CN107155897B - A kind of muskmelon anther induced medium and Anther culture breeding method - Google Patents
A kind of muskmelon anther induced medium and Anther culture breeding method Download PDFInfo
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- CN107155897B CN107155897B CN201710589435.8A CN201710589435A CN107155897B CN 107155897 B CN107155897 B CN 107155897B CN 201710589435 A CN201710589435 A CN 201710589435A CN 107155897 B CN107155897 B CN 107155897B
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- 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
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- 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
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Abstract
The invention discloses a kind of muskmelon anther induced medium and Anther culture breeding methods, belong to fruits and vegetables and cultivate planting technology field.The muskmelon anther induced medium that the present invention is researched and developed contains a great number of elements, microelement, molysite, organic principle, inorganic additive, plant growth regulator, coagulator and other additives etc..Muskmelon Anther culture breeding method and step of the invention include: bud materials with pretreatment, the preparation of culture medium, anther inoculation, anther Fiber differentiation, anther differentiation culture, domestication and transplant etc..Fiber differentiation is carried out to muskmelon anther using the present invention, antherderived callus inductivity is can effectively improve, greatly accelerates muskmelon Anther culture breeding process.
Description
Technical field
The present invention relates to a kind of muskmelon anther induced medium and Anther culture breeding methods, and in particular to one kind effectively improves flower
Medicine Callus induction rate, the muskmelon anther induced medium and Anther culture breeding method for greatly accelerating muskmelon Anther culture breeding process belong to
Planting technology field is cultivated in fruits and vegetables.
Background technique
Muskmelon (melon), alias sweet melon, wear intestines melon, fruit melon and ripe melon at muskmelon, and cucurbit is belonged in Plant Taxonomy
Section (Cucurbitaceae) Cucumis (Cucumis) is 1 year raw herbaceous plant, is a kind of fruit being favored by people
Type vegetables.Muskmelon originates in India, the tropical desert area in Africa, bright about in period in the Northern Wei Dynasty as watermelon passes to China together
Towards beginning plantation extensively.It is famous with its unique flavor always as one of big fruit in the world ten.
Contain a large amount of carbohydrate and citric acid, carrotene and vitamin B, C etc., and water in mature melon fruit
Divide plentifully, can relieve summer heat heat-clearing, quench one's thirst of promoting the production of body fluid, relieving restlessness etc.；Contain invertase in nutrition muskmelon, it can be by insoluble protein qualitative change
At soluble protein, kidney patient can be helped to absorb nutrition, it is beneficial to nephrotic；Cucurbitacin B contained by muskmelon pedicel can be bright
It is aobvious to increase experimental liver glycogen accumulation, mitigate chronic liver injury, to prevent hepatic cell fattydegeneration and inhibit fibroplasia；Sweet tea
Muskmelon pedicel contains picrotoxin, and the crystallinity amaroid such as Cucurbitacin B, E can stimulate gastric mucosa, takes orally in right amount, can cause to vomit, but be not
Body absorbs, and without the drawbacks such as collapse and poisoning；Modern studies have found that muskmelon seed kills the effects of roundworm, filaria, it can be extensive
For treating malnutrition due to parasitic infestation illness.Extra-nutrition muskmelon cold cuts contain protein, fat, carbohydrate, inorganic salts etc., can supplement people
Energy and nutrient required for body help body recovery health.
As a kind of important melon industrial crops, muskmelon is one of big important fruit in the world ten, cultivated area and yield
Larger, world temperate zone to torrid areas is cultivated extensively.The states such as China, Russia, Spain, the U.S., Iran, Italy, Japan
Family generally cultivates, with Chinese yield highest.Since the 1980s, the cultivated area and yield of China's muskmelon occupy always
The first in the world.Therefore, China's muskmelon production occupies an important position in the world, and muskmelon is the dominant crop of the high-end agricultural in China,
To increasing peasant income and agricultural restructuring important in inhibiting.
