CN115005094A - Composition for improving artificial hybridization fertilization success rate of polygonatum sibiricum, and improving method and application thereof - Google Patents
Composition for improving artificial hybridization fertilization success rate of polygonatum sibiricum, and improving method and application thereof Download PDFInfo
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Abstract
The invention provides a composition for improving the artificial hybridization fertilization success rate of polygonatum sibiricum, and an improvement method and application thereof, and relates to the technical field of artificial hybridization. The invention designs a pollen culture medium aiming at pollen and a stigma treating agent aiming at stigma respectively, so as to achieve the purpose of improving the activity of polygonatum pollen and the stigma receptivity and further realize the success rate of hybridization, and meanwhile, indexes such as seed setting rate, hybrid seed germination and the like are obviously improved through technical improvement.
Description
Technical Field
The invention belongs to the technical field of artificial hybridization, and particularly relates to a composition for improving the artificial hybridization fertilization success rate of polygonatum sibiricum, and an improvement method and application thereof.
Background
The Polygonatum plants belong to the genus Polygonatum (Polygonatum) of the family Liliaceae (Liliaceae), are distributed from subtropical zones to near polar regions in the northern hemisphere, are widely used as traditional Chinese medicinal plants in China, are gradually depleted along with the mass harvest of field Polygonatum resources, are an important path for developing the Polygonatum pharmaceutical industry through artificial cultivation and breeding, are mainly subjected to vegetative propagation by using rhizome stems in the artificial cultivation process of the existing Polygonatum plants, but are not beneficial to genetic variation of wild species and optimization of cultivated varieties, are easy to cause the problems of medicinal material quality degradation, plant susceptibility to diseases and insect pests and the like, so that the development of auxiliary breeding such as artificial hybridization and the like is an important technical means for ensuring genetic diversity of Polygonatum and obtaining high-value new varieties, and the phenomena of low pollination rate, low fruit setting rate and incomplete seed development exist in Polygonatum hybridization under natural conditions, but no related research can promote Polygonatum hybridization at present, especially improving pollination rate and fruit setting rate.
Disclosure of Invention
In view of the above, the invention aims to provide a composition for improving the artificial hybridization fertilization success rate of polygonatum sibiricum, and an improvement method and application thereof, so that the pollen germination rate and the fertilization success rate are improved, and the seed setting rate, the hybrid seed germination and other indexes are obviously improved
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a composition for improving the success rate of artificial hybridization fertilization of polygonatum sibiricum, which comprises a pollen culture medium and a stigma treating agent which are packaged independently;
the pollen culture medium comprises the following components in concentration: 80-100 g/L of sucrose, 0.01-0.05 g/L of boric acid and 0.5-1 mg/L of 6-BA;
the column head treating agent comprises the following components in concentration: GA 3 50～70mg/L。
Preferably, the solvents of the pollen culture medium and the stigma treatment agent are sterile water.
The invention also provides a method for improving the artificial hybridization fertilization success rate of polygonatum sibiricum, which comprises the following steps: (1) peeling off stamens before paternal opening, collecting pollen after drying, immersing the dried pollen in a pollen culture medium in the composition, and culturing for 2-4 h;
(2) emasculation is carried out before female parents are opened, stigma treatment agents in the composition are used for wiping stigmas, then pollen after the culture in the step (1) is used for pollination, and bagging culture is carried out.
Preferably, the male parent in the step (1) comprises three-year-old healthy yellow sperm plants which grow vigorously, and 30-50% of flower buds of each plant are removed before flower buds of the male parent develop.
Preferably, the drying in the step (1) comprises drying in a dryer for 24-48 h.
Preferably, before the immersion in the step (1), the method further comprises wrapping the dried pollen with gauze, and immersing after disinfection;
the culture temperature is 25-28 ℃;
and before pollination, drying the cultured pollen for 6-8 h at the temperature of 20-28 ℃.
Preferably, the disinfection comprises soaking the gauze bag wrapped with the dry pollen in 75% alcohol by volume for 15-20 s.
The invention also provides application of the composition or the method in improving the seed setting rate and the seed germination rate of polygonatum sibiricum hybrid seeds.
