CN107494522B - Low-temperature storage method for sugarcane pollen - Google Patents
Low-temperature storage method for sugarcane pollen Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention provides a low-temperature storage method of sugarcane pollen, which meets the requirements of different storage time periods of the sugarcane pollen in a mode of adjusting the water content of fresh sugarcane pollen. The method comprises the following steps: reducing the water content of fresh sugarcane pollen to 30-25%, storing for 1-5 days at 0 deg.C, and reducing the water content of fresh sugarcane pollen to about 20-15%, storing for 5-30 days at-20 deg.C. The method can prolong the vigor time of the sugarcane pollen, solve the problem of flowering asynchronism of sugarcane parents, overcome the time and space limitations in sugarcane crossbreeding, improve the breeding efficiency, provide convenience for inter-regional and even international germplasm exchange, and have the advantages of flexible storage of the sugarcane pollen, reliable technology, low cost, safe operation, good effect and the like.
Description
Technical Field
The invention relates to a method for storing sugarcane pollen at a low temperature.
Background
Pollen is indispensable for completing sexual hybridization of plants, contains all gene types of species, has abundant genetic diversity, is one of important materials for germplasm storage and exchange, but under natural conditions, the service life of most plant pollen is very short, which brings great inconvenience to the hybridization breeding work and is also not beneficial to related plant genetics research.
In 1922 Knowlton first reported that the snapdragon pollen was viable after storage at-180 ℃, and it was found that reducing the water content of the pollen increased the survival rate of the snapdragon pollen in frozen storage. In 1938, Brandes and Sartoris contained pollen of a cold-resistant clever strain in a special container, and the pollen was transported from the first place of the United states to south America to be hybridized with POJ2725, so that hybrid seedlings were obtained, and short-term storage of sugarcane pollen was realized for the first time. However, over the next decade, there has been little progress in the technology of sugar cane pollen storage. Until 1961, King created a pollen freeze-drying process that allowed a much longer storage time, and then Coleman and Buzacott performed a cross-emergence test with freeze-dried sugarcane stored pollen, respectively, and yielded a small number of seedlings. But because of the problems of practicability, exact effect and the like of freeze-drying treatment, the research work of storing the sugarcane pollen is still silent. Research on ultra-low temperature storage of plant pollen is rising day by day after the 80 s. In 1979 Akihama et al established a Pollen pool (Pollen Bank) at Japanese fruit tree laboratory, and three Pollen ultralow temperature centers (Stanwood, et al, 1986; Anonymous 1987; Parfitta 1988) were successively established in the United states in the 80 s for Pollen ultralow temperature storage and the storage mechanism was investigated (Ganeshan S.et al, 1990). In 1983, Tai takes pollen materials of a plurality of cleft hand-tight strains as storage research and uses pollen in vitro germination for activity detection, and tests of continuous years prove that the pollen of wild sugarcane cleft hand-tight is stored at ultralow temperature of-80 ℃ and the activity can be stored for at least 30 days. The technology draws the attention of the sugarcane breeding world in China and is proved by the Li Fusheng teacher who is big in Yunnan agriculture by utilizing the material of the saccharum officinarum in 2005.
However, most sugarcane strain materials with high sugar and high resistance are used in cross breeding, and because the viable cell walls of the pollen of the sugarcane strain materials are thin and sensitive to the external climate change, the sugarcane pollen loses vigor after being scattered for 1 hour, and no specific storage technology and application report related to the sugarcane pollen is found in China. With the change of climatic conditions and planting environments, the phenomena of difficult flowering of sugarcane germplasm resource parent materials and delayed flowering of parent flowering phases become more serious, and the time and labor are wasted and the effect is not obvious when the flowering phases of the parents are adjusted by methods such as photoperiod induction, sugarcane planting phase adjustment, phytohormone spraying and the like, so that the selection of the matched materials of the sugarcane crossbreeding parents is limited, the breeding of new varieties is slow, and therefore, the research and development of the low-temperature storage technology of the sugarcane pollen is necessary.
Disclosure of Invention
The invention aims to provide a low-temperature storage method for sugarcane pollen, which can solve the problem that a viable male parent pollen material is provided when parents are out of date or are hybridized remotely in sugarcane crossbreeding and has the advantages of flexible storage of sugarcane pollen, reliable technology, low cost, safe operation, good effect and the like.
The purpose of the invention can be realized by the following technical scheme:
the first scheme is that the method for storing the sugarcane pollen at low temperature comprises the following steps:
1) treatment of sugar cane pollen donor parents: selecting healthy sugarcane plants which are normal in development and free from insect damage, cutting off sugarcane parents from the middle lower parts of stems one day in advance in the full blooming period of the flower ears, bringing the sugarcane parents back to a hybridization greenhouse, and culturing the sugarcane in a sulfurous acid solution.