Since muskmelon is cross-pollinatd plant, natural hybrization rate is high, therefore is had using conventional breeding means acquisition pure lines
The disadvantages of period is long, difficulty is big, inhereditary feature is unstable, and utilize monoploid technology Breeding can be to avoid these problems, greatly
It is big to improve breeding efficiency.Haploid breeding technology refers to makes the heterogeneous gamete of certain kinds develop into list by inducing parthenogenesis
Times body plant, is sheerly using chromosome doubling, carries out a kind of breeding method of breed breeding later.This technology can
Shorten the breeding time limit, improve breeding efficiency.But in nature, the probability of the spontaneous Haploid production plant of crop is extremely low, Bu Guotong
Crossing some artificial means can get monoploid, such as anther or isolated microspore culture technique.So far, it is not found in muskmelon
The haplobiont of spontaneous generation, so muskmelon flower training technology just becomes the important channel for obtaining muskmelon haplobiont.
Summary of the invention
It is lacked the purpose of the present invention is extremely low for current muskmelon flower training callus induction rate, flower training technology is incomplete
It falls into, provides a kind of muskmelon anther induction for effectively improving antherderived callus inductivity, greatly accelerating muskmelon Anther culture breeding process
Culture medium and Anther culture breeding method.
The technical solution adopted by the invention is as follows:
A kind of muskmelon anther induced medium, it is characterised in that: culture medium prescription is contained by following components in every liter of distilled water
Amount is respectively as follows: KNO380-90mg/L, MgSO4∙7H2O 140-160mg/L, KH2PO470-76mg/L, Ca (NO3)2∙4H2O
160-200mg/L, MnSO4∙4H2O 25-30mg/L, Na2- EDTA 11-14mg/L, FeSO4∙7H2O 12-15mg/L, H3BO3
8.0-8.5mg/L, ZnSO4∙7H2O 8.5-9.5mg/L, KI 0.45-0.55mg/L, AgNO315-19mg/L, Na2MoO4∙
2H2O 0.2-0.3mg/L, CuSO4∙5H2O 0.02-0.03mg/L, CoCl2∙6H2O 0.02-0.03mg/L, adenine 13-
17mg/L, glutamine 450-550mg/L, vitamin B1 0.9-1.1mg/L, vitamin B6 0.6-0.8mg/L, Vc
0.55-0.65mg/L, niacin 4.5-5.5mg/L, inositol 120-140mg/L, glycine 3.5-4.5mg/L, insulin
1.8-2.2mg/L, cyclic guanylic acid 0.15-0.25g/L, glutathione 30-35mg/L, ethylmethane sulfonate 1.0-1.4g/L,
Sucrose 42-48g/L, agar 6.5-7.5g/L, 6-BA 0.8-1.2mg/L, NAA 0.8-1.2mg/L, mannitol 14-16g/
L, coconut palm cream 27-33g/L, plant vulcanize kinetin PSK- α 18-22pmol/L.
The culture medium prescription is respectively as follows: KNO by following components optimum content in every liter of distilled water385mg/L, MgSO4
∙7H2O 150mg/L, KH2PO473mg/L, Ca (NO3)2∙4H2O 180mg/L, MnSO4∙4H2O 27.5mg/L, Na2-EDTA
12.5mg/L FeSO4∙7H2O 13.5mg/L, H3BO38.25mg/L ZnSO4∙7H2O 9.0mg/L, KI 0.5mg/L,
AgNO317mg/L, Na2MoO4∙2H2O 0.25mg/L, CuSO4∙5H2O 0.025mg/L, CoCl2∙6H2O 0.025mg/L, gland
Purine 15mg/L, glutamine 500mg/L, vitamin B1 1.0mg/L, vitamin B6 0.7mg/L, Vc 0.6mg/L, niacin
5.0mg/L, inositol 130mg/L, glycine 4.0mg/L, insulin 2.0mg/L, cyclic guanylic acid 0.2g/L, glutathione
32.5mg/L, ethylmethane sulfonate 1.2g/L, sucrose 45g/L, agar 7.0g/L, 6-BA 1.0mg/L, NAA 1.0mg/L,
Mannitol 15g/L, coconut palm cream 30g/L, plant vulcanize kinetin PSK- α 20pmol/L.
The adding method of the ethylmethane sulfonate are as follows: after preparing ethylmethane sulfonate solution, with 0.22 μ of sterilizing
The filtering with microporous membrane of m sterilizes, and when condensing to 45-55 DEG C after culture medium high pressure steam sterilization, taking-up is placed in superclean bench
In, sterile working is added ethylmethane sulfonate solution, shakes up condensation, inoculation in the time for 24 hours.
The secure ph of the culture medium are as follows: 5.4-5.8, best secure ph are 5.6.