Has the advantages that: the invention provides a composition for improving the artificial hybridization fertilization success rate of polygonatum sibiricum, which respectively designs a pollen culture medium aiming at pollen and a stigma treating agent aiming at stigma so as to achieve the purpose of improving the pollen activity and the stigma receptivity of polygonatum sibiricum to realize the hybridization success rate, and meanwhile, the indexes such as seed setting rate, hybrid seed germination and the like are obviously improved through technical improvement.
Detailed Description
The invention provides a composition for improving the success rate of artificial hybridization fertilization of polygonatum sibiricum, which comprises a pollen culture medium and a stigma treatment agent which are independently packaged;
the pollen culture medium comprises the following components in concentration: 80-100 g/L of sucrose, 0.01-0.05 g/L of boric acid and 0.5-1 mg/L of 6-BA;
the column head treating agent comprises the following components in concentration: GA 3 50～70mg/L。
The pollen culture medium of the invention preferably comprises the following components in concentration: 80-100 g/L of sucrose, 0.01-0.05 g/L of boric acid and 0.5-1.0 mg/L of 6-BA, more preferably 85-100 g/L of sucrose, 0.02-0.05 g/L of boric acid and 0.6-1.0 mg/L of 6-BA, and most preferably 85-95 g/L of sucrose, 0.02-0.04 g/L of boric acid and 0.6-1.0 mg/L of 6-BA. In the present invention, the pollen medium is preferably sterile water as a solvent, and the method of preparing the pollen medium is not particularly limited in the present invention. The culture medium provides nutrients and hormones for the in vitro germination of the pollen, wherein the sucrose provides a carbon source for the germination of the pollen; boric acid mainly accelerates the development of floral organs and stimulates pollen grains to germinate, and the boric acid has obvious effect on promoting the elongation of pollen tubes; 6-BA is a spectrum plant growth regulator, can promote division growth of cells, promotes the growth of pollen cells for further germination of the stereo pollen and realizes the elongation of the pollen tube.
The stigma treatment agent preferably comprises 50-70 mg/L GA 3 More preferably 55-70 mg/L of GA 3 Most preferably 60-65 mg/L of GA 3 . The solvent of the stigma treating agent is preferably sterile water, and the stigma is treated by the inventionThe method of preparing the agent is not particularly limited. GA 3 As a growth regulator, the growth and development of pistil stigma are mainly promoted in the scheme, normal growth of pollen tubes in the stigma is ensured during pollen pollination, and meanwhile, the fruit setting rate is improved, and the fruit bearing after pollination is ensured.
The sealwort of the invention is preferably a sealwort plant, and preferably comprises one or two of sealwort, polygonatum cyrtonema, polygonatum kingianum and polygonatum hubeiense for artificial hybridization.
The invention also provides a method for improving the artificial hybridization fertilization success rate of polygonatum sibiricum, which comprises the following steps: (1) peeling off stamens before paternal opening, collecting pollen after drying, immersing the dried pollen in a pollen culture medium in the composition, and culturing for 2-4 h;
(2) castration is carried out before female parent opens, stigma is wiped by using a stigma treating agent in the composition, then pollination is carried out by using pollen cultured in the step (1), and bagging culture is carried out.
According to the invention, stamens are stripped before paternal, pollen is collected after drying, and the dried pollen is immersed in a pollen culture medium in the composition and cultured for 2-4 h. According to the invention, three-year-old yellow sperm-producing plants with vigorous growth and healthy plants are preferably selected as male parents, 30-50% of buds of each plant are removed before blooming after the development of the flower buds of the parents, and well-developed flower buds are left to serve as hybridization materials. The invention preferably collects pollen in the morning before paternal opening, strips the petal with sterile tweezers, and shears the stamen with sterile scissors. According to the invention, the stamens are preferably dried, the drying is preferably carried out in a dryer, and the drying time is preferably 24-48 h. The pollen is preferably collected after the drying, and the collection preferably comprises filling the pollen into a pollen preservation promoting bottle by using a sterile brush and storing the pollen in a refrigerator for later use.
