CN109220781A - A kind of Cucumber Target Leaf Spot resistance breeding method - Google Patents
A kind of Cucumber Target Leaf Spot resistance breeding method Download PDFInfo
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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
- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
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- A—HUMAN NECESSITIES
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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
- A01H4/001—Culture apparatus for tissue culture
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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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The present invention provides a kind of Cucumber Target Leaf Spot resistance breeding methods, belong to fruits and vegetables breeding field.The technology path of this method includes: the selection of 1) breeding material；It 2) will cucumber variety be improved and the anti-source mixing breed of Cucumber Target Leaf Spot；3) anther Fiber differentiation is carried out respectively to the anti-source kind of cucumber variety and Cucumber Target Leaf Spot to be improved of selection；Anther Fiber differentiation is carried out respectively to the anti-source kind of cucumber variety and Cucumber Target Leaf Spot to be improved of selection；4) extraction of Raw toxin concentrate；5) identification of Raw toxin screening pressure；6) F1 generation cenospecies Anther Culture obtains regeneration plant after Raw toxin screening；7) acclimatization and transplants；8) brown spot Resistance Identification and offspring screen.Method of the invention improves Cucumber Target Leaf Spot screening efficiency, significant to shorten the breeding for disease resistance period, greatly accelerates Cucumber Target Leaf Spot breeding for disease resistance process.
Description
Technical field
The present invention relates to a kind of Cucumber Target Leaf Spot resistance breeding methods, belong to fruits and vegetables breeding field.
Background technique
Cucumber (scientific name:Cucumis sativusL. it) is also referred to as cucumber, green cucumber, the torrid zone originating from Himalayas southern foot
Rainforest area, is 1 year sprawling herbs plant of Curcurbitaceae Cucumis, has more than 2000 years cultivation histories in China.Cucumber fruits
For color in glossy dark green or emerald green, there is soft spinule on surface, and crisp, edible way multiplicity, is the vegetables and fruits being favored by people.
Cucumber has rich in nutritional ingredients such as carbohydrate, vitamin B2, vitamin C, vitamin E, carrotene, niacin, calcium, phosphorus, iron
Hypoglycemic, anti-oxidant, anti-aging, beauty and skin care, the strong body of weight-reducing and other effects, can be used as a kind of Chinese medicine, cure mainly pyreticosis,
Polydipsia, abscess of throat, acute conjunctivitis and scald.
The happiness of cucumber property is warm, intolerant to cold, is suitble to be grown in temperate zone and torrid areas, therefore China cucumber open country is planted in the past
It is very uneven to train Area distribution, is concentrated mainly on some weather conditions and the relatively good province of natural environment, as Shandong, Henan,
The ground such as Guangdong, Hainan.In recent years, with the development of agricultural sector structure adjustment and facilities horticulture, the production of China's dependent territory cucumber
It develops rapidly, has accounted for 42% or so of green cucumber area at present, wherein vinyl house area is about 23%, energy saving day
Light greenhouse area is about 17%, and glass heliogreenhouse is about 2%, do not limited by season and region using facility cultivation, it can be achieved that
Cucumber whole year production and supply.However due to high temperature and humidity in protecting field, and space is relatively closed, such as prevents and treats and improper easily breeds
Disease is simultaneously spread rapidly.
Cucumber Target Leaf Spot (also known as target spot) is a kind of leaf diseases of cucumber, and pathogen is the melon stick of Deuteromycotina
Spore mould [Corynespora cassiicola(Berk. & Curt.) Wei.].The pathogen can grow at 5 ~ 40 DEG C,
It can quickly be bred under conditions of being suitable for, and generate a large amount of conidiums, borrowed air-flow or fertilising, irrigate spread in china.Cucumber is brown
Pinta mostly contains the melon phase in cucumber and starts to fall ill, specific Symptoms be in, lower blade first fall ill, then to top developer blade,
Petiole, vines are spread to when serious.Blade fall ill when, initial stage bears taupe fleck on blade face, after be gradually extended to size
The not less neat filbert or brown scab of equal circle or subcircular edge, Lesion size 3-30mm, in 10-15mm
Type spot is more；Mid-term scab expands as round or irregular shape, easily perforates；Later period multiple adjacent scabs are often linked to be piece, disease, strong friendship
Boundary is obvious, is in deep yellow brown, and blade is withered and yellow and dead quickly.Also the ash that will appear ellipse when severe disease, in vines and petiole is brown
Color scab can cause whole strain withered when Spot expansion is larger.