A kind of muskmelon Anther culture breeding method, it is characterised in that: Anther culture breeding method operates as follows:
(1) bud materials and pretreatment
When in crop field, the muskmelon of conventional cultivation is in flowering stage, morning 9-11 point acquires the plant of robust growth, no disease and pests harm
Taking developmental stage to be in the bud of mid-late uninucleate stage in strain is test material, again with self-styled modeling after the bud of acquisition is wrapped up with wet gauze
Material bag covers, and is placed in low-temperature treatment 2d in 4 DEG C of refrigerators, is then transferred to Heat thermostability 2d under 36 DEG C of environment；
(2) preparation of culture medium
It is sub-packed in the triangular flask of 250ml after culture medium described in claim 1,2,3 and 4 is prepared, every bottle of Sheng
The culture medium of 50ml-70ml, sealing are placed on that temperature is 121 DEG C, pressure is the 20min that sterilizes under the conditions of 15kPa high steam, cold
When coagulating 45-55 DEG C, taking-up is placed in superclean bench, sterile working, and ethylmethane sulfonate solution is added, shakes up condensation, for 24 hours
Inoculation in time；
(3) anther is inoculated with
Pretreated bud is taken out, first 1-2 min is rinsed with flowing water, peels off anthocaulus with tweezers, is wrapped up with fritter gauze,
15s in 70% alcohol is immersed, is sterilized 8-10 minutes with being transferred in 0.1% mercuric chloride solution after aseptic water washing 2-3 times, then with sterile
Water rinses 3-4 times, and bud is put into the sterilizes culture dish for being covered with filter paper, anther is stripped under aseptic condition is inoculated into step 2 and match
In the induced medium made, every bottle 14-16 pieces of anther of access；
(4) anther Fiber differentiation
It is 26-28 DEG C, intensity of illumination 1400-1800lx, photoperiod 11h that culture medium after inoculation anther, which is placed in temperature,
It is cultivated under light/13h dark environment, induces antherderived callus；
(5) anther differentiation culture
After antherderived callus length to diameter 2.5cm, selects translucent, loosely organized callus and transfer into differentiation culture
Differentiation culture is carried out on base, callus gradually differentiates green seedling；
(6) domestication and transplanting
Green seedling growth is vigorous after anther differentiation culture 35d, and sealed membrane is opened domestication hardening at this time, takes test tube seedling after 7d
Out, root is cleaned up, is transplanted into greenhouse, conventional cultivation.
The muskmelon bud selection standard of the mid-late uninucleate stage of the step (1) are as follows: the vertical stem length degree of measurement muskmelon bud is indulged
Stem length degree is the bud of 3.0-3.6mm.
The condition of step (5) the differentiation culture are as follows: 25-27 DEG C of temperature, humidity 70%-80%, intensity of illumination 2000-
2500lux, photoperiod 12h light/12h are dark.
Muskmelon anther induced medium and Anther culture breeding method provided by the invention are the research in existing other vegetable seeds
Specificity requires to carry out the achievement of creative improvement for muskmelon Anther Culture on basis, and the present invention substantially increases muskmelon flower
Medicine callus induction rate establishes muskmelon Anther culture breeding program for the first time.
Muskmelon anther induced medium provided by the present invention is screened by a large amount of single-factors, multifactor experiment,
It is optimum combination that research, which demonstrates nutrient media components proportion of the invention, through a large number of experiments.Using training provided by the present invention
Feeding base can greatly improve muskmelon induction of anther callus efficiency, therefore industry promotional value with higher.
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated.