After the dried pollen is obtained, the dried pollen is preferably wrapped by gauze and immersed in a pollen culture medium prepared by using sterile water for dissolving after being disinfected, and the disinfection preferably comprises the steps of placing the gauze wrapped with the pollen in 75% alcohol by volume for soaking for 15-20 s, then completely immersing in a liquid culture medium, and culturing for 2-4 h in an incubator at 25-28 ℃.
In the invention, castration is carried out before female parent opens, stigma treatment agent in the composition is used for wiping stigma, and then pollen after culture in step (1) is used for pollination and bagging culture.
According to the invention, the female parent preferably selects a three-year-old yellow sperm plant with vigorous growth and healthy plant as the female parent, 30-50% of flower buds of each plant are removed before blooming after the flower buds of the parent develop, and the well-developed flower buds are left as a hybridization material. According to the emasculation method, stamens are preferably removed in the morning just before female parents open, medical absorbent cotton is dipped in stigma treatment fluid to lightly wipe stigmas before pollination, and then the cultured pollen is pollinated to the female parents by using an aseptic brush pen.
The invention preferably also comprises timely bagging and information marking after pollination, and then plant cultivation is carried out according to a conventional method.
The invention also provides application of the composition or the method in improving the seed setting rate and the seed germination rate of polygonatum sibiricum hybrid seeds.
The application of the present invention is preferably the same as described above and will not be described further herein.
The composition for improving the success rate of artificial hybridization fertilization of polygonatum sibiricum provided by the invention, the improving method and the application thereof are described in detail with reference to the following examples, but they should not be construed as limiting the scope of the invention.
Example 1
Comparison of pollen viability and stigma
In a key laboratory nursery garden of the national forestry and grassland administration for protecting the biological diversity of southwest karst mountain land of Guizhou Master university in Guiyang City of Guizhou province, 20 experimental plants are respectively selected for four polygonatum cyrtonema, polygonatum sibiricum, Polygonatum kingianum and Polygonatum sibiricum Hubei, 10 flowers are selected from each experimental plant, an experimental group and a control group are arranged, and materials with consistent growth and development conditions are selected from the experimental group and the control group. The method comprises the steps of measuring the pollen germination rate by using a hanging drop culture method, measuring the pollen activity by using a TTC dyeing method, and measuring the stigmatisability by using a benzidine-hydrogen peroxide method.
Experimental groups: removing 30% of each plant bud before blooming after the parent bud grows, and leaving well-developed buds as hybridization materials; collecting pollen in the morning before paternal opening, peeling off petal with sterile forceps, and shearing off stamen with sterile scissors; packaging the taken stamens in a dryer, brushing pollen with a sterile writing brush after drying for 24 hours, filling the pollen into a pollen preservation promoting bottle, and storing the pollen preservation promoting bottle in a refrigerator for later use; preparing a pollen culture medium by using 80g/L of cane sugar, 0.05g/L of boric acid and 0.5mg/L of 6-BA according to mass fraction, and dissolving the prepared pollen culture medium in sterile water; wrapping the dried pollen with clean gauze, soaking in 75% ethanol for 15s, completely soaking in liquid culture medium, and culturing in 25 deg.C incubator for 2 h. Preparation of sterile water for preparing stigma treatment solution to prepare 50mg/L GA 3 The solution is ready for use;
and (5) bagging and marking information in time after pollination.
Control group: no treatment was performed, and hybridization was performed naturally.
As shown in Table 1, the experimental group can significantly improve the pollen germination rate and pollen activity, and significantly improve stigma receptivity, as compared with the control group.
TABLE 1 pollen viability and stigma receptivity comparisons
Note: -indicates no result; + indicates a result; , + + and + + + indicate significant results; +/-fingers have partial results and partial no results; lower case letters indicate significance of difference between the experimental group and the control group of the same index at the 0.05 level (same table below).
