CN103828710A - Efficient chrysanthemum cross-breeding method - Google Patents
Efficient chrysanthemum cross-breeding method Download PDFInfo
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- CN103828710A CN103828710A CN201410100386.3A CN201410100386A CN103828710A CN 103828710 A CN103828710 A CN 103828710A CN 201410100386 A CN201410100386 A CN 201410100386A CN 103828710 A CN103828710 A CN 103828710A
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Abstract
The invention discloses an efficient chrysanthemum cross-breeding method, and belongs to the field of biotechnological breeding. The method comprises the following steps: sterilizing hybrid seeds by 75 percent ethanol for 1 minute, sterilizing the hybrid seeds by 5 percent sodium hypochlorite for 50 minutes, sowing the seeds on a 15g.L<-1> sucrose-containing MS culture medium, and performing further expanding propagation and subculture to obtain a great number of aseptic seedlings by combining a tissue culture technology. According to the method, the germination rate and seedling rate of the hybrid seeds are reliably increased; moreover, for a part of chrysanthemum varieties with cross incompatibility, aseptic seedlings obtained by a small number of hybrid seeds can be subcultured and propagated in the current year to obtain a large group of strains, so that the breeding cycle is shortened, and chrysanthemum breeding methods are further enriched.
Description
Technical field
The invention belongs to biotechnology breeding field, be specifically related to the method for a kind of high efficiency chrysanthemum crossbreeding.
Background technology
Chrysanthemum is to originate in the ten great tradition famous flowers of China, is also one of most important flowers in the world, apart from the cultivation history of modern existing more than 1600 year, forms by natural hybrizations such as Mao Huaju, mother chrysanthemum and pale reddish brown mother chrysanthemums and through artificial long-term breeding.In the period Tang Dynasty, Chinese Chrysanthemum is imported Japan into through Korea, and middle period the 17th century begins, and Chinese Chrysanthemum is imported the countries such as Holland, France, Britain into, has now become one of the world's four large cut-flowers.At present, the Dendranthema morifolium Varieties of China's seed selection approximately has more than 4000, and global kind sum, on 20,000, is one of flowers that commodity output value is the highest in the world.Chrysanthemum not only has very consequence in cut-flower and potted flower, and in Landscape Application, has also brought into play great effect, therefore strengthens chrysanthemum breeding and has very high economic and social benefit.
Chrysanthemum is typical many source hybrids, and the offspring of growing directly from seeds very easily produces the separation of the proterties such as pattern, flower type, and the seedling offspring who therefore utilizes hybridization or open pollination to obtain carries out the Main Means that chrysanthemum breeding of new variety is current chrysanthemum breeding.But because the ripening rate difference of different cultivars is very big, the Seed-setting Rate of Varieties of type such as flower such as part kind honeycomb type, rosette form etc. that especially lobe is higher is low, seed is few, plumpness is low, the germination rate of field sowing and planting percent are also lower, the a large amount of tree seedling offsprings of more difficult acquisition, have greatly affected breeding efficiency.In addition, after field sowing tree seedling growth slower, be difficult to then obtain compared with the strain of large group, need after primary election then, in final election in second and third year and preferably, also extended breeding cycle.Given this, the filial generation combination low for part ripening rate, seed plumpness is poor, adopt aseptic seeding method, can overcome on the one hand the impact of poor environment, improve germination rate and planting percent, it is numerous that the seedling that can utilize aseptic seeding to obtain simultaneously under controllable environment carries out subculture expansion, obtains the strain compared with large group in then, can significantly shorten breeding cycle, improve breeding efficiency.
Summary of the invention
The object of invention be on current chrysanthemum breeding, part hybrid combination progeny seed is few, plumpness is low, the germination rate of conventional field sowing and also lower this present situation of planting percent, expand numerous by seed disinfection, aseptic seeding and subculture, a set of method simple to operation is provided, to improve germination rate, the planting percent of chrysanthemum hybrid seed, and in the clonal plant that forms then filial generation, thereby shortening breeding cycle, improves breeding efficiency, practicality and scientific outstanding.
The object of the invention is to realize in the following manner:
A method for high efficiency chrysanthemum crossbreeding, the method comprises the following steps:
1) sterilization of hybrid seed:
Hybrid seed is rinsed after 15～20min with running water, soak 24～26h through sterile water, then using again percent by volume 75% ethanol disinfection 1min, then with after mass percent 5% clorox sterilization 50min, use aseptic water washing 3～4 times;
2) aseptic seeding:
The seed of having sterilized is inoculated in containing 15gL
-1in the MS medium of sucrose, after 15d, the seed of germination is proceeded to MS medium;
3) acquisition of hybrid strain:
After MS medium culture 10～15d, by 3～5 sections of seedling stem section cuttings, proceed to respectively and in subculture medium, carry out subculture and expand numerous cultivation, in the time that seedling grows to 2～3cm, moved to and on root media, carry out culture of rootage; After 40d, hardening is transplanted.