A kind of muskmelon anther induced medium and Anther culture breeding method, it is characterised in that: Anther culture breeding method is by following step
Rapid operation:
(1) bud materials and pretreatment
When in crop field, the muskmelon of conventional cultivation is in flowering stage, morning 9-11 point acquires the plant of robust growth, no disease and pests harm
Taking developmental stage to be in the bud of mid-late uninucleate stage in strain is test material, the muskmelon bud selection standard of mid-late uninucleate stage are as follows: measurement
The vertical stem length degree of muskmelon bud indulges the bud that stem length degree is 3.0-3.6mm；Again with certainly after the bud of acquisition is wrapped up with wet gauze
It seals plastic bag cover to rise, is placed in low-temperature treatment 2d in 4 DEG C of refrigerators, is then transferred to Heat thermostability 2d under 36 DEG C of environment；
(2) preparation of culture medium
Muskmelon anther induced medium is prepared, culture medium prescription is respectively as follows: by following components content in every liter of distilled water
KNO380-90mg/L, MgSO4∙7H2O 140-160mg/L, KH2PO470-76mg/L, Ca (NO3)2∙4H2O 160-200mg/
L, MnSO4∙4H2O 25-30mg/L, Na2- EDTA 11-14mg/L, FeSO4∙7H2O 12-15mg/L, H3BO3 8.0-
8.5mg/L, ZnSO4∙7H2O 8.5-9.5mg/L, KI 0.45-0.55mg/L, AgNO315-19mg/L, Na2MoO4∙2H2O
0.2-0.3mg/L, CuSO4∙5H2O 0.02-0.03mg/L, CoCl2∙6H2O 0.02-0.03mg/L, adenine 13-17mg/L,
Glutamine 450-550mg/L, vitamin B1 0.9-1.1mg/L, vitamin B6 0.6-0.8mg/L, Vc 0.55-
0.65mg/L, niacin 4.5-5.5mg/L, inositol 120-140mg/L, glycine 3.5-4.5mg/L, insulin 1.8-
2.2mg/L, cyclic guanylic acid 0.15-0.25g/L, glutathione 30-35mg/L, ethylmethane sulfonate 1.0-1.4g/L, sucrose
42-48g/L, agar 6.5-7.5g/L, 6-BA 0.8-1.2mg/L, NAA 0.8-1.2mg/L, mannitol 14-16g/L, coconut palm cream
27-33g/L, plant vulcanize kinetin PSK- α 18-22pmol/L, pH value are as follows: 5.4-5.8；
Culture medium prescription is respectively as follows: KNO by following components optimum content in every liter of distilled water385mg/L, MgSO4∙7H2O
150mg/L, KH2PO473mg/L, Ca (NO3)2∙4H2O 180mg/L, MnSO4∙4H2O 27.5mg/L, Na2-EDTA 12.5mg/
L, FeSO4∙7H2O 13.5mg/L, H3BO38.25mg/L ZnSO4∙7H2O 9.0mg/L, KI 0.5mg/L, AgNO3 17mg/
L, Na2MoO4∙2H2O 0.25mg/L, CuSO4∙5H2O 0.025mg/L, CoCl2∙6H2O 0.025mg/L, adenine 15mg/L,
Glutamine 500mg/L, vitamin B1 1.0mg/L, vitamin B6 0.7mg/L, Vc 0.6mg/L, niacin 5.0mg/L, flesh
Alcohol 130mg/L, glycine 4.0mg/L, insulin 2.0mg/L, cyclic guanylic acid 0.2g/L, glutathione 32.5mg/L, first
Base sulfonic acid 1.2g/L, sucrose 45g/L, agar 7.0g/L, 6-BA 1.0mg/L, NAA 1.0mg/L, mannitol 15g/
L, coconut palm cream 30g/L, plant vulcanize kinetin PSK- α 20pmol/L, and best secure ph is 5.6；
It is sub-packed in after culture medium is prepared in the triangular flask of 250ml, the culture medium of every bottle of Sheng 50ml-70ml, after sealing
It is placed in that temperature is 121 DEG C, pressure is the 20min that sterilizes under the conditions of 15kPa high steam, when condensing to 45-55 DEG C, taking-up is placed in super
In net workbench, ethylmethane sulfonate solution, the adding method of ethylmethane sulfonate are as follows: prepare methyl is added in sterile working
After sulfonic acid solution, is sterilized with 0.22 μm of filtering with microporous membrane of sterilizing, condensed to after culture medium high pressure steam sterilization
At 45-55 DEG C, taking-up is placed in superclean bench, sterile working, and ethylmethane sulfonate solution is added, shakes up condensation, for 24 hours the time
Interior inoculation；Condensation is shaken up, inoculation in the time for 24 hours；
(3) anther is inoculated with
Pretreated bud is taken out, first 1-2 min is rinsed with flowing water, peels off anthocaulus with tweezers, is wrapped up with fritter gauze,
15s in 70% alcohol is immersed, is sterilized 8-10 minutes with being transferred in 0.1% mercuric chloride solution after aseptic water washing 2-3 times, then with sterile
Water rinses 3-4 times, and bud is put into the sterilizes culture dish for being covered with filter paper, anther is stripped under aseptic condition is inoculated into step 2 and match
In the induced medium made, every bottle 14-16 pieces of anther of access；
(4) anther Fiber differentiation
It is 26-28 DEG C, intensity of illumination 1400-1800lx, photoperiod 11h that culture medium after inoculation anther, which is placed in temperature,
It is cultivated under light/13h dark environment, induces antherderived callus；
(5) anther differentiation culture
After antherderived callus length to diameter 2.5cm, selects translucent, loosely organized callus and transfer into differentiation culture
Differentiation culture is carried out on base, breaks up the condition of culture are as follows: 25-27 DEG C of temperature, humidity 70%-80%, intensity of illumination 2000-
2500lux, photoperiod 12h light/12h are dark, and callus gradually differentiates green seedling；
(6) domestication and transplanting
Green seedling growth is vigorous after anther differentiation culture 35d, and sealed membrane is opened domestication hardening at this time, takes test tube seedling after 7d
Out, root is cleaned up, is transplanted into greenhouse, conventional cultivation.