Example 2
Hybridization of Polygonatum cyrtonema and Polygonatum kingianum
In the nursery garden of key laboratories of the national forestry and grassland institute for protection of biological diversity in southwest karst mountain land of Guiyang university in Guiyang City of Guizhou province, the method of the embodiment is adopted, 50% of buds of each plant are removed before blooming after the flower buds of the parents develop, and well-developed flower buds are left to serve as hybridization materials; just before paternal patencyCollecting pollen in the morning, peeling off petal with sterile forceps, and shearing off stamen with sterile scissors; packaging the taken stamens in a dryer, brushing pollen with a sterile writing brush after drying for 48 hours, filling the pollen into a pollen preservation promoting bottle, and storing the pollen preservation promoting bottle in a refrigerator for later use; preparing a pollen culture medium by using 100g/L of sucrose, 0.01g/L of boric acid and 1mg/L of 6-BA according to mass fraction, and dissolving with sterile water to prepare; wrapping dried pollen with clean gauze, soaking in 75% ethanol for 20s, completely soaking in liquid culture medium, culturing at 28 deg.C for 4 hr, preparing 70mg/L GA with sterile water 3 The solution is reserved, and the two species of polygonatum cyrtonema and polygonatum kingianum are subjected to artificial hybridization. 3210 co-pollinated flowers, 1660 artificial pollination and 1550 natural pollination. After pollination, the plants grow well, and the adverse symptoms such as withering of leaves and the like do not appear. And fruit setting rates were counted at 20 days and 50 days after pollination (table 2), and the fruit setting rates at 20 days and 50 days after artificial cross pollination were significantly higher than those of natural pollination.
TABLE 2 measurement of the fruit setting rate of Polygonatum cyrtonema and Polygonatum kingianum hybridization
Example 3
Hybridization of rhizoma Polygonati with Hubei rhizoma Polygonati
In the key laboratory nursery garden of the national forestry and grassland institute for protecting the biological diversity of southwest karst mountain land of Guizhou university in Guiyang City of Guizhou province, 40% of flower buds of each plant are removed before the flower buds of the parent plant are developed and well developed flower buds are left to serve as hybridization materials; collecting pollen in the morning before paternal opening, peeling off petal with sterile forceps, and shearing off stamen with sterile scissors; packaging the taken stamens in a dryer, brushing pollen with a sterile writing brush after drying for 36 hours, filling the pollen into a pollen preservation promoting bottle, and storing the pollen preservation promoting bottle in a refrigerator for later use; preparing a pollen culture medium by using 90g/L of sucrose, 0.02g/L of boric acid and 0.8mg/L of 6-BA in terms of mass fraction, and dissolving with sterile water to prepare; wrapping the dried pollen with clean gauze, soaking in 75% alcohol for 18s, and completely soaking in liquid culture mediumCulturing in 26 deg.C incubator for 3 hr, preparing 60mg/L GA with sterile water 3 The solution is reserved, and the two species of the sealwort and the Hubei sealwort are hybridized artificially. 2140 flowers pollinated by co-crossing, 1080 flowers pollinated by artificial pollination and 1060 flowers pollinated by natural pollination. After pollination, the plants grow well, and the adverse symptoms such as withering of leaves and the like do not appear. And fruit setting rates were counted at 20 days and 50 days after pollination (table 3), and the fruit setting rates at 20 days and 50 days after artificial cross pollination were significantly higher than those of natural pollination.
TABLE 3 measurement of the fruit setting rate of Polygonatum cyrtonema and Polygonatum kingianum hybridization
Example 4
Hybridization of Polygonatum cyrtonema and Hubei Polygonatum cyrtonema
In the nursery garden of key laboratories of the national forestry and grassland institute for protecting the biological diversity of southwest karst mountain land of Guiyang university in Guiyang City of Guizhou province, the method of the embodiment is adopted, 35% of buds of each plant are removed before the buds of the parent plant are developed and the well-developed buds are left to serve as hybridization materials; collecting pollen in the morning before paternal opening, peeling off petal with sterile forceps, and shearing off stamen with sterile scissors; packaging the taken stamens in a dryer, brushing pollen with a sterile writing brush after drying for 36 hours, filling the pollen into a pollen preservation promoting bottle, and storing the pollen preservation promoting bottle in a refrigerator for later use; preparing a pollen culture medium, namely 85g/L of sucrose, 0.02g/L of boric acid and 0.6mg/L of 6-BA according to mass fraction, and dissolving the prepared culture medium in sterile water; wrapping dried pollen with clean gauze, soaking in 75% ethanol for 15s, completely soaking in liquid culture medium, culturing at 25 deg.C for 2.5h, preparing stigma treatment solution with sterile water to obtain 55mg/L GA 3 The solution is reserved, and the two species of polygonatum cyrtonema and polygonatum hubei are hybridized artificially. 2940 flowers pollinated in co-crossing mode and 1540 artificial pollinated flowersAnd 1400 pollination in nature. After pollination, the plants grow well, and the adverse symptoms such as withering of leaves and the like do not appear. And fruit setting rates were counted at 20 days and 50 days after pollination (table 4), and the fruit setting rates at 20 days and 50 days after artificial cross pollination were significantly higher than those of natural pollination.
