CN103828710B - A kind of method of high efficiency chrysanthemum crossbreeding efficiency - Google Patents
A kind of method of high efficiency chrysanthemum crossbreeding efficiency Download PDFInfo
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- CN103828710B CN103828710B CN201410100386.3A CN201410100386A CN103828710B CN 103828710 B CN103828710 B CN 103828710B CN 201410100386 A CN201410100386 A CN 201410100386A CN 103828710 B CN103828710 B CN 103828710B
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Abstract
The present invention is the method for a kind of high efficiency chrysanthemum crossbreeding, belongs to biotechnology breeding field, and the present invention selects filial generation seed, after 75% ethanol disinfection 1min and 5% hypochlorite disinfectant 50min, is seeded in containing 15gL
-1on the MS medium of sucrose, and conjunctive tissue culture technique, expand numerous and squamous subculture further, obtain a large amount of aseptic seedling.This method improves hybrid seed germination rate and planting percent conscientiously, and part is existed to the Dendranthema morifolium Varieties of cross incompatibility, can the aseptic seedling obtained by a small amount of filial generation seed be expanded numerous in carrying out subculture then, to obtain the strain compared with large group, thus shortening breeding cycle, and chrysanthemum breeding means are enriched further.
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 the ten great tradition famous flowers of original China, be also one of most important flowers in the world, apart from the cultivation history of modern existing more than 1600 year, is formed through artificial long-term breeding by natural hybrizations such as Mao Huaju, mother chrysanthemum and pale reddish brown mother chrysanthemums.In the period Tang Dynasty, Chinese Chrysanthemum imports Japan into through Korea, and middle period the 17th century begins, and Chinese Chrysanthemum imports the countries such as Holland, France, Britain into, has now become one of large cut-flower in the world four.At present, the Dendranthema morifolium Varieties of China's seed selection about 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, also played great effect, therefore strengthens chrysanthemum breeding and has very high economic and social benefit.
Chrysanthemum is typical many source hybrids, and Seedling tree very easily produces the separation of the proterties such as pattern, flower pattern, and the seedling offspring therefore utilizing hybridization or open pollination to obtain carries out the Main Means that chrysanthemum breeding of new variety is current chrysanthemum breeding.But due to the ripening rate very different of different cultivars, the Seed-setting Rate of Varieties of the part kind flower pattern such as honeycomb type, rosette form 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 offspring of more difficult acquisition, greatly have impact on breeding efficiency.In addition, after field sowing, tree seedling growth is comparatively slow, is difficult to then obtain the strain compared with large group, after needing primary election then, in final election in second and third year with preferred, also extends breeding cycle.Given this, the filial generation combination low for part ripening rate, seed plumpness is poor, adopt aseptic seeding method, the impact of poor environment can be overcome on the one hand, improve germination rate and planting percent, it is numerous that the seedling that simultaneously can aseptic seeding be utilized to obtain under controllable environment carries out subculture expansion, in obtaining the strain compared with large group then, significantly can shorten breeding cycle, improve breeding efficiency.
Summary of the invention
The object of invention be few for current chrysanthemum breeding upper part hybrid combination progeny seed, plumpness is low, the germination rate of conventional field sowing and planting percent also this present situation lower, 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 forming the clonal plant of filial generation then, thus shortening breeding cycle, improve breeding efficiency, practicality and science are given prominence to.
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:
By hybrid seed with after tap water 15 ~ 20min, soak 24 ~ 26h through sterile water, then using percent by volume 75% ethanol disinfection 1min again, then after using mass percent 5% hypochlorite disinfectant 50min, with 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 seedling stem section cutting 3 ~ 5 sections, proceed to respectively in subculture medium and carry out subculture and expand numerous cultivation, when seedling grows to 2 ~ 3cm, moved on root media and 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 DEG C of Refrigerator stores.The surfactant TritonX-100 of liquor natrii hypochloritis's volume 0.3% can be added in liquor natrii hypochloritis.
The temperature of subculture and culture of rootage is 25 ± 2 DEG C, illumination 14h, light intensity 1500 ~ 2000lx.Subculture number can be determined as required during squamous subculture, thus make every seed obtain several strain not waiting individuality to dozens of.Subculture expands numerous medium adopted of cultivating and can be MS+6-BA0.2mgL
-1medium, the medium that culture of rootage adopts can for adding growth hormone NAA0.1mgL
-11/2MS medium.