In said method, hybrid seed dries in the shade after gathering, and is placed in kraft paper bag, 4 ℃ of Refrigerator stores.In liquor natrii hypochloritis, can add the surfactant TritonX-100 of liquor natrii hypochloritis's volume 0.3%.
The temperature of subculture and culture of rootage is 25 ± 2 ℃, illumination 14h, light intensity 1500～2000lx.When subculture is cultivated, can determine as required subculture number, thereby make the acquisition of every seed several to the dozens of individual strain such as not.The medium that subculture expands numerous cultivation employing can be MS+6-BA0.2mgL
-1medium, the medium that culture of rootage adopts can be for adding growth hormone NAA0.1mgL
-11/2MS medium.
Said method specifically comprises the following steps:
1) preservation of hybrid seed and sterilization
After being gathered, chrysanthemum hybrid seed dries in the shade, 4 ℃ of Refrigerator stores; Seed rinses after 15min with running water, soaks 24h through sterile water.Then use percent by volume 75% ethanol disinfection 1min, then with using aseptic water washing 3 times after mass percent 5% clorox sterilization 50min.
2) aseptic seeding
The seed of having sterilized is inoculated in containing 15gL
-1the MS medium of sucrose, 20 seeds of each culture dish inoculation, after 15d, proceed to MS medium culture by the seed of germination.
3) acquisition of hybrid generation's strain
After MS medium culture 10～15d, by 3～5 sections of seedling stem section cuttings, proceed to respectively MS+6-BA0.2mgL
-1medium in carry out subculture and expand numerous cultivation, and determine subculture number as required, thereby it is several to the dozens of individual strain such as or not every seed is obtained.In the time that seedling grows to 2～3cm, move to and add NAA0.1mgL
-11/2MS medium on strong sprout taking root, after about 40d, hardening is transplanted, and can obtain hybrid strain.The temperature of aseptic seeding, subculture and culture of rootage is 25 ± 2 ℃, illumination 14h, light intensity 1500～2000lx.
Aseptic seeding technology and aseptic seeding system are not appeared in the newspapers before the present invention in chrysanthemum breeding practice.Groping in process of this technology, different disinfectant (H are comprehensively compared
2o
2, clorox), disinfecting time, type of culture medium (MS, B5, N6), inorganic salt concentration (1/4MS, 1/2MS, MS), sucrose concentration (15gL
-1, 30gL
-1, 45gL
-1) etc. the sterilization of factor on seed and the impact of culture effect, by design orthogonal experiment, filter out best pretreating method for seed, sterilization method and germination medium, thereby set up a whole set of chrysanthemum aseptic seeding system.Wherein, for this significant variable of disinfecting time, disinfecting time 5min, 10min are set respectively, 15min, waits at interval of 5min, until 1h, carefully compare the growth conditions of emergence rate, pollution rate and seedling in seeding process, final definite optimum disinfecting time, had both guaranteed that pollution rate was 0, guaranteed again percentage of seedgermination higher (> 50%).Therefore, the present invention is a kind of method that improves chrysanthemum crossbreeding efficiency, aseptic seeding technology is combined with group culturation rapid propagating technology, apply in crossbreeding practice, both can improve hybrid seed germination rate and planting percent, can obtain filial generation strain in then again, be convenient to compared with the proterties of large group observation and comparison filial generation, thereby shortening breeding cycle, improves breeding efficiency.
Chrysanthemum aseptic seeding provided by the invention and subculture expand numerous method, have following good effect compared with existing field sowing technology:
1) compared with conventional seed planting method, the hybrid combination offspring that the inventive method is low for ripening rate, seed is few, plumpness is low, utilizes the aseptic seeding method under controllable environment, can improve percentage of seedgermination more than 20.8%, and planting percent reaches 100%, thereby expand seed selection offspring scale.The excellent parent hybrid combination lower for ripening rate, progeny seed is less is a kind of effectively breeding supplementary means and technology.
2) further utilizing group culturation rapid propagating technology to carry out subculture by the aseptic seedling that aseptic seeding technology is obtained expands numerous, can make every hybrid seed obtain as required then number strain and even tens of strain not etc. compared with the strain of large group, in completing primary election then, carry out production pilot scale for superior strain Second Year and lay a good foundation, can shortening the breeding cycle more than 1 year.
3) the inventive method is simple, with low cost, and result accurately and reliably.