TABLE 4 measurement of fruit setting rate of Polygonatum cyrtonema and Polygonatum kingianum hybridization
Example 5
Hybridization of rhizoma Polygonati with Polygonatum kingianum
In the key laboratory nursery garden of the national forestry and grassland institute for protecting the biological diversity of southwest karst mountain land of Guizhou university in Guiyang City of Guizhou province, 45% of flower buds of each plant are removed before the flower buds of the parent plant are developed and well developed flower buds are left to serve as hybridization materials; collecting pollen in the morning before paternal opening, peeling off petal with sterile forceps, and shearing off stamen with sterile scissors; packaging the taken stamens in a dryer, brushing pollen with a sterile writing brush after drying for 40 hours, filling the pollen into a pollen preservation promoting bottle, and storing the pollen preservation promoting bottle in a refrigerator for later use; preparing a pollen culture medium by using 90g/L of cane sugar, 0.02g/L of boric acid and 0.7mg/L of 6-BA according to mass fraction, and dissolving the prepared pollen culture medium by using sterile water; wrapping dried pollen with clean gauze, soaking in 75% ethanol for 15s, completely soaking in liquid culture medium, culturing at 25 deg.C for 2 hr, preparing stigma treatment solution with sterile water to obtain 55mg/L GA 3 The solution is reserved, and the two species of polygonatum kingianum and polygonatum kingianum are subjected to artificial hybridization. 1200 co-pollinated flowers, 580 artificial pollinated flowers and 620 natural pollinated flowers. After pollination, the plants grow well, and no adverse symptoms such as withered leaves and the like occur. And fruit setting rates were counted at 20 days and 50 days after pollination (table 5), and the fruit setting rates at 20 days and 50 days after artificial cross pollination were significantly higher than those of natural pollination.
TABLE 5 measurement of fruit setting rate of Polygonatum cyrtonema and Polygonatum kingianum hybridization
Example 6
Determination of hybrid seed Germination
In the nursery garden of key laboratories of the national forestry and grassland institute for protection of biological diversity in southwest karst mountain land of Guiyang university in Guiyang City of Guizhou province, the method of the embodiment is adopted, 50% of buds of each plant are removed before blooming after the flower buds of the parents develop, and well-developed flower buds are left to serve as hybridization materials; collecting pollen in the morning before paternal opening, peeling off petals with sterile forceps, and shearing off stamens with sterile scissors; packaging the taken stamens in a dryer, brushing pollen with a sterile writing brush after drying for 48 hours, filling the pollen into a pollen preservation promoting bottle, and storing the pollen preservation promoting bottle in a refrigerator for later use; preparing a pollen culture medium by using 100g/L of cane sugar, 0.01g/L of boric acid and 1mg/L of 6-BA according to mass fraction, and dissolving the prepared pollen culture medium in sterile water; wrapping dried pollen with clean gauze, soaking in 75% alcohol for 20s, completely soaking in liquid culture medium, culturing at 28 deg.C for 4 hr, preparing stigma treatment solution with 70mg/L GA in sterile water 3 Solutions were prepared by randomly picking up four sets of hybridization combinations constructed in examples 2 to 5, and selecting 840 seeds from the experimental and control sets in each hybridization combination. The test paper method was used to perform seed germination experiments to determine germination rate, germination vigor and germination index (table 6). The result shows that the seed hybridized by the method of the invention can obviously improve stigma granting performance in the aspects of germination rate, germination vigor and germination index.