Said method specifically comprises the following steps:
1) preservation of hybrid seed and sterilization
Dry in the shade after chrysanthemum hybrid seed is gathered, 4 DEG C of Refrigerator stores; Seed, with after tap water 15min, soaks 24h through sterile water.Then use percent by volume 75% ethanol disinfection 1min, then use aseptic water washing 3 times with after mass percent 5% hypochlorite disinfectant 50min.
2) aseptic seeding
The seed of having sterilized is inoculated in containing 15gL
-1the MS medium of sucrose, 20 seeds inoculated by each culture dish, after 15d, the seed of germination proceeded to MS medium culture.
3) acquisition of hybrid generation's strain
After MS medium culture 10 ~ 15d, by seedling stem section cutting 3 ~ 5 sections, proceed to MS+6-BA0.2mgL respectively
-1medium in carry out subculture and expand numerous cultivation, and determine subculture number as required, thus every seed obtained severally do not wait individual strain to dozens of.When 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 DEG C, illumination 14h, light intensity 1500 ~ 2000lx.
Aseptic seeding technology and aseptic seeding system are not appeared in the newspapers before this invention in chrysanthemum breeding practice.Grope in process in this technology, the different disinfectant (H of Integrated comparative
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, thus 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 the emergence rate in seeding process, pollution rate and seedling, finally determine optimum disinfecting time, both ensured that pollution rate is 0, ensure percentage of seedgermination higher (> 50%) again.Therefore, the present invention is a kind of method improving chrysanthemum crossbreeding efficiency, aseptic seeding technology is combined with group culturation rapid propagating technology, apply in crossbreeding practice, both can improving hybrid seed germination rate and planting percent, in obtaining filial generation strain then, the proterties compared with large group observation and comparison filial generation can be convenient to again, thus shortening breeding cycle, improve 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%, thus expand seed selection offspring scale.The excellent parent hybrid combination that, progeny seed lower for ripening rate is less is a kind of effective breeding supplementary means and technology.
2) it is numerous that the aseptic seedling by aseptic seeding technology being obtained utilizes group culturation rapid propagating technology to carry out subculture expansion further, every hybrid seed can be made to obtain number strain and even the tens of strain strain compared with large group not etc. then as required, while completing primary election then, carry out production pilot scale for superior strain Second Year to lay a good foundation, can more than 1 year shortening the breeding cycle.
3) the inventive method is simple, with low cost, and result accurately and reliably.
Accompanying drawing explanation
Fig. 1 is each step plant display figure that aseptic seeding and subculture expand numerous acquisition filial generation strain
(material: utilize Cut Flower Chrysanthemum Morifolium kind ' Feelinggreendark ' and ' Mary ', the seed obtained through hybridization for male parent with ' Mary ').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 being expanded 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
Dry in the shade after chrysanthemum hybrid seed is gathered, be placed in kraft paper bag, 4 DEG C of Refrigerator stores.
Seed, with after tap water 15min, soaks 24h through sterile water.Then on superclean bench, use percent by volume 75% ethanol disinfection 1min, then use mass percent 5% hypochlorite disinfectant 50min.Wherein, the surfactant TritonX-100 adding liquor natrii hypochloritis's volume 0.3% is needed in liquor natrii hypochloritis.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, and ware is built with containing 15gL
-1the MS medium of sucrose, 20 seeds inoculated by each culture dish.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 seedling stem section cutting 3 ~ 5 sections, proceed to MS+ basic element of cell division 6-BA0.2mgL respectively
-1medium in carry out subculture and expand numerous cultivation, subculture number 1 ~ 3 time.When 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 and is cultivated in the matrix mixed according to volume ratio 2:2:1 by Nutrition Soil, vermiculite, perlite, after two weeks, can be colonizated in field, obtains hybrid strain.
The temperature of subculture and culture of rootage is 25 ± 2 DEG C, illumination 14h, light intensity 1500 ~ 2000lx.
Embodiment 2:
Are dried in the shade after the seed collection that potted plant little chrysanthemum kind ' Zhong Mountain is early white ', ' Nanjing sunlight ' open pollination are obtained, be placed in kraft paper bag, 4 DEG C of Refrigerator stores.