In recent years, harm of the Cucumber Target Leaf Spot on cucumber production has exacerbation trend.The disease is in open country and protecting field
Occur, it is especially serious to occur in protecting field.Defoliation yield is fallen when serious up to 5-10% when open country main harm blade, generally morbidity
Leaf rate seriously affects plant photosynthesis up to 60%, to cause the underproduction；Protecting field due to high temperature and humidity environmental quality, more
Be conducive to pathogen breeding, and blade face moisture condensation, illumination is insufficient, day and night temperature is small can all aggravate disease occurrence degree, if prevention and control
Improper, which can rapidly develop and spread, it is only necessary to which defoliation yield caused by 1 week or so the disease can be developed to 90% by 5%, cause whole
A plant is dead, and cucumber yield loss is very big.
In order to prevent and treat Cucumber Target Leaf Spot, method of the cultural control in conjunction with chemical prevention is mostly used in production greatly.Agricultural is anti-
It controls and typically refers to take some positive cultivation steps, as protection is irrigated and reinforced to seed disinfection, efficent rotation, the rational application of fertilizer
Ground cultivation management etc.；Chemical prevention refers to that spraying chemical agent at the initial stage of a disease is treated.But these method control efficiency
Sometimes not satisfactory, and spray chemical residues and problem of environmental pollution brought by chemical agent and more should not be underestimated.Therefore,
In order to develop highly efficient and productive and environmentally protective agricultural, promote the sustainable development of China cucumber industry, it is necessary to pay much attention to cultivate
With popularization disease-resistant variety, brown spot resistance is improved to the breeding objective important as one.
The disease resistant and breeding method of cucumber routine be using introduce a fine variety, selection and use, the conventional breedings approach breeding such as crossbreeding
A collection of vertical resistance and the preferable excellent variety of horizontal resistance and strain out need long-term big but a disadvantage is that breeding cycle is long
The artificial crop field screening of amount, time-consuming and laborious, inefficiency are not able to satisfy the demand of cucumber breeding for disease resistance more and more.Monoploid
Breeding is one of modern biotechnology breeding technique, i.e., induces Haploid production using plant tissue culture technique, then pass through certain
Kind means double genome, to make plant recovery normal chromosomal number, therefore the individual that haploid breeding is cultivated is all
For homozygote, individual self progeny is not in trait segregation situation in planting process from now on.In the practices of breeding, it utilizes
Haploid breeding technology can not only be quickly obtained pure lines, shorten breeding cycle, and available more widely variations,
Especially recessive character is easier to be showed, so that the type of abundant breeding basic material, accelerates breeding process, with routine
Breeding method is compared, and has significant advantage.And Anther Culture is that artificial induction obtains the effective of monoploid and homozygous polyploid
The emphasis that one of approach and the present invention study.
Summary of the invention
Cucumber Target Leaf Spot resistance screening efficiency, shortening breeding for disease resistance week can be improved the purpose of the present invention is to provide a kind of
Phase, to accelerate the Cucumber Target Leaf Spot resistance breeding method of cucumber breeding for disease resistance process.