2) Pollen collection: the next morning, at 7:30, the flowering time of is observed, pollen is collected in time and poured into a stainless steel sieve of 40 meshes to 50 meshes, and the pollen is sieved and then is put into a centrifuge tube for later use.
3) Pollen storage: and (3) mixing the fresh pollen and the allochroic silica gel particles obtained in the step (2) according to a mass ratio of 1: 7-1: 8, fully mixing, putting into a 2ml freezing tube, and standing for 8-12 min in a refrigerating chamber at 4 ℃ to reduce the water content of the pollen to 30-25%; clamping the silica gel particles by using tweezers, and storing the silica gel particles in a refrigerator at 0 ℃ for 1-5 days.
The optimization scheme is as follows: further comprising the steps of:
4) when the pollen needs to germinate and identify, the stored pollen is directly placed in a liquid culture medium and germinates at the temperature of 28-32 ℃.
The second scheme is that the method for storing the sugarcane pollen at the low temperature comprises the following steps:
1) treatment of sugar cane pollen donor parents: the same scheme as the first scheme.
2) Pollen collection: the same scheme as the first scheme.
3) Pollen storage: and (3) mixing the fresh pollen and the allochroic silica gel particles obtained in the step (2) according to a mass ratio of 1: 4-1: 5, fully mixing, putting into a 2ml freezing tube, standing for 15-18 min in a refrigerating chamber at 4 ℃ to reduce the water content of the pollen to 20% -15%; and clamping the silica gel particles by using forceps, sealing and storing in a refrigerator at the temperature of-20 ℃ for 5-30 days.
The optimization scheme is as follows: further comprising the steps of:
4) when the pollen needs to germinate and identify, the stored pollen is spread in a wet box, is rehydrated for 30min at 4 ℃, is placed in a liquid culture medium, and germinates at 28-32 ℃.
The invention has the following prominent substantive characteristics and remarkable progress:
(1) the invention overcomes the difficulty that the sugarcane pollen is difficult to store all the time, establishes the low-temperature storage technology of the sugarcane pollen for the first time, comprises pollen collection, drying, storage, rehydration and the like, and can prolong the activity of the pollen within 30 d.
(2) The invention provides possibility for the research of sugarcane crossbreeding and the creation of new combination, and has important application value for the breeding of new sugarcane varieties. For example, the pollen of late flower variety (line) and early flower variety (line) can not be naturally hybridized; can carry out distant hybridization of sugarcane and saccharum officinarum, Heyaowang, festuca arundinacea and the like; intercrossing of sugar cane parents that are not encountered in flowering; the application of the invention can greatly enrich the combination range of sugarcane crossbreeding, break the space-time limitation of sugarcane spica hybridization and enrich the genetic diversity of sugarcane resources.
(3) The low-temperature storage method of the sugarcane pollen is simple and easy to operate, has low cost, can effectively ensure the vitality of the sugarcane pollen in hybridization, and has important practical and popularization significance in sugarcane crossbreeding.
Drawings
FIG. 1 is a diagram showing germination of low-temperature stored pollen in sugarcane stigma.
Detailed Description
The invention is further illustrated by the following specific examples:
the first embodiment is a low-temperature storage method of sugarcane pollen, and the method comprises the following steps:
step one, treating a sugar cane pollen donor parent: cutting the sugarcane female parent with the pollen back in the afternoon of the previous day and putting the sugarcane female parent with the pollen into the sulfurous acid stem-nourishing solution to ensure that the sugarcane stem provides sufficient nutrients for the flower spike to bloom.
Step two, pollen collection: the next morning, at 7:30, the flowering time of is observed, pollen is collected in time, impurities such as anthers and the like are removed by using a 40-50-mesh stainless steel sieve, and pure pollen is quickly put into a dry centrifuge tube to be sealed and stored in a dark place.
Step three, pollen storage: weighing the fresh pollen in the step two, fully mixing the fresh pollen and allochroic silica gel according to the mass ratio of 1: 7-1: 8, putting the mixture into a 2ml freezing storage tube, and standing the mixture for 8-12 min in a refrigerating chamber at 4 ℃ to reduce the water content of the pollen to 30% -25%. Then clamping the silica gel particles by using tweezers, sealing and storing at normal temperature, 4 ℃, 0 ℃ and-20 ℃ respectively, and determining the germination rate by pollen in vitro germination at certain intervals.