Embodiment 1
A kind of muskmelon anther induced medium, is prepared according to formula as below:
Following components content is respectively as follows: KNO in every liter of distilled water385mg/L, MgSO4∙7H2O 150mg/L, KH2PO4
73mg/L, Ca (NO3)2∙4H2O 180mg/L, MnSO4∙4H2O 27.5mg/L, Na2- EDTA 12.5mg/L, FeSO4∙7H2O
13.5mg/L H3BO38.25mg/L ZnSO4∙7H2O 9.0mg/L, KI 0.5mg/L, AgNO317mg/L, Na2MoO4∙2H2O
0.25mg/L, CuSO4∙5H2O 0.025mg/L, CoCl2∙6H2O 0.025mg/L, adenine 15mg/L, glutamine
500mg/L, vitamin B1 1.0mg/L, vitamin B6 0.7mg/L, Vc 0.6mg/L, niacin 5.0mg/L, inositol 130mg/
L, glycine 4.0mg/L, insulin 2.0mg/L, cyclic guanylic acid 0.2g/L, glutathione 32.5mg/L, methane sulfonic acid second
Ester 1.2g/L, sucrose 45g/L, agar 7.0g/L, 6-BA 1.0mg/L, NAA 1.0mg/L, mannitol 15g/L, coconut palm cream
30g/L, plant vulcanize kinetin PSK- α 20pmol/L, and best secure ph is 5.6.
It is sub-packed in after culture medium is prepared in the triangular flask of 250ml, the culture medium of every bottle of Sheng 50ml-70ml, after sealing
It is placed in that temperature is 121 DEG C, pressure is the 20min that sterilizes under the conditions of 15kPa high steam, when condensing to 45-55 DEG C, taking-up is placed in super
In net workbench, ethylmethane sulfonate solution, the adding method of ethylmethane sulfonate are as follows: prepare methyl is added in sterile working
After sulfonic acid solution, is sterilized with 0.22 μm of filtering with microporous membrane of sterilizing, condensed to after culture medium high pressure steam sterilization
At 45-55 DEG C, taking-up is placed in superclean bench, sterile working, and ethylmethane sulfonate solution is added, shakes up condensation, for 24 hours the time
Interior inoculation；Condensation is shaken up, inoculation in the time for 24 hours.