In order to achieve the goal above, technical scheme is as follows:
A kind of Cucumber Target Leaf Spot resistance breeding method, which is characterized in that method includes the following steps:
1) selection of breeding material
It selects agronomy and good yield characteristics but brown spot resistance is insufficient when purpleflower violet herb main breed is as cucumber product to be improved
Kind, it selects to show as the cucumber variety of highly resistance as the anti-source of Cucumber Target Leaf Spot through brown spot Resistance Identification in Cucumber Germplasm library
Kind；Just plant cucumber variety and Cucumber Target Leaf Spot to be improved anti-source kind season；
It 2) will cucumber variety be improved and the anti-source mixing breed of Cucumber Target Leaf Spot
Cucumber variety to be improved with the positive season plantation of step 1) is female parent, and the anti-source kind of Cucumber Target Leaf Spot is that male parent is hybridized,
Obtain F1 generation cenospecies；
3) anther Fiber differentiation is carried out respectively to the anti-source kind of cucumber variety and Cucumber Target Leaf Spot to be improved of selection
In cucumber flowering stage, it is anti-to acquire cucumber variety and Cucumber Target Leaf Spot to be improved that microspore development period is mid-late uninucleate stage
The wet gauze package of bud is placed on 4 DEG C of Cold pretreatment 48h, then carried out disinfection to bud by the bud of source kind,
It aseptically strips anther to be inoculated in induced medium, induces callus respectively under specific condition of culture；
4) extraction of Raw toxin concentrate
Melon stick spore mold species are transferred in PDA culture medium, is placed in 25 DEG C of constant incubators and expands numerous 7 days；It is inoculated with 5 diameters
The bacteria cake of about 4mm is into 250mLPS culture solution, 28 DEG C of shaking table shaken cultivations, 14 days acquisition bacterium solutions；The bacterium solution of acquisition was carried out
Filter, rotary evaporation is carried out at 60 DEG C for obtained filtrate until filtrate reaches the 1/10 of original volume, with 4500r/min in centrifuge
In carry out centrifugation and go to precipitate, isometric ether is added in the supernatant of acquisition and is extracted, is repeated 3 times, 3 ether are merged
Extraction phase, 45 DEG C of progress rotary evaporations remove ether on a rotary evaporator, and the slurry of acquisition adds water to be settled to 1mL to be
Raw toxin concentrate is placed in 4 DEG C of refrigerators and saves；
5) identification of Raw toxin screening pressure
Raw toxin concentrate is diluted, the Raw toxin dilution of various concentration is made into, and is added to the anther point prepared
Change in culture medium, the Raw toxin for making differential medium contain gradient concentration, the cucumber product to be improved that then will be obtained in step S2
The callus of kind and the anti-source kind of Cucumber Target Leaf Spot is transferred to respectively on above-mentioned differential medium, in 23-27 DEG C of temperature, illumination
It is cultivated under conditions of intensity 1500-2000Lx, daily illumination 12-14h, filters out the two phenylacetic acid and exist
The Raw toxin concentration of the corresponding addition of 2.0% or so differential medium, using the average value of two Raw toxin concentration as cucumber anther
Differentiation culture Raw toxin screening pressure；
6) F1 generation cenospecies Anther Culture obtains regeneration plant after Raw toxin screening
Positive season plants F1 generation cenospecies, when plant enters flowering stage, chooses the plant to grow fine and carries out anther Fiber differentiation,
Operating method is identical as step 2, and the callus of acquisition is transferred to the differential medium for being added to brown spot Raw toxin screen pressing
On cultivated, differential medium formula and condition of culture are identical as step 4), obtain adventitious bud；When the adventitious bud differentiated is long
At being transferred on root media after the sprout of 2-4cm high, in 26-28 DEG C of temperature, intensity of illumination 2400-2800Lx, every daylight
According to 26-34d is cultivated under conditions of 14-16h, flower training regeneration plant is obtained；
7) acclimatization and transplants
When flower training regeneration plant root system length to 4-6 item, root long 3-5cm, moves it in greenhouse and refined using natural lighting
Seedling does not open bottle cap hardening 3d, opens bottle cap hardening 5d；Then bloom control is removed out of bottle, cleans the training for being attached to root
It is transplanted into matrix after supporting base, carries out conventional cultivation management in suitable condition；
8) brown spot Resistance Identification and offspring screen
Mature cucumber bloom control carried out the acquisition of single plant sowing do not suit to train family, further after cultivation, to its disease resistance into
Row inoculated identification, filter out brown spot highly resistance and agronomy and yield traits and breeding trunk parent similar in single plant, then through 1-2
Cucumber Target Leaf Spot resistance new lines are obtained for pedigree selection.
The discrimination method in microspore development period in the step 3) are as follows: identified first according to bud form size, selected
Length is the closure bud of 0.6 ~ 1.8cm, further pulverizes 2 ~ 4 pieces of anther picking in bud, releases pollen, use acetic acid
Microscopy confirmation microspore development period is mid-late uninucleate stage after fuchsin dyeing.
The method that bud sterilizes in the step 3) are as follows: bud tap water is rinsed into 30min, is then transferred into ultra-clean work
Make on platform, first the soaking disinfection 60s in 75% ethyl alcohol, with aseptic water washing 1-2 times, then is transferred to 0.1% HgCl2Middle soaking disinfection
10min, with aseptic water washing 3-5 times；Bud surface moisture is blotted with aseptic filter paper after rinsing well.
