CN105850745B - A kind of matrimony vine flower pesticide inducing culture and Anther culture breeding method - Google Patents
A kind of matrimony vine flower pesticide inducing culture and Anther culture breeding method Download PDFInfo
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- CN105850745B CN105850745B CN201610314204.1A CN201610314204A CN105850745B CN 105850745 B CN105850745 B CN 105850745B CN 201610314204 A CN201610314204 A CN 201610314204A CN 105850745 B CN105850745 B CN 105850745B
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- 244000241838 Lycium barbarum Species 0.000 title claims abstract description 105
- 235000015459 Lycium barbarum Nutrition 0.000 title claims abstract description 105
- 239000000575 pesticide Substances 0.000 title claims abstract description 40
- 238000009395 breeding Methods 0.000 title claims abstract description 35
- 230000001939 inductive effect Effects 0.000 title claims abstract description 21
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 26
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 18
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- 238000000034 method Methods 0.000 claims abstract description 11
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- 238000012360 testing method Methods 0.000 claims abstract description 8
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 238000011081 inoculation Methods 0.000 claims abstract description 4
- 239000002609 medium Substances 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- 229940045719 antineoplastic alkylating agent nitrosoureas Drugs 0.000 claims description 16
- 235000015463 Lycium carolinianum Nutrition 0.000 claims description 11
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 10
- 241000196324 Embryophyta Species 0.000 claims description 10
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- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 claims description 5
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- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims description 5
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- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 5
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- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 2
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Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of matrimony vine flower pesticide inducing culture and Anther culture breeding method, belong to the field of tissue culture of matrimony vine.The matrimony vine flower pesticide callus inducing medium that is screened of the present invention contains a great number of elements, trace element, molysite and complexing agent, organic principle, inorganic additive, plant growth regulator, physiological activator, carbon source, coagulator and other additives etc..The matrimony vine Anther culture breeding method and step of the present invention includes：Bud draw materials with low-temperature treatment, the preparation of culture medium, flower pesticide inoculation, alternating temperature Fiber differentiation, break up and take root, tame and transplant etc..Fiber differentiation is carried out to matrimony vine flower pesticide using the present invention, antherderived callus inductivity can be effectively improved, the matrimony vine antherderived callus quality obtained is high, can form a large amount of excellent test tube seedlings in a short time, greatly accelerate matrimony vine breeding process.The present invention can be not only used for matrimony vine Anther culture breeding, can also be applied to the fields such as matrimony vine rapid propagation in vitro, preserving seed.
Description
Technical field
The present invention relates to a kind of matrimony vine flower pesticide inducing culture and Anther culture breeding method, and in particular to one kind can be carried effectively
High matrimony vine flower pesticide callus induction rate, the measured matrimony vine flower pesticide inducing culture of flower training matter and Anther culture breeding method, belong to Chinese holly
The field of tissue culture of Qi.
Background technology
Matrimony vine（Lycium Barbarum. L）, it is Solanaceae（Solanaceae）Lycium（Lycium）Plant, the whole world
80 kinds are there are about, are distributed widely in South and North America, Eurasia, Africa and Austronesia.China's Lycium record has 7
Individual kind and 2 mutation, i.e. matrimony vine and its mutation northern Lycium chinense, lycium barbarum and its mutation yellow fruit matrimony vine, Lycium barbarum, black fruit Chinese holly
Qi, cut calyx matrimony vine, Yunnan matrimony vine, column casing matrimony vine.Lycium is mainly distributed on three areas in northwest and North China, first, sweet
The respectful band of Zhangye one, product claim " baogan medlar "；Second, Zhongweiof Ningxia, northwest China, in ground, the product such as peaceful claim " FRUCTUS LYCII BARBARI "；Third, Tianjin
Area, product claim " fructus lycii sinensis ".Wherein, lycium barbarum quality is optimal, only lycium barbarum quilt《Chinese Pharmacopoeia》Include as medicine
With the base plant of matrimony vine.At present, main matrimony vine kind of planting is mainly the series cultivated using lycium barbarum as breeding material in production
Mainly there are the ground such as Ningxia, Gansu, Qinghai, Xinjiang, the Inner Mongol in kind, the larger area of planting scale.