TABLE 6 determination of hybrid seed Germination
Comparative example 1 changing pollen culture solution conditions
In a key laboratory nursery garden of the national forestry and grassland administration for biological diversity protection in southwest karst mountain land of the university of Guizhou Master, Guiyang, Guizhou province, 30 experimental plants with the same growth vigor are respectively selected for four polygonatum sibiricum, polygonatum kingianum and Polygonatum Hubei, 10 flowers are selected for each plant, a first treatment group, a second treatment group, a third treatment group and a control group are arranged, and other parameters are the same except that the composition and concentration of a pollen culture medium (liquid) are different.
Processing group one: the pollen is implemented by adopting the scheme, 50 percent of buds of each plant are removed before blooming after the parent buds develop, and well-developed buds are left to be used as hybrid materials; collecting pollen in the morning before paternal opening, peeling off petals with sterile forceps, and shearing off stamens with sterile scissors; packaging the taken stamens in a dryer, brushing off pollen by using a sterile brush pen after drying for 48 hours, filling the pollen into a pollen storage promoting bottle, and storing the pollen in a refrigerator for later use; preparing a pollen culture medium by using 100g/L of cane sugar, 0.01g/L of boric acid and 6-BA1mg/L of sterile water according to mass fraction, and dissolving the prepared solution in the sterile water; wrapping dried pollen with clean gauze, soaking in 75% ethanol for 20s, completely soaking in liquid culture medium, culturing at 28 deg.C for 4 hr, preparing 70mg/L GA with sterile water 3 The solution is ready for use;
and a second treatment group: adjusting the sucrose concentration of the pollen culture solution to 60 g/L;
and (3) treatment group III: adjusting the concentration of 6-BA in the pollen culture solution to 0.3 mg/L;
treatment group four: adjusting the concentration of 6-BA in the pollen culture solution to 1.8 mg/L;
control group: no treatment was performed.
And (3) measuring the pollen germination rate by using a hanging drop culture method, and measuring the pollen vitality by using a TTC dyeing method. The results are shown in table 7, where treatment group one is significantly higher in pollen germination rate and pollen activity than the other treatment groups.
TABLE 7 Effect of changing pollen culture solution conditions on pollen viability
Note: lower case letters indicate significance of difference between groups of the same index at the 0.05 level (the same table below).
Comparative example 2 changing pollen culture solution conditions
In a key laboratory nursery garden of the national forestry and grassland administration for protection of biological diversity in southwest karst mountain land of the university of Guizhou Master, Guiyang, Guizhou province, four polygonatum kingianum, polygonatum sibiricum, polygonatum kingianum and polygonatum hubeiense are respectively selected and randomly set up hybridization combinations, each hybridization combination is provided with a first treatment group, a second treatment group, a third treatment group, a fourth treatment group and a control group, and except that the compositions of pollen culture solutions are different, the other parameters are the same.
Processing group one: the pollen is cultured by adopting the pollen culture solution in the scheme, 50 percent of each plant bud is removed before blooming after the parent bud grows, and the well-developed bud is left to be used as a hybridization material; collecting pollen in the morning before paternal opening, peeling off petals with sterile forceps, and shearing off stamens with sterile scissors; packaging the taken stamens in a dryer, brushing pollen with a sterile writing brush after drying for 48 hours, filling the pollen into a pollen preservation promoting bottle, and storing the pollen preservation promoting bottle in a refrigerator for later use; preparing a pollen culture medium by using 100g/L of cane sugar, 0.01g/L of boric acid and 6-BA1mg/L of sterile water according to mass fraction, and dissolving the prepared solution in the sterile water; wrapping dried pollen with clean gauze, soaking in 75% ethanol for 20s, completely soaking in liquid culture medium, culturing at 28 deg.C for 4 hr, preparing 70mg/L GA with sterile water 3 The solution is ready for use;
and a second treatment group: the concentration of the sucrose in the pollen culture solution is adjusted to 60 g/L;
and (4) treatment group three: the concentration of 6-BA in the pollen culture solution in the scheme is adjusted to be 0.3 mg/L;
treatment group four: in the scheme, the concentration of 6-BA in the pollen culture solution is adjusted to be 1.8mg/L, and other conditions of the experimental group and the control group are consistent. After pollination, the plants grow well, and the adverse symptoms such as withering of leaves and the like do not appear. And fruit setting rates were counted at 20 days and 50 days after pollination (table 8), with the fruit setting rate at 20 days and 50 days in treatment group one being significantly higher than that in the other treatment group and the control group.