The formula of callus inducing medium in the step 3) are as follows: N6+ cysteine hydrochloride 25mg/L+ biotin
20 mg/L+N- methyl-N-nitrosourea 1.1mg/L of 0.8mg/L+ glutathione 0.5g/L+ polyvinyl alcohol 0.8mg/L+ glycine betaine
+ 2,4-D 1.0mg/L+ tetramethyl glutaric acid 0.2mg/L+ polyvinylpyrrolidone 0.3g/L+mashed potatoes 33mg/L+ tea polyphenols
10mg/L, pH value are 5.6 ~ 6.0.
Specific condition of culture refers to 23-27 DEG C of temperature in the step 2, is protected from light culture.
The formula of differential medium in the step 5) are as follows: KNO32200mg/L, NH4NO3564mg/L, MgSO4·7H2O
280mg/L, NaH2PO4150mg/L, CaCl2·2H2O 255mg/L, MnSO4·4H2O 22.3mg/L, ZnSO4·7H2O
5.5mg/L, H3BO3 6.4mg/L, Na2MoO4·2H2O 0.25mg/L, CoCl2·6H2O 0.025mg/L, CuSO4·5H2O
0.25mg/L, Fe-Na2EDTA 33.6mg/L, K2Cr2O7 0.019mg/L, inositol 85mg/L, niacin 1.4mg/L, glycine
2.0mg/L, tryptophan 3.4mg/L, beef extract 27mg/L, thiamine hydrochloride 0.3mg/L, puridoxine hydrochloride 0.5mg/L, pantothenic acid
1.2mg/L, curcumin 4.9mg/L, L- selenium-methyl selenium substituted aminothiopropionic 0.11mg/L, L-carnitine 5.5mg/L, rosemary mention
Take liquid 33mL/L, humic acid 40mg/L, 6-BA2.0 mg/L, health polyphenol 0.5mg/L, brassin lactones 0.2mg/L, benzene peptamine
Sour 0.2mg/L, pH value are 5.6 ~ 6.0.
The formula of root media in the step 6) are as follows: 1/2MS+IBA0.2mg/L+ choline chloride 0.05mg/L, pH value
It is 5.6 ~ 6.0.
The condition for being suitable in the step 7) refers to day temperature control at 20-30 DEG C, and nocturnal temperature is controlled in 15-20
DEG C, intensity of illumination is controlled in 2500-5000Lx, and humid control is in 60-80%.
The rosemary extracting solution first boils 0.5h the preparation method comprises the following steps: rosemary plant is put into distilled water, then
60-70 DEG C of extraction 2.5h of water temperature is kept, juice is filtered to remove solid residue, then with sterilizing filter mistake in superclean bench
It is spare that sterile extracting solution is made in filter.
Compared with the prior art, the present invention has the following advantages:
1, the present invention feature sensitive to brown spot germ Raw toxin in atomization using cucumber anther callus, first
Raw toxin screening is carried out to cucumber parents callus using the differential medium containing gradient concentration Crude toxin, is determined
Suitable Raw toxin screens pressure, then screens as standard to F1 generation cenospecies, to improve the screening of disease-resistant material
Efficiency.
2, present invention optimizes cucumber anther-cultural systems, and especially find out a kind of suitable for slightly malicious in addition pathogen
Cucumber anther differentiation culture program under the conditions of element, provides guarantee for the screening of disease-resistant material.
3, breeding method of the invention combines haploid breeding technology, and F-1 hybrids obtain homozygosis by Anther Culture
Flower training family, screened using further brown spot Resistance Identification and offspring, 2-3 can be obtained Cucumber Target Leaf Spot resistance
New lines greatly accelerate the process of Cucumber Target Leaf Spot breeding for disease resistance compared with conventional breeding methods, to cucumber breeding for disease resistance
Development with very high promotion be worth.
Specific embodiment
The present invention is further illustrated combined with specific embodiments below, to better understand the invention.
Embodiment 1
2014 to 2016 years, technical solution according to the invention carried out Cucumber Target Leaf Spot resistance breeding, the technology path of the program
It is as follows:
1) selection of breeding material
Select the main Protectorate cultivation cucumber variety in Shandong Province ' Shandong sage peak 1 ' as cucumber variety to be improved, ' Lu Shengding
Peak 1 ' plant strain growth gesture is stronger, and branchiness is weak, and based on main stem knot melon, long rodlike, the skin bottle green of sugar-preserved gourd, thorn white, thorn is close,
Meat is clear and melodious, thick flavor, and quality is good, precocious, relatively cold-resistant, and compared with low light tolerance, fertilizer favorite water is sufficient, but not high to brown spot resistance；Choosing
Select excellent No. 3 of cucumber variety ' saliva ' as the anti-source kind of Cucumber Target Leaf Spot, which is bred as by Tianjin Ke Run cucumber research institute, foxiness
Highly resistance is shown as in sick Resistance Identification；Positive season plantation ' Shandong sage peak 1 ' and ' excellent No. 3 of saliva '.