Matrimony vine is China's traditional conventional Chinese medicine simply, first recorded in《Sheng Nong's herbal classic》, top grade is listed in, calls it：" long term usage is hard
Muscles and bones, make light of one's life by commiting suicide not old, resistance to cold and heat ".Chinese wolfberry fruit is also the functional health food of a kind of " integration of drinking and medicinal herbs ", and commonly uses simply
Liver-kidney tonifying Chinese medicine, its color is scarlet, sweet and sour.The fruit of Chinese wolfberry is sweet, it is mild-natured, return liver and kidney channel, have that nourishing liver and kidney, strengthening the essence is bright
Mesh, moisten the lung and relieve the cough, anti-aging and other effects.In recent years, as attention of the people to health care, the research of matrimony vine processing are more next
It is more deep.Traditional matrimony vine product, as the technique of wolfberry juice, medlar vinegar and Lycium chinense wine is gradually improved；LBP-X, flavones and seed oil
Product is extracted, matrimony vine dried product and the exploitation of matrimony vine joint product have turned into the focus of matrimony vine processing industry research.
With the further exploitation of matrimony vine medicinal health value, the cultivated area of matrimony vine constantly expands, and is increasingly becoming northwest
One of regional main economic seeds.Conventional breeding methods can not meet the needs of modern agriculture is to matrimony vine breed improvement, and train
The new method for educating matrimony vine new varieties is seldom.At present, monoploid plant is obtained by using Anther Culture in method for breeding haploidy
There is extremely early stable separation offspring, the shortening breeding time limit through doubling the Anther culture breeding of generation zygoid after strain, simplify seed selection
The advantages that program, matrimony vine varieties distribution is extremely important.
In the research of matrimony vine Anther culture breeding, Polish scholar Zenkteler is earliest using the saltbush leaf Chinese holly for being distributed in south-eastern Europe
Qi has induced pollen plant.China's matrimony vine haploid breeding starts from the 1980s, Plant Inst. cares for refined honor
Researcher obtains lycium barbarum pollen plant, Fan Yinghan etc. and obtains two kinds of lycium plant flower pesticide lists times by embryoid approach first
Body, Cao Youlong etc. obtain regeneration plant by matrimony vine flower pesticide callus cell suspension culture, and these researchs are matrimony vine flower pesticide
Cultivate being laid a good foundation for the discovery of influence factor, homozygous Anther culture breeding material and mutant strain.
At present, although matrimony vine Anther Culture achieves no small progress, in general, matrimony vine anther culture technique remains
Embryoid induction rate is relatively low, flower training technology is not perfect the defects of, matrimony vine Anther Culture is limited in heredity and breeding
Using and development.But with the continuous adjustment to deepen continuously with culture medium of research, matrimony vine Anther Culture should be towards more
The direction of various limiting factors is overcome to develop.Further investigate the influence factor of matrimony vine Anther Culture（Minimal medium, hormone, work
Property carbon, temperature, mannitol pretreatment）, optimize the condition of matrimony vine Anther Culture, establish the technology of a set of suitable matrimony vine Anther Culture
System, to improving matrimony vine breeding efficiency and promoting the fast development of China's wolfberry industry to have great importance.
The content of the invention
The purpose of the present invention is for flower training callus induction rate existing for current matrimony vine Anther culture breeding is relatively low, Hua Peiji
The incomplete defect of art, there is provided one kind can effectively improve matrimony vine flower pesticide callus induction rate, the flower training measured matrimony vine of matter
Flower pesticide inducing culture and Anther culture breeding method.
