollen is preserved as follows: subpackaging the sterilized cassava pollen obtained in the step (2) into a freezing storage tube, and storing the freezing storage tube filled with the pollen in an environment with the temperature lower than 10 ℃; otherwise exactly as in example 2;
and a fourth group: removing the sterilization step; meanwhile, the pollen preservation is that the sterilized cassava pollen obtained in the step (2) is subpackaged into a freezing storage tube, and the freezing storage tube filled with the pollen is preserved in an environment with the temperature lower than 10 ℃; otherwise the procedure was exactly as in example 2.
TABLE 4 Effect of different preservation methods on pollen Activity (I)
4. The activities of the above groups of pollen after 12 months of storage were continuously compared and the data recorded are shown in table 5:
TABLE 5 Effect of different preservation methods on pollen Activity (II)
As can be seen from the data in tables 4 and 5, the preservation method of the present application can maintain the activity of pollen, and the fourth group does not sterilize pollen, which results in the activity of pollen being continuously destroyed by microorganisms during the cold storage process, but the cold storage temperature cannot meet the requirement for maintaining activity.
The above description is intended to describe in detail the preferred embodiments of the present invention, but the embodiments are not intended to limit the scope of the claims of the present invention, and all equivalent changes and modifications made within the technical spirit of the present invention should fall within the scope of the claims of the present invention.
Claims (3)
1. A cassava pollen collecting and preserving method is characterized by comprising the following steps:
(1) collecting male flower pollen: selecting a parent serving as a male parent, observing the flowering phase of the parent, collecting male flowers at 11:30 am when the male flowers are bright in color but do not bloom, and bringing the collected male flowers back to a laboratory;
(2) and (3) sterilization: uniformly spraying medical alcohol on the surface of the cassava male flowers collected in the step (1), and then placing under an ultraviolet lamp for irradiating for 15-25min to obtain sterilized cassava male flowers;
(3) drying pollen: collecting pollen of the sterilized male cassava flowers, drying the collected cassava pollen until the water content is 10-20%, classifying the collected cassava pollen according to varieties on a super-clean workbench, and shaking off the yellow pollen on the sterilized kraft paper for collection;
(4) and (3) pollen preservation: respectively filling the cassava pollen obtained in the step (3) into a freezing storage tube, and storing at low temperature;
(5) and (3) pollen recovery: taking out the preserved pollen from the freezing chamber, and thawing for 0.5h at normal temperature to finish pollen resuscitation;
the low-temperature preservation in the step (4) is specifically as follows: storing the pollen which needs to be used within 3 months in a freezing storage tube filled with the pollen at the temperature lower than 10 ℃;
the low-temperature preservation in the step (4) is specifically as follows: the pollen to be used after 3 months is stored in a freezing chamber below-20 ℃ after the freezing tube filled with the pollen is placed in liquid nitrogen for 7-9 s.
2. The method for collecting and preserving cassava pollen according to claim 1, wherein the pollen collection in step (3) is carried out in a specific manner: placing the sterilized male cassava flowers in a sterile room, drying at the temperature of 24-26 ℃ and under the illumination intensity of 100-.
3. The method for collecting and preserving cassava pollen according to claim 1, wherein the medical alcohol in the step (2) is 70-75% by volume.
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