TABLE 8 Effect of changing pollen culture solution conditions on fruit set percentage
Comparative example 3 changing the conditions of the column head treating liquid
In a key laboratory nursery garden of the national forestry and grassland administration for protecting the biological diversity of southwest karst mountain land of Guizhou Master university in Guiyang City of Guizhou province, 10 flowers are selected from each of 30 experimental plants with the same growth vigor of Polygonatum sibiricum, Polygonatum kingianum and Polygonatum Hubei, a treatment group I, a treatment group II, a treatment group III and a control group are arranged, the pollen of the experimental group I is implemented by adopting the scheme, 50% of flower buds of each plant are removed before the flower buds of the parent plants are developed and well developed flower buds are left to serve as hybridization materials; collecting pollen in the morning before paternal opening, peeling off petal with sterile forceps, and shearing off stamen with sterile scissors; packaging the taken stamens in a dryer, brushing pollen with a sterile writing brush after drying for 48 hours, filling the pollen into a pollen preservation promoting bottle, and storing the pollen preservation promoting bottle in a refrigerator for later use; preparing a pollen culture medium by using 100g/L of cane sugar, 0.01g/L of boric acid and 6-BA1mg/L of sterile water according to mass fraction, and dissolving the prepared solution in the sterile water; wrapping dried pollen with clean gauze, soaking in 75% ethanol for 20s, completely soaking in liquid culture medium, culturing at 28 deg.C for 4 hr, preparing 70mg/L GA with sterile water 3 The solution is ready for use; treatment group two the column head treatment fluid GA 3 Adjusting to 40mg/L, treating the column head treating solution GA in group III 3 The concentration was adjusted to 85mg/L, and the control group was not treated at all, and the conditions were the same for each group except for the above change. The stigmatic feasibility was determined by the benzidine-hydrogen peroxide assay. The results show that treatment group one is significantly higher in stigma receptivity than the other treatment groups (table 9).
TABLE 9 Effect of changing pollen culture conditions on pollen viability
Note: -indicates no result; + indicates a result; , + + and + + + indicate significant results; the +/-finger has partial results and partial no results.
Comparative example 4 changing the conditions of the column head treating liquid
In a seedling nursery of a key laboratory of the national forestry and grassland institute of protecting the biological diversity of southwest karst mountain land of Guiyang City, Guizhou province, four polygonatum sibiricum, Polygonatum kingianum and Polygonatum Hubei are respectively selected to be randomly provided with a hybridization combination, each hybridization combination is provided with a first treatment group, a second treatment group, a third treatment group and a control group, the pollen of the first treatment group is implemented by adopting the scheme, 50% of flower buds of each plant are removed before the flower buds of the parent plants bloom after the development, and well-developed flower buds are left to serve as hybridization materials; collecting pollen in the morning before paternal opening, peeling off petals with sterile forceps, and shearing off stamens with sterile scissors; packaging the taken stamens in a dryer, brushing pollen with a sterile writing brush after drying for 48 hours, filling the pollen into a pollen preservation promoting bottle, and storing the pollen preservation promoting bottle in a refrigerator for later use; preparing a pollen culture medium by using 100g/L of cane sugar, 0.01g/L of boric acid and 1mg/L of 6-BA according to mass fraction, and dissolving the prepared pollen culture medium in sterile water; wrapping dried pollen with clean gauze, soaking in 75% ethanol for 20s, completely soaking in liquid culture medium, culturing at 28 deg.C for 4 hr, preparing 70mg/L GA with sterile water 3 The solution is ready for use; treating group two column head treating fluid GA 3 Adjusting to 40mg/L, treating the column head treating solution GA in group III 3 The concentration was adjusted to 85mg/L, and the control group was not treated at all, and the conditions were the same for each group except for the above change. After pollination, the plants grow well, and the adverse symptoms such as withering of leaves and the like do not appear. And fruit setting rates were counted at 20 days and 50 days after pollination (table 10), with the fruit setting rate at 20 days and 50 days in treatment group one being significantly higher than that in the other treatment group and the control group.