The technical solution adopted by the present invention is as follows：
A kind of matrimony vine flower pesticide inducing culture, it is characterised in that be formulated by every liter of distilled water by following material：
KNO3900~1100mg/L, NH4NO3900~1100mg/L, KH2PO4270~330mg/L, Ca (NO3)2∙4H2O 330~
360mg/L, KCl 60~70mg/L, Na2-EDTA∙2H2O 70~80mg/L, FeSO4∙7H2O 50~60mg/L, MnSO4∙
4H2O 4.0~5.0mg/L, ZnSO4∙7H2O 2.1~2.3mg/L, H3BO31.1~1.3mg/L of 2.3~2.7mg/L, KI,
CuSO4∙5H2O 0.02~0.03mg/L, CoCl 6H2O 0.02~0.03mg/L, NaMoO4∙2H20.2~0.3mg/L of O,
AgNO323~27mg/L, 90~110mg/L of inositol, 0.09~0.11mg/L of vitamin B1, vitamin B6 0.09~
0.11mg/L, 0.45~0.55mg/L of nicotinic acid, vitamin C 28~32 μm of ol/L, 2.3~2.7mg/L of insulin, folic acid
0.18~0.22mg/L, 1.8~2.2mg/L of glycine, 0.3~0.4mg/L of proline, 0.35~0.45g/ of aspartic acid
L, N- methyl -30~36mg/L of nitroso ureas, 25~30g/L of maltose, 15~20g/L of fructose, 6.5~7.5g/ of agar
L；2,4-D 0.5~0.7mg/L, multiple phthalein 0.2~0.3mg/L of nucleic acid, 0.5~0.6mg/L of alar-85,25~35g/L of mannitol, water
Solve 0.25~0.35g/L of albumen, 0.35~0.45g/L of activated carbon, 35~45g/L of boxthorn root extract solution.
It is by the optimum content containing the preparation of following material in every liter of distilled water：KNO31000mg/L, NH4NO3
1000mg/L, KH2PO4300mg/L, Ca (NO3)2∙4H2O 345mg/L, KCl 65mg/L, Na2-EDTA∙2H2O 75mg/L,
FeSO4∙7H2O 55mg/L, MnSO4∙4H2O 4.5mg/L, ZnSO4∙7H2O 2.2mg/L, H3BO32.5mg/L, KI 1.2mg/
L, CuSO4∙5H2O 0.025mg/L, CoCl 6H2O 0.025mg/L, NaMoO4∙2H2O 0.25mg/L, AgNO325mg/L, flesh
Alcohol 100mg/L, vitamin B1 0.1mg/L, vitamin B6 0.1mg/L, nicotinic acid 0.5mg/L, 30 μm of ol/L of vitamin C, pancreas
Island element 2.5mg/L, folic acid 0.2mg/L, glycine 2.0mg/L, proline 0.35mg/L, aspartic acid 0.4g/L, N- first
Base-nitroso ureas 33mg/L, maltose 27.5g/L, fructose 17.5g/L, agar 7g/L；2,4-D 0.6mg/L, multiple phthalein core
Sour 0.25mg/L, alar-85 0.55mg/L, mannitol 30g/L, protein hydrolysate 0.3g/L, activated carbon 0.4g/L, boxthorn root extraction
Liquid 40g/L.
The acquisition methods of described boxthorn root extract solution are：30g is weighed after boxthorn root is cleaned, is cut into pieces, is added
100ml distilled water, boils 30min, is stood with 4~6 layers of filtered through gauze, supernatant will be taken after to be precipitated.
The adding method of described inorganic additive N- methyl-nitroso ureas is：Prepare N- methyl-nitroso ureas solution
Afterwards, sterilized with 0.22 μm of filtering with microporous membrane of sterilizing, when 45~55 DEG C are condensed to after culture medium high pressure steam sterilization, taken
Go out to be placed in superclean bench, sterile working, add N- methyl-nitroso ureas solution, shake up condensation, be inoculated with 48h.
The secure ph of described culture medium prescription is：5.6~6.0, optimal secure ph is 5.8.