Test melon variety is selected as red city 10 and imperial sweet tea No.1, and Anther culture breeding method operates as follows:
Bud materials and pretreatment: when in crop field, the muskmelon of conventional cultivation is in flowering stage, the acquisition growth of morning 9-11 point
Taking developmental stage to be in the bud of mid-late uninucleate stage on healthy and strong, no disease and pests harm plant is test material, the muskmelon flower of mid-late uninucleate stage
Flower bud selection standard are as follows: the vertical stem length degree of measurement muskmelon bud indulges the bud that stem length degree is 3.0-3.6mm；The bud of acquisition is used
It is covered again with self-sealing plastic bag after wet gauze package, is placed in low-temperature treatment 2d in 4 DEG C of refrigerators, is then transferred to heat shock under 36 DEG C of environment
Handle 2d；
Anther inoculation: taking out pretreated bud, first rinses 1-2 min with flowing water, peels off anthocaulus with tweezers, use fritter
Gauze package, immerses 15s in 70% alcohol, sterilizes 9 minutes with being transferred in 0.1% mercuric chloride solution after aseptic water washing 2-3 times, then
With aseptic water washing 3-4 times, bud is put into the sterilizes culture dish for being covered with filter paper, anther is stripped under aseptic condition and is inoculated into step
In rapid 2 prepared induced medium, every bottle 14-16 pieces of anther of access；
Anther Fiber differentiation: it is 27 DEG C, intensity of illumination 1600lx, photoperiod that the culture medium after inoculation anther, which is placed in temperature,
It is cultivated under the environment of 11h light/13h dark, induces antherderived callus, count induction of anther callus rate, antherderived callus group
Inductivity (%)=anther callus number/inoculation anther number × 100% is knitted, actual count obtains red city 10 and imperial sweet tea No.1 flower
Medicine callus induction rate is respectively 35.4% and 28.8%；
Anther differentiation culture: after antherderived callus length to diameter 2.5cm, translucent, loosely organized callus is selected
It transfers and carries out differentiation culture on differential medium, break up the condition of culture are as follows: 26 DEG C of temperature, humidity 75%, intensity of illumination
2250lux, photoperiod 12h light/12h are dark；Callus gradually differentiates green seedling；
Domestication and transplanting: green seedling growth is vigorous after anther differentiation culture 35d, and sealed membrane is opened domestication hardening, 7d at this time
Test tube seedling is taken out afterwards, root is cleaned up, is transplanted into greenhouse, conventional cultivation.
Embodiment 2
In the R&D process of muskmelon anther Fiber differentiation of the invention, the single-factor for having carried out nutrient media components, which is matched, has a competition
It tests, the single factor experiment constituent species of culture medium prescription are respectively as follows: adenine, Vc, insulin, cyclic guanylic acid, methane sulfonic acid second
Ester, coconut palm cream and plant vulcanize kinetin PSK- α.Each test compositional difference only limits one kind, other components and 1 phase of embodiment
Together.Test melon variety is also selected as red city 10 and imperial sweet tea No.1, the preparation method of culture medium, Anther culture breeding method with reality
It is identical to apply example 1.
Single-factor proportioning test specifically matches and test result such as following table 1- table 7.
The single factor experiment changed by the muskmelon anther induced medium component of table 1 to table 7 is it can be found that culture medium prescription
Single factor experiment constituent species in the increase of insulin, ethylmethane sulfonate and coconut palm cream proportion produce significant unfavorable effect
It answers；And adenine, Vc, cyclic guanylic acid and plant vulcanization kinetin PSK- α increases cannot dramatically increase culture medium induction imitate
Fruit reaches the minimum of optimal value in culture medium inducing effect since the increase of these additives can improve the preparation cost of culture medium
Proportional quantity is the optimum proportioning after comprehensive production cost.It can be seen that carrying out increasing or decreasing for any nutrient media components is
Unfavorable, while illustrating that component proportion of the invention is optimal.
Embodiment 3
In order to which muskmelon anther induced medium more provided by the present invention and other culture mediums are cured muskmelon anther embryo
The difference of injured tissue induced efficiency, in addition we have selected 2 kinds of anther induced mediums, test variety also use red city 10 and
Imperial sweet tea No.1, anther separation method, tissue culture operating method, cultural method are same as Example 1, after embryo callus is formed
Induction of anther callus rate of the red city 10 with the anther of imperial sweet tea No.1 in 2 kinds of culture mediums is counted respectively.
Other 2 kinds of anther Fiber differentiation based formulas are as follows:
C17-2 culture medium: C17-2 minimal medium+20g/L sucrose+7g/L agar+2mg/L 2,4-D+1mg/L excitement
Element, pH are 5.8(documents from " sorghum anther and inflorescence culture evoked callus and the regeneration plant such as F S Wen
Strain ")；
MS culture medium :+3% sucrose+1.0mg/LNAA+1.0mg/L KT+10% coconut palm cream of MS minimal medium, pH 5.8
(documents are from Gao Xiuyun etc. " Plantlets of Tomato Obtained From Anther Culture In Vitro ").
Culture medium comparative experiments
It will be lured using anther induced medium provided by the present invention with other 2 kinds of culture mediums provided by comparative example
It leads red city 10 and imperial sweet tea No.1 anther callus counts resulting induction of anther callus rate and is compared, comparison result
As shown in table 8 below.