It 2) will cucumber variety be improved and the anti-source mixing breed of Cucumber Target Leaf Spot
' Shandong sage peak 1 ' with the positive season plantation of step 1) is female parent, and ' excellent No. 3 of saliva ' is that male parent is hybridized, it is miscellaneous to obtain F1 generation
Hand over kind.
3) anther Fiber differentiation is carried out respectively to the anti-source kind of cucumber variety and Cucumber Target Leaf Spot to be improved of selection
In cucumber flowering stage, the flower at ' Shandong sage peak 1 ' and ' excellent No. 3 of saliva ' that microspore development period is mid-late uninucleate stage is acquired
Flower bud (the wherein discrimination method in microspore development period are as follows: first according to bud form size identify, select length be 0.6 ~
2 ~ 4 pieces of anther picking in bud are further pulverized, pollen are released, after being dyed with aceto-camine by the closure bud of 1.8cm
Microscopy confirms that microspore development period is mid-late uninucleate stage), the wet gauze package of bud is placed on 4 DEG C of Cold pretreatments
Then bud tap water is rinsed 30min, is then transferred on superclean bench by 48h, first the soaking disinfection in 75% ethyl alcohol
60s with aseptic water washing 1-2 times, then is transferred to 0.1% HgCl2Middle soaking disinfection 10min, with aseptic water washing 3-5 times；It rinses
Bud surface moisture is blotted with aseptic filter paper after clean, anther is aseptically stripped and is inoculated in induced medium, in temperature
23-27 DEG C of degree induces callus under the conditions of being protected from light respectively；
The wherein formula of induced medium are as follows: N6+ cysteine hydrochloride 25mg/L+ biotin 0.8mg/L+ glutathione
20 mg/L+N- methyl-N-nitrosourea 1.1mg/L+2,4-D 1.0mg/L+ of 0.5g/L+ polyvinyl alcohol 0.8mg/L+ glycine betaine
Tetramethyl glutaric acid 0.2mg/L+ polyvinylpyrrolidone 0.3g/L+mashed potatoes 33mg/L+ tea polyphenols 10mg/L, pH value are
5.8。
4) extraction of Raw toxin concentrate
Melon stick spore mold species are transferred in PDA culture medium, is placed in 25 DEG C of constant incubators and expands numerous 7 days；It is inoculated with 5 diameters
The bacteria cake of about 4mm is into 250mLPS culture solution, 28 DEG C of shaking table shaken cultivations, 14 days acquisition bacterium solutions；The bacterium solution of acquisition was carried out
Filter, rotary evaporation is carried out at 60 DEG C for obtained filtrate until filtrate reaches the 1/10 of original volume, with 4500r/min in centrifuge
In carry out centrifugation and go to precipitate, isometric ether is added in the supernatant of acquisition and is extracted, is repeated 3 times, 3 ether are merged
Extraction phase, 45 DEG C of progress rotary evaporations remove ether on a rotary evaporator, and the slurry of acquisition adds water to be settled to 1mL to be
Raw toxin concentrate is placed in 4 DEG C of refrigerators and saves.