A kind of matrimony vine Anther culture breeding method, it is characterised in that operate as follows：
（1）Bud is drawn materials and low-temperature treatment
When in crop field, the matrimony vine of conventional cultivation is in initial bloom stage or full-bloom stage, the morning 9 points~11 points collection robust growths, nothings
Developmental stage is taken to be in the bud of monokaryon middle and advanced stage as examination material on the plant of pest and disease damage, after the bud of collection is wrapped up with wet gauze
Covered again with self-sealing plastic bag, be placed in 4 DEG C of 2~7d of refrigerator low-temperature treatment；
（2）The preparation of culture medium
It is sub-packed in after culture medium described in claim 1,2,3,4 and 5 is prepared in 200ml triangular flask, every bottle of Sheng
40ml~60ml culture medium, be placed in after sealing temperature be 121 DEG C, pressure be the 20min that sterilizes under the conditions of 15kPa high steams,
When condensing to 45~55 DEG C, taking-up is placed in superclean bench, sterile working, is added N- methyl-nitroso ureas solution, is shaken up cold
It is solidifying, it is standby in 48h；
（3）Flower pesticide is inoculated with
The bud after low-temperature treatment is taken out, first 1~2 min is rinsed with flowing water, anthocaulus is peelled off with tweezers, in 70% alcohol
10s is soaked, drips Tween-80 solution immersion 10min with aseptic water washing 2~3 times, then with 0.1% mercuric chloride+2, aseptic water washing 4~
Bud is put into after 6 times in the sterilizing culture dish for being covered with filter paper, aseptically strips flower pesticide with sterilized tweezers, be inoculated with
In on the inducing culture that step 2 prepares, the flower pesticide of 4~6 buds of every bottle of inoculation；
（4）Alternating temperature Fiber differentiation
The culture medium after flower pesticide will be inoculated with and be placed in 1.5~2.5d of light culture in the environment of 25~27 DEG C of temperature, be then transferred to
2~3d of Heat thermostability under 37~39 DEG C of high temperature, dark conditions, be then transferred to temperature be 27~29 DEG C, humidity be 75%~80% black
Fiber differentiation under dark condition, induces antherderived callus；
（5）Break up and take root
After callus length to diameter 2cm, the callus for selecting faint yellow loose type is transferred and carried out on differential medium
Differentiation culture, callus gradually differentiate green seedling, and green seedling band root of the seedling length higher than 2.5cm is cut, transferred on root media
Strong plantlets and rootage；
（6）Domestication and transplanting
When test tube seedling grows to 3~5 leaf age, sealed membrane is opened into domestication hardening, taken out test tube seedling after 7~10d, by root
Portion cleans up, and is transplanted into the nutritive cube containing turfy soil, often watering, keeps humidity, after seedling is grown up, is transplanted to big
Field, conventional cultivation.
The step（5）The condition of culture for breaking up and taking root is：24~26 DEG C of temperature, humidity are that 70%~80%, illumination is strong
It is dark to spend 1800~2300lux, photoperiod 12h light/12h.
The step（5）The formula of middle root media is：MS minimal medium+0.5g/L activated carbon+1.0mg/L multiple-effect
Azoles.
Matrimony vine flower pesticide inducing culture and Anther culture breeding method specificity provided by the invention have been directed to matrimony vine Anther Culture
Requirement.Fiber differentiation is carried out to matrimony vine flower pesticide using the present invention, antherderived callus inductivity can be effectively improved, the Chinese holly obtained
Qi antherderived callus quality is high, can form a large amount of excellent test tube seedlings in a short time, greatly accelerate matrimony vine breeding process.This hair
It is bright to can be not only used for matrimony vine Anther culture breeding, the fields such as matrimony vine rapid propagation in vitro, preserving seed can also be applied to.
Embodiment
With reference to case study on implementation, the invention will be further described, is not intended to limit the present invention.
Embodiment
Prepare culture medium provided by the present invention：
The peaceful Qi of matrimony vine kind 3 and yellow fruit matrimony vine are chosen within 2014 as Anther Culture flower pesticide donor, prepares institute of the present invention
Matrimony vine inducing culture is stated, Anther Culture is carried out using the Anther culture breeding method of the present invention, comprised the following steps that：