The results are shown in table 1: all the inactivated components are stored for 1d at normal temperature; the pollen also loses activity quickly under the environment of minus 20 ℃, and the water content of 30-25 percent of the pollen is easy to freeze below 0 ℃ so that the sugarcane pollen is frozen and died; the activity of the product is still 36.2% after being stored for 2 days at 4 ℃, and then the product is rapidly reduced and inactivated after being stored for 5 days; the pollen activity is still as high as 51.3 percent after being stored for 5 days at the temperature of 0 ℃. Considering the characteristic that the sugarcane pollen is sensitive to the external environment, from the practical application angle of artificial pollination, the pollen with the germination rate of 51.3 percent still has strong hybrid pollination advantages after being stored for 5 days, so that pollination can be seen within 5 days, and the storage way of selecting the pollen with the water content of 30 to 25 percent at the temperature of 0 ℃ is more suitable.
TABLE 1 in vitro germination rates of sugarcane pollen at different storage temperatures
The second embodiment is a low-temperature storage method of sugarcane pollen, and the method comprises the following steps:
step one, the parent treatment of the sugar cane pollen donor is the same as the embodiment one.
And step two, pollen collection is carried out in the same way as in the first embodiment.
And step three, storing the pollen, namely weighing the fresh pollen in the step two, fully mixing the fresh pollen and the allochroic silica gel particles according to the mass ratio of 1: 4-1: 5, putting the mixture into a 2ml freezing storage tube, and standing the mixture for 15-18 min in a refrigerating chamber at 4 ℃ to reduce the water content of the pollen to 20% -15%. Then clamping the silica gel particles by using tweezers, sealing and storing at normal temperature, 4 ℃, 0 ℃ and-20 ℃ respectively, and determining the germination rate by pollen in vitro germination at certain intervals.
The results are shown in table 2: as can be seen from Table 2, when the water content of the pollen is reduced to 20% -15%, the pollen is completely inactivated within 1d at normal temperature; the pollen activity is up to 70.3% after 1d of storage at 4 ℃, but is completely inactivated after 10 d; the germination rate of 18.3% still remains after 10 days of storage at 0 ℃, and no germination phenomenon exists after 20 days of in vitro germination through pollen.
The method comprises the steps of storing the pollen at the temperature of minus 20 ℃, spreading the stored pollen in a wet box, rehydrating the stored pollen at the temperature of 4 ℃ for 30min, then placing the pollen in a liquid culture medium, germinating at the temperature of 28-32 ℃, wherein the germination rate is 61.9% after 1d, 53.6% after 10d, 39.7% after 20d, and still reaching 26.1% after 30d, so that the pollen with the water content of 20-15% is selected to be stored at the temperature of minus 20 ℃, and the pollen activity can be kept for more than 30 days.
TABLE 2 in vitro germination rates of sugarcane pollen at different storage temperatures
Experimental validation example: sugarcane pollen collecting and storing method
1. Test material
In 2015 and 2016, ROC22 planted in a Hainan sugarcane breeding field is used as a pollen viability research material; the male sterile line Yunzhan 99-601 is used as the female parent material. When the flower spike of the experimental material is drawn to the size of a small pen tube, the root system of the stem node is induced to grow by adopting a high-pressure stem wrapping method, a layer of 1 cm-thick wet nutrient soil is uniformly coated among three stem nodes in the middle section of the sugarcane stem, and the wet nutrient soil is tightly wrapped and compacted by white plastic cloth to prevent the nutrient soil from evaporating too fast. Cutting the stem of the ear which blooms in the same growth vigor in the next day in the evening from 12 last ten days, returning the stem to a greenhouse, and culturing the stem with clear water to prevent each plant of the bunch of powder from being independently placed in hybridization cells separated by cloth.
2. Pollen collection
In the morning of 7:30, the flowering time of is observed, ROC22 pollen is collected in time, and the pollen is poured into a stainless steel sieve of 40 meshes to 50 meshes and then is subpackaged into 2ml centrifuge tubes.
3. Pollen storage
spreading fresh flower powder in a centrifuge tube on dry filter paper, keeping the thickness within 0.6mm, covering a layer of dry filter paper, putting the filter paper in a culture dish, sealing the filter paper with a sealing film, and storing the filter paper in a refrigerator at 4 ℃ in a dark place for 24 hours;
② mixing fresh pollen and allochroic silica gel in a centrifugal tube according to the mass ratio of 1: 7-1: 8, putting the mixture into a 2ml freezing tube, standing the mixture in a 4 ℃ refrigerating chamber for 8-12 min to reduce the water content of the pollen to 30-25%, clamping silica gel particles with tweezers, sealing, and storing the sealed mixture in a 0 ℃ refrigerator for 5 days.
and thirdly, fully mixing the fresh pollen and the allochroic silica gel in the centrifugal tube according to the mass ratio of 1: 4-1: 5, then putting the mixture into a 2ml freezing tube, standing the mixture for 15-18 min in a 4 ℃ refrigerating chamber to reduce the water content of the pollen to 20% -15%, then clamping the silica gel particles with tweezers, sealing the sealed silica gel particles, and then storing the sealed silica gel particles in a refrigerator at the temperature of-20 ℃ for 30 days.