5) identification of Raw toxin screening pressure
Raw toxin concentrate is diluted, the Raw toxin that concentration is 0,2.5%, 5.0%, 7.5%, 10.0%, 12.5% is made into and dilutes
Liquid, and it is added in the anther differential medium prepared (the formula of differential medium are as follows: KNO32200mg/L, NH4NO3
564mg/L, MgSO4·7H2O 280mg/L, NaH2PO4150mg/L, CaCl2·2H2O 255mg/L, MnSO4·4H2O
22.3mg/L ZnSO4·7H2O 5.5mg/L, H3BO3 6.4mg/L, Na2MoO4·2H2O 0.25mg/L, CoCl2·6H2O
0.025mg/L, CuSO4·5H2O 0.25mg/L, Fe-Na2EDTA 33.6mg/L, K2Cr2O7 0.019mg/L, inositol 85mg/
L, niacin 1.4mg/L, glycine 2.0mg/L, tryptophan 3.4mg/L, beef extract 27mg/L, thiamine hydrochloride 0.3mg/L, salt
Sour pyridoxol 0.5mg/L, pantothenic acid 1.2mg/L, curcumin 4.9mg/L, L- selenium-methyl selenium substituted aminothiopropionic 0.11mg/L are left-handed
Carnitine 5.5mg/L, rosemary extracting solution 33mL/L, humic acid 40mg/L, 6-BA2.0 mg/L, health polyphenol 0.5mg/L, rape
Plain lactone 0.2mg/L, benzene peptide amino acid 0.2mg/L, pH value 5.8；Wherein rosemary extracting solution the preparation method comprises the following steps: by rosemary
Plant, which is put into distilled water, first boils 0.5h, then keeps 60-70 DEG C of extraction 2.5h of water temperature, it is residual that juice is filtered to remove solid
Slag, then filter with sterilizing filter that sterile extracting solution is made is spare in superclean bench), so that differential medium is contained gradient dense
The callus at ' the Shandong sage peak 1 ' that obtains in step S2 and ' excellent No. 3 of saliva ' is then transferred to by the Raw toxin of degree respectively
It states on differential medium, is trained under conditions of 23-27 DEG C of temperature, intensity of illumination 1500-2000Lx, daily illumination 12-14h
It supports, the Raw toxin concentration that the two phenylacetic acid corresponds to addition in 2.0% or so differential medium is filtered out, by two
The average value of a Raw toxin concentration breaks up culture Raw toxin screening pressure as cucumber anther, as a result see the table below 1；
It can be seen from the above result that the anther differentiation rate at ' Shandong sage peak 1 ', at 2.0% or so, differential medium adds thick
Toxin concentration is about 5.5%, and for the anther differentiation rate of ' excellent No. 3 of saliva ' at 2.0% or so, the Raw toxin of differential medium addition is dense
Degree about 10.0% takes the average value 7.75% of the two as brown spot Raw toxin screening pressure, uses in follow-up test.
6) F1 generation cenospecies Anther Culture obtains regeneration plant after Raw toxin screening
Positive season plants F1 generation cenospecies, when plant enters flowering stage, chooses the plant to grow fine and carries out anther Fiber differentiation,
Operating method is identical as step 2, and the callus of acquisition is transferred to the differential medium for being added to brown spot Raw toxin screen pressing
On cultivated, differential medium formula and condition of culture are identical as step 4), obtain adventitious bud；When the adventitious bud differentiated is long
At being transferred on root media after the sprout of 2-4cm high, (prescription of rooting medium is 1/2MS+IBA0.2mg/L+ choline chloride
0.05mg/L, pH value 5.8), under conditions of 26-28 DEG C of temperature, intensity of illumination 2400-2800Lx, daily illumination 14-16h
26-34d is cultivated, flower training regeneration plant is obtained.
7) acclimatization and transplants
When flower training regeneration plant root system length to 4-6 item, root long 3-5cm, moves it in greenhouse and refined using natural lighting
Seedling does not open bottle cap hardening 3d, opens bottle cap hardening 5d；Then bloom control is removed out of bottle, cleans the training for being attached to root
It is transplanted into matrix after supporting base, day temperature control is at 20-30 DEG C, and at 15-20 DEG C, intensity of illumination control exists for nocturnal temperature control
2500-5000Lx, humid control carry out conventional cultivation management in 60-80%.
8) brown spot Resistance Identification and offspring screen
Mature cucumber bloom control progress single plant sowing acquisition 146 is not suited and trains family, it is disease-resistant to its further after cultivation
Property carry out inoculated identification, filter out brown spot highly resistance and agronomy and yield traits and breeding trunk parent similar in single plant, then pass through
1-2 selects finally to obtain 3 Cucumber Target Leaf Spot resistance new lines for pedigree.
Comparative example 1
In order to analyze differential medium of the invention in the case where adding Raw toxin, influence to cucumber anther differentiation effect:
Replacement step 5) in differential medium, and add the Extracted toxin liquid of same concentrations gradient, differential medium citation
Offer " cucumber (Cucumis sativus L.) Anther Culture " and in formula MS+ 0.1mg/L NAA+3.0mg/L 6-BA+3%
+ 0.8% agar of sucrose, by result statistics such as the following table 2.