TABLE 10 Effect of changing pollen culture conditions on fruit set percentage
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. A composition for improving the success rate of artificial hybridization fertilization of rhizoma polygonati is characterized by comprising a pollen culture medium and a stigma treating agent which are packaged independently;
the pollen culture medium comprises the following components in concentration: 80-100 g/L of sucrose, 0.01-0.05 g/L of boric acid and 0.5-1 mg/L of 6-BA;
the column head treating agent comprises the following components in concentration: GA 3 50～70mg/L。
2. The composition of claim 1, wherein the solvents for the pollen culture medium and the stigma treatment agent are sterile water.
3. A method for improving the success rate of artificial hybridization fertilization of rhizoma polygonati is characterized by comprising the following steps: (1) peeling stamens before paternal, collecting pollen after drying, immersing the dried pollen in a pollen culture medium in the composition of claim 1 or 2, and culturing for 2-4 h;
(2) emasculation is carried out before female parent opening, stigma treatment agent in the composition of claim 1 or 2 is used for wiping stigma, pollen after culturing in step (1) is used for pollination, and bagging culture is carried out.
4. The method according to claim 3, wherein the male parent in step (1) comprises a vigorous, healthy three-year-old plant of spermatogonium, and 30-50% of the flower buds of each plant are removed before the flower buds of the male parent develop.
5. The method of claim 3, wherein the drying in step (1) comprises drying in a dryer for 24-48 hours.
6. The method as claimed in claim 3, wherein before the immersing in step (1), the method further comprises wrapping the dried pollen with gauze, and immersing after sterilizing;
the culture temperature is 25-28 ℃;
and before pollination, drying the cultured pollen for 6-8 h at the temperature of 20-28 ℃.
7. The method of claim 6, wherein the sterilizing comprises soaking the gauze bag containing the dried pollen in 75% alcohol by volume for 15-20 s.
8. Use of the composition of claim 1 or 2 or the method of any one of claims 3 to 7 for increasing seed set rate and seed germination rate of polygonatum sibiricum hybrid seeds.
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| ZA2022/11716A ZA202211716B (en) | 2022-06-29 | 2022-10-27 | Composition and method for improving fertilization success rate of artificial hybridization of polygonatum, and use thereof |
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1470247A1 (en) * | 1987-04-16 | 1989-04-07 | Институт экологической генетики АН МССР | Method of germinating hybrid tomato pollen |
| JP2009178083A (en) * | 2008-01-30 | 2009-08-13 | Mizuho Chemical:Kk | Powder composition for solution pollination |
| CN102517249A (en) * | 2011-12-15 | 2012-06-27 | 新疆农业大学 | Culture medium for improving germination and growth of Luntai white apricot blossom pollen |
| CN110074111A (en) * | 2019-05-15 | 2019-08-02 | 黑龙江八一农垦大学 | A kind of inorganic agent and method improving millet Cross fertile rate |
| US20210092920A1 (en) * | 2019-10-01 | 2021-04-01 | Monsanto Technology Llc | Cross pollination through liquid-mediated delivery of pollen to enclosed stigmas of flowers from recipient plants |
-
2022
- 2022-06-29 CN CN202210755586.7A patent/CN115005094B/en active Active
- 2022-10-27 ZA ZA2022/11716A patent/ZA202211716B/en unknown
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| SU1470247A1 (en) * | 1987-04-16 | 1989-04-07 | Институт экологической генетики АН МССР | Method of germinating hybrid tomato pollen |
| JP2009178083A (en) * | 2008-01-30 | 2009-08-13 | Mizuho Chemical:Kk | Powder composition for solution pollination |
| CN102517249A (en) * | 2011-12-15 | 2012-06-27 | 新疆农业大学 | Culture medium for improving germination and growth of Luntai white apricot blossom pollen |
| CN110074111A (en) * | 2019-05-15 | 2019-08-02 | 黑龙江八一农垦大学 | A kind of inorganic agent and method improving millet Cross fertile rate |
| US20210092920A1 (en) * | 2019-10-01 | 2021-04-01 | Monsanto Technology Llc | Cross pollination through liquid-mediated delivery of pollen to enclosed stigmas of flowers from recipient plants |
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