During sterilization, after seed first uses tap water 15min, soak 24h through sterile water.Then on superclean bench, use percent by volume 75% ethanol disinfection 1min, then use mass percent 5% hypochlorite disinfectant 50min.Wherein, the surfactant TritonX-100 adding liquor natrii hypochloritis's volume 0.3% is needed in liquor natrii hypochloritis.Finally use aseptic water washing 3 times, each washing time is about 2min.
Be inoculated in culture dish by the seed of having sterilized, ware is built with containing 15gL
-1the MS medium of sucrose, 20 seeds inoculated by each culture dish.After 15d, the seed of germination is proceeded to MS medium.
Take conventional seed planting as contrast, the seed being about to dry in the shade is broadcast in 72 hole dishes, and a seed broadcast by each cave dish, and plug media mixes with Nutrition Soil, vermiculite, perlite (2:2:1).
The illumination cultivation room seed broadcast being placed on 24 ~ 26 DEG C is carried out according to embodiment 1 " acquisition of hybrid generation's strain " step, and wherein, subculture number 1 time, waters twice water every day.Germination rate and planting percent is added up respectively at May 6 and July 8.Result shows, the germination rate of ' Zhong Mountain early white ', ' Nanjing sunlight ' aseptic seeding comparatively conventional seed planting improves 29.1% and 20.8% respectively, and meanwhile, planting percent improves 29.2% and 14.6% respectively.Proof utilizes the inventive method, can conscientiously improve percentage of seedgermination and planting percent.
Germination rate and the planting percent of table 1 aseptic seeding and conventional seed planting compare
Embodiment 3:
In November, 2012 is maternal to cultivate chrysanthemum kind ' Feelinggreendark ', ' Mary ' carries out conventional manual's 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 full seed 12, another 6 inabundant mature and plump.For ensureing germination rate, the planting percent of seed, and in obtaining the strain colony of filial generation then, take " preservation of seed and the sterilization " described in the embodiment of the present invention 1 and " aseptic seeding " step, 18 seeds are all sprouted successfully, germination rate 100%; Carry out " acquisition of the hybrid generation's strain " step described in embodiment 1 again, subculture number 2 ~ 3 times, obtains 17 offspring's strains, planting percent 94.44%, and obtains totally 120 individual plants, and each individual plant forms the strain that 5 ~ 15 strains do not wait.In July, 2013, offspring's strain colony is transplanted in Agricultural University Of Nanjing that " Chinese Chrysanthemum Germ-plasma resources protection " center ", obtaining pattern then autumn is green superior strain 11, has established good basis for obtaining green variety further.
The inventive method improves hybrid seed germination rate and planting percent conscientiously, and part is existed to the Dendranthema morifolium Varieties of cross incompatibility, can the aseptic seedling obtained by a small amount of filial generation seed be expanded numerous in carrying out subculture then, obtain the strain compared with large group, thus shortening breeding cycle, and chrysanthemum breeding means are enriched further.
Material used in the present invention or cultivar origin or channel as follows:
Claims (6)
1. a method for high efficiency chrysanthemum crossbreeding, is characterized in that the method comprises the following steps:
1) sterilization of hybrid seed:
By hybrid seed with after tap water 15 ~ 20min, soak 24 ~ 26h through sterile water, then using percent by volume 75% ethanol disinfection 1min again, then after using mass percent 5% hypochlorite disinfectant 50min, with aseptic water washing 3 ~ 4 times; The surfactant TritonX-100 of liquor natrii hypochloritis's volume 0.3% is added in liquor natrii hypochloritis;
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 seedling stem section cutting 3 ~ 5 sections, proceed to respectively in subculture medium and carry out subculture and expand numerous cultivation, when seedling grows to 2 ~ 3cm, moved on root media and 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 DEG C 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 DEG C, illumination 14h, light intensity 1500 ~ 2000lx.
4. the method for chrysanthemum crossbreeding according to claim 1, determines subculture number when it is characterized in that squamous subculture as required, thus makes every seed obtain several strain not waiting individuality to dozens of.
5. the method for chrysanthemum crossbreeding according to claim 1, it is characterized in that subculture expands numerous medium cultivating employing is MS+6-BA0.2mgL
-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.
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| TW201446126A (en) * | 2013-06-13 | 2014-12-16 | Univ Asia | Uniform germination method for castor seed |
| CN106258936B (en) * | 2016-08-11 | 2018-08-28 | 南京农业大学 | A kind of early blossoming, high yield type tea, medicine dual-purpose Dendranthema morifolium Varieties hybrid selection method |
| 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 |