Accompanying drawing explanation
Fig. 1 is each step plant demonstration figure that aseptic seeding and subculture expand numerous acquisition filial generation strain
(material: utilize Cut Flower Chrysanthemum Morifolium kind ' Feeling green dark ' and ' Mary ', take ' Mary ' as male parent through hybridization obtain seed).Wherein:
A: the hybrid seed of having sterilized is inoculated in containing 15gL
-1the MS medium of sucrose, scale=1cm;
B: after aseptic seeding 7d, seed sprouting, scale=1cm;
After C:15d, the seed of germination proceeds to MS medium, scale=0.5cm;
D.10, after～15d, seedling stem section proceeds to MS+6-BA0.2mgL
-1medium, subculture expand numerous, scale=1cm;
E.10d, after, stem section moves to 1/2MS+NAA0.1mgL
-1medium on take root, scale=1cm;
F.40d after, hardening, scale=1cm.
Embodiment
The method that expands numerous raising chrysanthemum crossbreeding efficiency by aseptic seeding and subculture provided by the present invention, its embodiment is as follows:
Embodiment 1
1) preservation of seed and sterilization
After chrysanthemum hybrid seed is gathered, dry in the shade, be placed in kraft paper bag, 4 ℃ of Refrigerator stores.
Seed rinses after 15min with running water, soaks 24h through sterile water.Then on superclean bench with percent by volume 75% ethanol disinfection 1min, then with the mass percent 5% clorox 50min that sterilizes.Wherein, in liquor natrii hypochloritis, need to add the surfactant TritonX-100 of liquor natrii hypochloritis's volume 0.3%.Finally use aseptic water washing 3 times, each washing time is about 2min.
2) aseptic seeding
The seed of having sterilized is inoculated in culture dish, in ware, is equipped with containing 15gL
-1the MS medium of sucrose, 20 seeds of each culture dish inoculation.After 15d, the seed of germination is proceeded to MS medium.
3) acquisition of hybrid generation's strain
After MS medium culture 10～15d, by 3～5 sections of seedling stem section cuttings, proceed to respectively MS+ basic element of cell division 6-BA0.2mgL
-1medium in carry out subculture and expand numerous cultivation, subculture number 1～3 time.In the time that seedling grows to 2～3cm, moved to and added growth hormone NAA0.1mgL
-11/2MS medium on take root.Hardening after about 40d, aseptic seedling is transplanted in the matrix being mixed according to volume ratio 2:2:1 by Nutrition Soil, vermiculite, perlite and is cultivated, and after two weeks, can be colonizated in field, obtains hybrid strain.
The temperature of subculture and culture of rootage is 25 ± 2 ℃, illumination 14h, light intensity 1500～2000lx.
Embodiment 2:
After the seed collection that potted plant little chrysanthemum kind ' Zhong Mountain is early white ', ' Nanjing sunlight ' open pollination are obtained, dry in the shade, be placed in kraft paper bag, 4 ℃ of Refrigerator stores.
When sterilization, seed first rinses after 15min with running water, soaks 24h through sterile water.Then on superclean bench with percent by volume 75% ethanol disinfection 1min, then with the mass percent 5% clorox 50min that sterilizes.Wherein, in liquor natrii hypochloritis, need to add the surfactant TritonX-100 of liquor natrii hypochloritis's volume 0.3%.Finally use aseptic water washing 3 times, each washing time is about 2min.
The seed of having sterilized is inoculated in culture dish, in ware, is equipped with containing 15gL
-1the MS medium of sucrose, 20 seeds of each culture dish inoculation.After 15d, the seed of germination is proceeded to MS medium.
Take conventional seed planting as contrast, the seed that is about to dry in the shade is broadcast in 72 hole dishes, and each cave dish is broadcast a seed, and plug media is to mix with Nutrition Soil, vermiculite, perlite (2:2:1).
Carry out according to embodiment 1 " acquisition of hybrid generation's strain " step the illumination cultivation chamber that the seed of broadcasting is placed on to 24～26 ℃, and wherein, subculture number 1 time, waters water every day twice.Respectively at adding up germination rate and planting percent on May 6 and July 8.Result shows, the germination rate of ' Zhong Mountain is early white ', ' Nanjing sunlight ' aseptic seeding has improved respectively 29.1% and 20.8% compared with conventional seed planting, and meanwhile, planting percent has improved respectively 29.2% and 14.6%.Proof is utilized the inventive method, can conscientiously improve percentage of seedgermination and planting percent.