By upper table 8 it can be found that muskmelon anther induced medium provided by the invention and Anther culture breeding method are to muskmelon product
The induction of anther callus rate mean value of the red city of kind 10 and imperial sweet tea No.1 is up to 32.1%, it can be found that muskmelon flower of the invention
Medicine induced medium and Anther culture breeding method specificity require to carry out the achievement of creative improvement for muskmelon Anther Culture, for the first time
Establish muskmelon Anther culture breeding program.Muskmelon anther callus can be greatly improved using culture medium provided by the present invention to lure
Lead efficiency, therefore industry promotional value with higher.
Those skilled in the art can be according to the present disclosure with the art technology grasped in the present invention
Appearance makes replacement or modification, but these replacement or modifications are all not regarded as a departure from present inventive concept, these replacement or modifications
In claimed interest field.
Claims (6)
1. a kind of muskmelon Anther culture breeding method, it is characterised in that: melon variety is red city 10 and imperial sweet tea No.1, and muskmelon flower is cultivated
Kind method operates as follows:
(1) bud materials and pretreatment
When in crop field, the muskmelon of conventional cultivation is in flowering stage, morning 9-11 point is acquired on the plant of robust growth, no disease and pests harm
Taking developmental stage to be in the bud of mid-late uninucleate stage is test material, uses self-sealing plastic bag again after the bud of acquisition is wrapped up with wet gauze
It covers, is placed in low-temperature treatment 2d in 4 DEG C of refrigerators, be then transferred to Heat thermostability 2d under 36 DEG C of environment；
(2) preparation of culture medium
It is sub-packed in the triangular flask of 250ml after muskmelon anther induced medium is prepared, the culture of every bottle of Sheng 50ml-70ml
Base, sealing are placed on that temperature is 121 DEG C, pressure is the 20min that sterilizes under the conditions of 15kPa high steam, when condensing to 45-55 DEG C,
Taking-up is placed in superclean bench, sterile working, and ethylmethane sulfonate solution is added, shakes up condensation, inoculation in the time for 24 hours；
(3) anther is inoculated with
Pretreated bud is taken out, first 1-2 min is rinsed with flowing water, peels off anthocaulus with tweezers, is wrapped up with fritter gauze, is immersed
15s in 70% alcohol is sterilized 8-10 minutes with being transferred in 0.1% mercuric chloride solution after aseptic water washing 2-3 times, is then rushed with sterile water
It washes 3-4 times, bud is put into the sterilizes culture dish for being covered with filter paper, anther is stripped under aseptic condition be inoculated into step 2 and prepare
Induced medium on, every bottle 14-16 pieces of anther of access；
(4) anther Fiber differentiation
It is 26-28 DEG C, intensity of illumination 1400-1800lx, photoperiod 11h light/13h that culture medium after inoculation anther, which is placed in temperature,
It is cultivated under dark environment, induces antherderived callus；
(5) anther differentiation culture
After antherderived callus length to diameter 2.5cm, selects translucent, loosely organized callus and transfer on differential medium
Differentiation culture is carried out, callus gradually differentiates green seedling；
(6) domestication and transplanting
Green seedling growth is vigorous after anther differentiation culture 35d, and sealed membrane is opened domestication hardening at this time, takes out test tube seedling after 7d,
Root is cleaned up, greenhouse, conventional cultivation are transplanted into；
The formula of the muskmelon anther induced medium is that following components content is respectively as follows: in every liter of distilled water
KNO3 80-90mg/L, MgSO4∙7H2O 140-160mg/L, KH2PO4 70-76mg/L, Ca (NO3)2∙4H2O 160-
200mg/L, MnSO4
∙4H2O 25-30mg/L, Na2- EDTA 11-14mg/L, FeSO4∙7H2O 12-15mg/L, H3BO38.0-8.5mg/L
ZnSO4∙7H2O 8.5-9.5mg/L, KI 0.45-0.55mg/L, AgNO3 15-19mg/L, Na2MoO4∙2H2O 0.2-0.3mg/
L, CuSO4∙5H2O 0.02-0.03mg/L, CoCl2∙6H2O 0.02-0.03mg/L, adenine 13-17mg/L, glutamine
450-550mg/L, vitamin B1 0.9-1.1mg/L, vitamin B6 0.6-0.8mg/L, Vc 0.55-0.65mg/L, niacin
4.5-5.5mg/L, inositol 120-140mg/L, glycine 3.5-4.5mg/L, insulin 1.8-2.2mg/L, cyclic guanylic acid
0.15-0.25g/L, glutathione 30-35mg/L, ethylmethane sulfonate 1.0-1.4g/L, sucrose 42-48g/L, agar
6.5-7.5g/L, 6-BA 0.8-1.2mg/L, NAA 0.8-1.2mg/L, mannitol 14-16g/L, coconut palm cream 27-33g/L, plant
Vulcanize kinetin PSK- α 18-22pmol/L.