4. Pollen viability assay
4.1 in situ pollen Germination detection
In the morning of 8:00, 120 newly-opened male sterile line Yunzan 99-601 (the flower ears are subjected to emasculation treatment at 50 ℃ for 5min in advance one day) fresh stigma are selected, 30 pollen stored in three ways are respectively awarded, wherein the pollenized stigma is used as a control, and the pollen at-20 ℃ needs to be subjected to rehydration treatment at 4 ℃ for 30min and then is used.
fixing pollen germination detection method, removing external glumes of the hybridization stigma and ovary after pollination for 4h, respectively fixing in FAA solution (89 ml of 50% ethanol, 6ml of glacial acetic acid and 5ml of formaldehyde solution are mixed), wherein the fixing time needs more than 24h, washing with ② water, pre-washing the material fixed in the FAA solution with purified ② water for three times, 2min ③, softening and transparentizing, namely softening and transparentizing the washed material with 8mol/L NaOH for 2h, then washing with ② water for three times, 2min ③, dyeing with 0.1% aniline blue (0.1 mol/L K is used), and removing the external glumes with ovary and ovary3PO4prepared) dyeing for more than 12h, ⑤ microscopic examination, namely placing the treated sugarcane ovary and stigma on a glass slide during microscopic examination, and dropwise adding a drop of phosphate buffer (0.2M, pH6.8, NaH)2PO4And Na2HPO4Or KH2PO4And K2HPO4) Cover with cover glass and press lightly with tweezers. And (3) performing microscopic examination and photography on the glass slide under near ultraviolet light by using a fluorescence microscope, wherein the wavelength is 315-380 nm. In the experiment, 10 stigma columns are observed in each treatment to ensure the accuracy of the experiment.
4.2 determination of pollen viability by hybrid pollination
Respectively granting 3 kinds of pollen stored in different modes to male sterile line Guangdong sugar 94-128 (the flower spike is subjected to emasculation treatment at 50 ℃ for 5min one day in advance), treating 3 spikes each time, continuously pollinating for 3 days, taking selfing as a control, then cutting off the small spikes which do not flower and pollinate, bagging, transferring to the sun, waiting for maturity under a water culture condition, and after the seeds are mature, counting the emergence rate after sowing for 20 days according to a conventional method.
5 analysis of results
5.1 in situ pollen Germination detection
The sugarcane stigma surface secretion has high viscosity, sugarcane stored pollen can be attached to the stigma surface in a large amount at first no matter whether the sugarcane stored pollen is alive or not, the viable pollen can germinate gradually under the induction of the viscous secretion, the pollen tube slowly extends, the pollen tube is inserted into the stigma and slowly extends to the ovary bottom, the ungerminated pollen is lost in the process of washing the stigma with clear water, and the stored pollen vitality can be judged by observing the adhesion of the stigma pollen and the elongation of the pollen tube. As seen in FIG. 1, the stored pollens of sugarcane all germinated after pollination for 4h, indicating that the stored pollens all had different degrees of vigor; no pollen is seen on the surface of the inbred stigma in a reverse view, which shows that the sugarcane 99-601 selected in the test is male sterile or has poor pollen development, and the accuracy of the test can be greatly improved. According to the number of the attached pollen on a single stigma, the pollen is stored at 4 ℃ for 24h and more than 0 ℃ for 5d and stored at 20 ℃ for 30 d; the pollen tube has been extended to the vicinity of the ovary at the bottom of the pollen tube after pollination for 4 hours after storage for 24 hours at 4 ℃ and 5 days at 0 ℃ for 4 hours over the extended length of the pollen tube, while the pollen tube extends only to 2/3 of the stigma after storage for 30 days at-20 ℃, and the growth rate of the pollen tube is lower than that of the two stored pollen tubes. The result shows that the sugarcane pollen can achieve the purpose of prolonging the pollen vitality through three different storage modes, but as the storage time is prolonged, the number of viable pollen is reduced, and the pollen vitality is also reduced.