The germination rate of table 1 aseptic seeding and conventional seed planting and planting percent comparison
Embodiment 3:
In November, 2012, ' Feeling green dark ' was as maternal take cultivation chrysanthemum kind, ' Mary ' carries out conventional artificial hybridization for male parent, because seed setting rate of the female parent is low, and male parent is honeycomb type, lobe is high, pollen is few, causes filial generation ripening rate extremely low, and 428 inflorescences of artificial pollination only obtain 18 hybrid seeds, wherein 12 of full seeds, another 6 inabundant mature and plump.For guaranteeing germination rate, the planting percent of seed, and in the strain colony that obtains then filial generation, take " preservation of seed and sterilization " and " aseptic seeding " step described in the embodiment of the present invention 1,18 seeds are all sprouted successfully, germination rate 100%; Carry out " acquisition of the hybrid generation's strain " step described in embodiment 1, subculture number 2～3 times, has obtained 17 offspring's strains again, planting percent 94.44%, and obtain totally 120 individual plants, each individual plant forms the strain that 5～15 strains do not wait.In July, 2013, offspring's strain colony is transplanted to " Chinese Chrysanthemum germ plasm resource is preserved " center ", obtains then pattern autumn and be 11 of green superior strains, has established good basis for further obtaining green variety in Agricultural University Of Nanjing.
The inventive method has improved hybrid seed germination rate and planting percent conscientiously, and there is the Dendranthema morifolium Varieties of cross incompatibility for part, the aseptic seedling that the filial generation seed by a small amount of can be obtained expands numerous in carrying out subculture then, obtain compared with the strain of large group, thereby shortening breeding cycle, and chrysanthemum breeding means are further enriched.
Material used in the present invention or cultivar origin or channel are as follows:
Claims (7)
1. a method for high efficiency chrysanthemum crossbreeding, is characterized in that the method comprises the following steps:
1) sterilization of hybrid seed:
Hybrid seed is rinsed after 15～20min with running water, soak 24～26h through sterile water, then using again percent by volume 75% ethanol disinfection 1min, then with after mass percent 5% clorox sterilization 50min, use aseptic water washing 3～4 times;
2) aseptic seeding:
The seed of having sterilized is inoculated in containing 15gL
-1in the MS medium of sucrose, after 15d, the seed of germination is proceeded to MS medium;
3) acquisition of hybrid strain:
After MS medium culture 10～15d, by 3～5 sections of seedling stem section cuttings, proceed to respectively and in subculture medium, carry out subculture and expand numerous cultivation, in the time that seedling grows to 2～3cm, moved to and on root media, carry out culture of rootage; After 40d, hardening is transplanted.
2. the method for chrysanthemum crossbreeding according to claim 1, is characterized in that in the method, hybrid seed dries in the shade after gathering, and is placed in kraft paper bag, 4 ℃ of Refrigerator stores.
3. the method for chrysanthemum crossbreeding according to claim 1, is characterized in that the temperature of subculture and culture of rootage is 25 ± 2 ℃, illumination 14h, light intensity 1500～2000lx.
4. the method for chrysanthemum crossbreeding according to claim 1, is characterized in that when subculture is cultivated determining as required subculture number, thereby it is several to the dozens of individual strain such as or not every seed is obtained.
5. the method for chrysanthemum crossbreeding according to claim 1, is characterized in that it is MS+6-BA0.2mgL that subculture expands the medium that numerous cultivation adopts
-1medium.
6. the method for chrysanthemum crossbreeding according to claim 1, is characterized in that the medium that culture of rootage adopts is interpolation growth hormone NAA0.1mgL
-11/2MS medium.
7. the method for chrysanthemum crossbreeding according to claim 1, is characterized in that adding in liquor natrii hypochloritis the surfactant TritonX-100 of liquor natrii hypochloritis's volume 0.3%.
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Cited By (3)
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| CN104221527A (en) * | 2013-06-13 | 2014-12-24 | 亚洲大学 | Homogeneous germination method for castor seeds |
| CN106258936A (en) * | 2016-08-11 | 2017-01-04 | 南京农业大学 | A kind of early blossoming, high yield type tea, the selection cross method of medicine dual-purpose Dendranthema morifolium Varieties |
| CN111317700A (en) * | 2020-04-02 | 2020-06-23 | 安赛搏(重庆)生物技术有限公司 | Method for preparing cosmetic base water by fermenting rice young bud raw materials |
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| CN111317700A (en) * | 2020-04-02 | 2020-06-23 | 安赛搏(重庆)生物技术有限公司 | Method for preparing cosmetic base water by fermenting rice young bud raw materials |
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Address after: 211225 Jiangsu Nanjing Lishui District Baima Town National Agricultural Science and Technology Park Nanjing Agricultural University base Patentee after: Nanjing Agricultural University Address before: Nanjing City, Jiangsu Province, No. 1 210095 Weigang Patentee before: Nanjing Agricultural University |