2. muskmelon Anther culture breeding method according to claim 1, it is characterised in that: the mid-late uninucleate stage of the step (1)
Muskmelon bud selection standard are as follows: measurement muskmelon bud vertical stem length degree, indulge stem length degree be 3.0-3.6mm bud.
3. muskmelon Anther culture breeding method according to claim 1, it is characterised in that: the item of step (5) the differentiation culture
Part are as follows: 25-27 DEG C of temperature, humidity 70%-80%, intensity of illumination 2000-2500lux, photoperiod 12h light/12h are dark.
4. muskmelon Anther culture breeding method according to claim 1, it is characterised in that: the culture medium prescription is steamed by every liter
Following components optimum content is respectively as follows: KNO in distilled water385mg/L, MgSO4∙7H2O 150mg/L, KH2PO473mg/L, Ca
(NO3)2∙4H2O 180mg/L, MnSO4∙4H2O 27.5mg/L, Na2- EDTA 12.5mg/L, FeSO4∙7H2O 13.5mg/L,
H3BO38.25mg/L ZnSO4∙7H2O 9.0mg/L, KI 0.5mg/L, AgNO317mg/L, Na2MoO4∙2H2O 0.25mg/L,
CuSO4∙5H2O 0.025mg/L, CoCl2∙6H2O 0.025mg/L, adenine 15mg/L, glutamine 500mg/L, vitamin
B1 1.0mg/L, vitamin B6 0.7mg/L, Vc 0.6mg/L, niacin 5.0mg/L, inositol 130mg/L, glycine 4.0mg/
L, insulin 2.0mg/L, cyclic guanylic acid 0.2g/L, glutathione 32.5mg/L, ethylmethane sulfonate 1.2g/L, sucrose
45g/L, agar 7.0g/L, 6-BA 1.0mg/L, NAA 1.0mg/L, mannitol 15g/L, coconut palm cream 30g/L, plant vulcanization excitement
Plain PSK- α 20pmol/L.
5. muskmelon Anther culture breeding method according to claim 1, it is characterised in that: the addition of the ethylmethane sulfonate
Method are as follows: after preparing ethylmethane sulfonate solution, sterilized with 0.22 μm of filtering with microporous membrane of sterilizing, to culture medium high pressure
When condensing to 45-55 DEG C after steam sterilizing, taking-up is placed in superclean bench, sterile working, and ethylmethane sulfonate solution is added,
Condensation is shaken up, inoculation in the time for 24 hours.
6. muskmelon Anther culture breeding method according to claim 1, it is characterised in that: the secure ph of the culture medium
Are as follows: 5.4-5.8.
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| CN104686371A (en) * | 2015-04-04 | 2015-06-10 | 王开 | Efficient induction medium formula of sorghum anther |
| CN105660405A (en) * | 2016-02-16 | 2016-06-15 | 江苏强农农业技术服务有限公司 | Formula of inducing medium for tomato anther culture breeding |
| CN106489728A (en) * | 2015-09-08 | 2017-03-15 | 无锡南理工科技发展有限公司 | A kind of Anthers of Hordeum Vulgare inducing culture based formulas |
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| CN106489728A (en) * | 2015-09-08 | 2017-03-15 | 无锡南理工科技发展有限公司 | A kind of Anthers of Hordeum Vulgare inducing culture based formulas |
| CN105660405A (en) * | 2016-02-16 | 2016-06-15 | 江苏强农农业技术服务有限公司 | Formula of inducing medium for tomato anther culture breeding |
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| 甜瓜花药离体培养研究;马刘峰;《中国优秀博硕士学位论文全文数据库（硕士）农业科技辑》;20161115;摘要，2.1 试验材料，2.2 试验方法，第三章 结果与分析 |
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