（1）Bud is drawn materials and low-temperature treatment
When in crop field, the peaceful Qi of conventional cultivation 3 and yellow fruit matrimony vine are in initial bloom stage or full-bloom stage, 9 points~11 points of the morning adopts
It is examination material to collect the bud for taking developmental stage to be in monokaryon middle and advanced stage on the plant of robust growth, no disease and pests harm, by the bud of collection
Covered again with self-sealing plastic bag after being wrapped up with wet gauze, be placed in 4 DEG C of 2~7d of refrigerator low-temperature treatment；
（2）The preparation of culture medium
According to optimum formula：KNO31000mg/L, NH4NO31000mg/L, KH2PO4300mg/L, Ca (NO3)2∙4H2O
345mg/L, KCl 65mg/L, Na2-EDTA∙2H2O 75mg/L, FeSO4∙7H2O 55mg/L, MnSO4∙4H2O 4.5mg/L,
ZnSO4∙7H2O 2.2mg/L, H3BO32.5mg/L, KI 1.2mg/L, CuSO4∙5H2O 0.025mg/L, CoCl 6H2O
0.025mg/L, NaMoO4∙2H2O 0.25mg/L, AgNO325mg/L, inositol 100mg/L, vitamin B1 0.1mg/L, dimension life
Plain B6 0.1mg/L, nicotinic acid 0.5mg/L, vitamin C 30 μm of ol/L, insulin 2.5mg/L, folic acid 0.2mg/L, glycine
2.0mg/L, proline 0.35mg/L, aspartic acid 0.4g/L, N- methyl-nitroso ureas 33mg/L, maltose 27.5g/
L, fructose 17.5g/L, agar 7g/L；2,4-D 0.6mg/L, multiple phthalein nucleic acid 0.25mg/L, alar-85 0.55mg/L, mannitol
30g/L, protein hydrolysate 0.3g/L, activated carbon 0.4g/L, boxthorn root extract solution 40g/L（The acquisition methods of boxthorn root extract solution
For：30g is weighed after boxthorn root is cleaned, is cut into pieces, 100ml distilled water is added, 30min is boiled, with 4~6 layers of filtered through gauze
Stand, supernatant will be taken after to be precipitated）；Matrimony vine flower pesticide inducing culture is prepared by this formula, pH value control is 5.8.Will culture
Basigamy is sub-packed in 200ml triangular flask after making, and every bottle of Sheng 40ml~60ml culture medium, temperature is placed in after sealing as 121
DEG C, pressure be to sterilize 20min under the conditions of 15kPa high steams, when condensing to 45~55 DEG C, taking-up is placed in superclean bench,
Sterile working, N- methyl-nitroso ureas solution is added, shake up condensation, it is standby in 48h；
（3）Flower pesticide is inoculated with
The bud after low-temperature treatment is taken out, first 1~2 min is rinsed with flowing water, anthocaulus is peelled off with tweezers, in 70% alcohol
10s is soaked, drips Tween-80 solution immersion 10min with aseptic water washing 2~3 times, then with 0.1% mercuric chloride+2, aseptic water washing 4~
Bud is put into after 6 times in the sterilizing culture dish for being covered with filter paper, aseptically strips flower pesticide with sterilized tweezers, be inoculated with
In on the inducing culture that step 2 prepares, the flower pesticide of 4~6 buds of every bottle of inoculation；
（4）Alternating temperature Fiber differentiation
The culture medium after flower pesticide will be inoculated with and be placed in 1.5~2.5d of light culture in the environment of 25~27 DEG C of temperature, be then transferred to
2~3d of Heat thermostability under 37~39 DEG C of high temperature, dark conditions, be then transferred to temperature be 27~29 DEG C, humidity be 75%~80% black
Fiber differentiation under dark condition, antherderived callus is induced, count induction of anther callus rate；
（5）Break up and take root
After callus length to diameter 2cm, the callus for selecting faint yellow loose type is transferred and carried out on differential medium
Differentiation culture, callus gradually differentiate green seedling, now count anther callus plantlet differentiation rate；By seedling length higher than 2.5cm's
Green seedling band root is cut, strong plantlets and rootage on root media of transferring（The formula of root media is：MS minimal mediums+0.5g/
L activated carbon+1.0mg/L paclobutrazols）, condition of culture, which controls, is：24~26 DEG C of temperature, humidity are 70%~80%, intensity of illumination
1800~2300lux, photoperiod 12h light/12h are dark；
（6）Domestication and transplanting
When test tube seedling grows to 3~5 leaf age, sealed membrane is opened into domestication hardening, taken out test tube seedling after 7~10d, by root
Portion cleans up, and is transplanted into the nutritive cube containing turfy soil, often watering, keeps humidity, after seedling is grown up, is transplanted to big
Field, conventional cultivation, transplanting survival rate is counted after one month.
The peaceful Qi of matrimony vine kind 3 and yellow fruit matrimony vine flower pesticide callus induction rate, plantlet differentiation rate and transplanting survival rate system
It is as shown in the table to count result.