5.2 determination of pollen viability by hybrid pollination
And (3) completing an artificial pollination hybridization test of 12 combined stored pollen in 2015-2016 hybridization seasons, wherein 3 are female parent selfing control combinations, and all seedlings are obtained in 9 individual artificial pollination combinations. In 3 repeated combinations of sugarcane 99-601 selfing control, the number of seedlings is slightly more than that of only 1 combination, and the number of the other repeated seedlings is less than 10, which shows that the male killing effect by using warm water in the test is ideal. The 3 repeated seedlings of the hybridization combination of ROC22 pollen pollination stored for 24h at 4 ℃ by taking sugarcane 99-601 as a female parent reach more than 1000, which shows that the stored pollen has excellent vitality; the emergence rates of three hybrid combined flower spikes stored at 0 ℃ for 5d pollination are over 600 plants, wherein the repeated emergence rate of one of the three hybrid combined flower spikes is up to 962 plants, which indicates that the storage effect is good; in three repeated combinations of pollen and cane 99-601 hybridized after being stored for 30 days at-20 ℃, the emergence rate is as high as more than 300 plants and is far higher than that of a control combination, and thus, a part of pollen can be normally pollinated and fruited after the ROC22 pollen is stored for 30 days.
In conclusion, the two low-temperature storage technologies of the sugarcane pollen are effective methods for prolonging the vitality of the sugarcane pollen, more filial generations can be obtained through artificial pollination, the space-time limitation of flowering sterility is broken, and the sugarcane cross breeding efficiency is improved.
TABLE 3 storage pollen pollination fructification statistics
Claims (4)
1. The low-temperature storage method of the sugarcane pollen is characterized by comprising the following steps:
1) treatment of sugar cane pollen donor parents: selecting healthy sugarcane plants which are normal in development and free from insect damage, cutting off sugarcane parents from the middle lower parts of stems one day in advance in the full blooming period of the flower ears, bringing the sugarcane parents back to a hybridization greenhouse and raising the sugarcane in a sulfurous acid solution;
2) pollen collection: the next morning, at 7:30, flowering time is observed, pollen is collected in time and poured into a stainless steel sieve of 40 meshes to 50 meshes, and the pollen is sieved and then is put into a centrifuge tube for later use;
3) pollen storage: and (3) mixing the fresh pollen and the allochroic silica gel particles obtained in the step (2) according to a mass ratio of 1: 7-1: 8, fully mixing, putting into a 2ml freezing tube, and standing for 8-12 min in a refrigerating chamber at 4 ℃ to reduce the water content of the pollen to 30-25%; then clamping the silica gel particles by using tweezers, and storing the silica gel particles in a refrigerator at 0 ℃ for 1-5 days.
2. The method for low temperature storage of sugar cane pollen according to claim 1 further comprising the steps of:
4) and (3) when the pollen needs germination identification, directly placing the pollen stored in the step (3) into a liquid culture medium, and germinating at the temperature of 28-32 ℃.
3. The low-temperature storage method of the sugarcane pollen is characterized by comprising the following steps:
1) treatment of sugar cane pollen donor parents: selecting healthy sugarcane plants which are normal in development and free from insect damage, cutting off sugarcane parents from the middle lower parts of stems one day in advance in the full blooming period of the flower ears, bringing the sugarcane parents back to a hybridization greenhouse and raising the sugarcane in a sulfurous acid solution;
2) pollen collection: the next morning, at 7:30, flowering time is observed, pollen is collected in time and poured into a stainless steel sieve of 40 meshes to 50 meshes, and the pollen is sieved and then is put into a centrifuge tube for later use;
3) pollen storage: and (3) mixing the fresh pollen and the allochroic silica gel particles obtained in the step (2) according to a mass ratio of 1: 4-1: 5, fully mixing, putting into a 2ml freezing tube, standing for 15-18 min in a refrigerating chamber at 4 ℃ to reduce the water content of the pollen to 20% -15%; then clamping the silica gel particles by using tweezers, and storing the silica gel particles in a refrigerator at the temperature of-20 ℃ for 5-30 days.
4. The method for low temperature storage of sugar cane pollen according to claim 3 further comprising the steps of:
4) when the pollen needs to germinate and identify, the pollen stored in the step (3) is spread in a wet box, is rehydrated for 30min at 4 ℃, is placed in a liquid culture medium, and germinates at 28-32 ℃.
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Application publication date: 20171222 Assignee: Inner Mongolia Zhongxing Seed Industry Co.,Ltd. Assignor: Nanfan Seed Industry Research Institute Guangdong Academy of Sciences Contract record no.: X2023980034574 Denomination of invention: Low temperature storage method for sugarcane pollen Granted publication date: 20200519 License type: Common License Record date: 20230411 |