By upper table it can be found that matrimony vine flower pesticide inducing culture provided by the invention and Anther culture breeding method are to matrimony vine kind
Rather Qi 3 and the induction of anther callus rate average of yellow fruit matrimony vine are up to 20.9%, and antherderived callus plantlet differentiation rate average is up to
49.5%, transplanting survival rate reaches 97.7%, it can be found that the matrimony vine flower pesticide inducing culture and Anther culture breeding method of the present invention are non-
Often the requirement of suitable matrimony vine Anther Culture, our result of study have filled up the matrimony vine Breeding by anther culture of industry application value
The blank of technology, and the present invention can be not only used for matrimony vine Anther culture breeding, can also be applied to matrimony vine rapid propagation in vitro, kind quality guarantee
The field such as deposit.
Those skilled in the art can be according to present disclosure and the art technology grasped in the present invention
Appearance makes replacement or modification, but these are replaced or modification is all not regarded as a departure from present inventive concept, and these are replaced or modification
In claimed interest field.
Claims (5)
- A kind of 1. matrimony vine Anther culture breeding method, it is characterised in that matrimony vine flower pesticide inducing culture is pressed in every liter of distilled water by following Material is formulated：KNO3900~1100mg/L, NH4NO3900~1100mg/L, KH2PO4270~330mg/L, Ca (NO3)2∙4H2O 330~360mg/L, KCl60~70mg/L, Na2-EDTA∙2H2O 70~80mg/L, FeSO4∙7H2O 50~ 60mg/L, MnSO4∙4H2O 4.0~5.0mg/L, ZnSO4∙7H2O 2.1~2.3mg/L, H3BO32.3~2.7mg/L, KI 1.1~1.3mg/L, CuSO4∙5H2O 0.02~0.03mg/L, CoCl2∙6H2O 0.02~0.03mg/L, Na2MoO4∙2H2O 0.2~0.3mg/L, AgNO323~27mg/L, 90~110mg/L of inositol, tie up 0.09~0.11mg/L of raw B1, vitamin B6 0.09~0.11mg/L, 0.45~0.55mg/L of nicotinic acid, vitamin C 28~32 μm of ol/L, 2.3~2.7mg/L of insulin, leaf Acid 0.18~0.22mg/L, 1.8~2.2mg/L of glycine, 0.3~0.4mg/L of proline, 0.35~0.45g/L of aspartic acid, N- methyl -30~36mg/L of nitroso ureas, 25~30g/L of maltose, 15~20g/L of fructose, 6.5~7.5g/L of agar；2,4-D 0.5~0.7mg/L, multiple phthalein 0.2~0.3mg/L of nucleic acid, 0.5~0.6mg/L of alar-85,25~35g/L of mannitol, protein hydrolysate 0.25~0.35g/L, 0.35~0.45g/L of activated carbon, 35~45g/L of boxthorn root extract solution；The acquisition methods of the boxthorn root extract solution are：30g is weighed after boxthorn root is cleaned, is cut into pieces, adds 100ml distillations Water, 30min is boiled, stood with 4~6 layers of filtered through gauze, supernatant will be taken after to be precipitated；The adding method of the N- methyl-nitroso ureas is：After preparing N- methyl-nitroso ureas solution, with 0.22 μ of sterilizing M filtering with microporous membrane sterilizing, after the matrimony vine flower pesticide inducing culture high pressure steam sterilization for being not added with N- methyl-nitroso ureas When condensing to 45~55 DEG C, taking-up is placed in superclean bench, sterile working, is added N- methyl-nitroso ureas solution, is shaken up cold It is solidifying, it is inoculated with 48h；The matrimony vine Anther culture breeding method, it is characterised in that operate as follows：（1）Bud is drawn materials and low-temperature treatmentWhen in crop field, the matrimony vine of conventional cultivation is in initial bloom stage or full-bloom stage, 9 points~11 points of the morning gathers robust growths, without disease pest The bud that developmental stage is in monokaryon middle and advanced stage is taken to be used again after the bud of collection is wrapped up with wet gauze to try material on harmful plant Self-sealing plastic bag covers, and is placed in 4 DEG C of 2~7d of refrigerator low-temperature treatment；（2）The preparation of culture mediumIt is sub-packed in after the matrimony vine flower pesticide inducing culture for being not added with N- methyl-nitroso ureas is prepared in 200ml triangular flask, Every bottle of Sheng 40ml~60ml, be placed in after sealing temperature be 121 DEG C, pressure be the 20min that sterilizes under the conditions of 15kPa high steams, it is cold When coagulating to 45~55 DEG C, taking-up is placed in superclean bench, sterile working, is added N- methyl-nitroso ureas solution, is shaken up cold It is solidifying, it is standby in 48h；（3）Flower pesticide is inoculated withThe bud after low-temperature treatment is taken out, first 1~2 min is rinsed with flowing water, peels off anthocaulus with tweezers, soaked in 70% alcohol 10s, drip Tween-80 solution immersion 10min, aseptic water washing 4~6 times with aseptic water washing 2~3 times, then with 0.1% mercuric chloride+2 Bud is put into afterwards in the sterilizing culture dish for being covered with filter paper, aseptically strips flower pesticide with sterilized tweezers, be inoculated in On the matrimony vine flower pesticide inducing culture that step 2 prepares, the flower pesticide of 4~6 buds of every bottle of inoculation；（4）Alternating temperature Fiber differentiationThe culture medium after flower pesticide will be inoculated with and be placed in 1.5~2.5d of light culture in the environment of 25~27 DEG C of temperature, then it is transferred to 37~ 2~3d of Heat thermostability under 39 DEG C of high temperature, dark conditions, be then transferred to temperature be 27~29 DEG C, humidity be 75%~80% dark bar Fiber differentiation under part, induces antherderived callus；（5）Break up and take rootAfter callus length to diameter 2cm, the callus for selecting faint yellow loose type is transferred and broken up on differential medium Culture, callus gradually differentiate green seedling, and green seedling band root of the seedling length higher than 2.5cm is cut, taken root on root media of transferring Strong sprout；（6）Domestication and transplantingWhen test tube seedling grows to 3~5 leaf age, sealed membrane is opened into domestication hardening, taken out test tube seedling after 7~10d, root is clear Wash clean, it is transplanted into the nutritive cube containing turfy soil, often watering, keeps humidity, after seedling is grown up, be transplanted to crop field, often Rule cultivation.
- 2. matrimony vine Anther culture breeding method according to claim 1, matrimony vine flower pesticide inducing culture in every liter of distilled water by containing There is following material to be formulated：KNO31000mg/L, NH4NO31000mg/L, KH2PO4300mg/L, Ca (NO3)2∙4H2O 345mg/L, KCl 65mg/L, Na2-EDTA∙2H2O 75mg/L, FeSO4∙7H2O 55mg/L, MnSO4∙4H2O 4.5mg/L, ZnSO4∙7H2O 2.2mg/L, H3BO32.5mg/L, KI 1.2mg/L, CuSO4∙5H2O 0.025mg/L, CoCl2∙6H2O 0.025mg/L, Na2MoO4∙2H2O 0.25mg/L, AgNO325mg/L, inositol 100mg/L, vitamin B1 0.1mg/L, dimension life Plain B6 0.1mg/L, nicotinic acid 0.5mg/L, vitamin C 30 μm of ol/L, insulin 2.5mg/L, folic acid 0.2mg/L, glycine 2.0mg/L, proline 0.35mg/L, aspartic acid 0.4g/L, N- methyl-nitroso ureas 33mg/L, maltose 27.5g/L, fruit Sugared 17.5g/L, agar 7g/L；2,4-D 0.6mg/L, multiple phthalein nucleic acid 0.25mg/L, alar-85 0.55mg/L, mannitol 30g/L, water Solve albumen 0.3g/L, activated carbon 0.4g/L, boxthorn root extract solution 40g/L.
- 3. matrimony vine Anther culture breeding method according to claim 1, it is characterised in that：Described matrimony vine flower pesticide inducing culture PH value be：5.6~6.0.
- 4. matrimony vine Anther culture breeding method according to claim 1, it is characterised in that：The step（5）Break up and take root Condition of culture is：24~26 DEG C of temperature, humidity are 70%~80%, 1800~2300lux of intensity of illumination, photoperiod 12h light/12h Secretly.
- 5. matrimony vine Anther culture breeding method according to claim 1, it is characterised in that：The step（5）Middle root media Formula be：MS minimal medium+0.5g/L activated carbon+1.0mg/L paclobutrazols.
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