It is in comparison sheet 1 and table 2 as a result, in the case where Raw toxin concentration is 0, ' Shandong sage peak 1 ' and ' excellent No. 3 of saliva '
Anther differentiation rate be not significantly different, and after being added to Raw toxin, in comparative example 1 ' Shandong sage peak 1 ' and ' excellent No. 3 of saliva '
Anther differentiation rate is extremely low or even partization, can not carry out follow-up test；Significant result difference illustrates differentiation culture of the invention
For base more suitable for breaking up culture in the cucumber anther of the condition for having Raw toxin, progress, reason may be differentiation culture of the invention
Be added in base tryptophan, curcumin, L- selenium-methyl selenium substituted aminothiopropionic, L-carnitine, brassin lactones, health polyphenol, fan change
The substances such as fragrant extracting solution can enhance cell activity, plant tissue resistance be improved, to make the callus group of brown spot resistance
Knitting being capable of normal differentiation.
It is understood that being merely to illustrate the present invention above with respect to specific descriptions of the invention and being not limited to this
Technical solution described in inventive embodiments, those skilled in the art should understand that, still the present invention can be carried out
Modification or equivalent replacement, to reach identical technical effect；As long as meet use needs, all protection scope of the present invention it
It is interior.
Claims (9)
1. a kind of Cucumber Target Leaf Spot resistance breeding method, which is characterized in that method includes the following steps:
1) selection of breeding material
It selects agronomy and good yield characteristics but brown spot resistance is insufficient when purpleflower violet herb main breed is as cucumber product to be improved
Kind, it selects to show as the cucumber variety of highly resistance as the anti-source of Cucumber Target Leaf Spot through brown spot Resistance Identification in Cucumber Germplasm library
Kind；Just plant cucumber variety and Cucumber Target Leaf Spot to be improved anti-source kind season；
It 2) will cucumber variety be improved and the anti-source mixing breed of Cucumber Target Leaf Spot
Cucumber variety to be improved with the positive season plantation of step 1) is female parent, and the anti-source kind of Cucumber Target Leaf Spot is that male parent is hybridized,
Obtain F1 generation cenospecies；
3) anther Fiber differentiation is carried out respectively to the anti-source kind of cucumber variety and Cucumber Target Leaf Spot to be improved of selection
In cucumber flowering stage, it is anti-to acquire cucumber variety and Cucumber Target Leaf Spot to be improved that microspore development period is mid-late uninucleate stage
The wet gauze package of bud is placed on 4 DEG C of Cold pretreatment 48h, then carried out disinfection to bud by the bud of source kind,
It aseptically strips anther to be inoculated in induced medium, induces callus respectively under specific condition of culture；
4) extraction of Raw toxin concentrate
Melon stick spore mold species are transferred in PDA culture medium, is placed in 25 DEG C of constant incubators and expands numerous 7 days；It is inoculated with 5 diameters
The bacteria cake of about 4mm is into 250mLPS culture solution, 28 DEG C of shaking table shaken cultivations, 14 days acquisition bacterium solutions；The bacterium solution of acquisition was carried out
Filter, rotary evaporation is carried out at 60 DEG C for obtained filtrate until filtrate reaches the 1/10 of original volume, with 4500r/min in centrifuge
In carry out centrifugation and go to precipitate, isometric ether is added in the supernatant of acquisition and is extracted, is repeated 3 times, 3 ether are merged
Extraction phase, 45 DEG C of progress rotary evaporations remove ether on a rotary evaporator, and the slurry of acquisition adds water to be settled to 1mL to be
Raw toxin concentrate is placed in 4 DEG C of refrigerators and saves；
5) identification of Raw toxin screening pressure
Raw toxin concentrate is diluted, the Raw toxin dilution of various concentration is made into, and is added to the anther point prepared
Change in culture medium, the Raw toxin for making differential medium contain gradient concentration, the cucumber product to be improved that then will be obtained in step S2
The callus of kind and the anti-source kind of Cucumber Target Leaf Spot is transferred to respectively on above-mentioned differential medium, in 23-27 DEG C of temperature, illumination
It is cultivated under conditions of intensity 1500-2000Lx, daily illumination 12-14h, filters out the two phenylacetic acid and exist
The Raw toxin concentration of the corresponding addition of 2.0% or so differential medium, using the average value of two Raw toxin concentration as cucumber anther
Differentiation culture Raw toxin screening pressure；
6) F1 generation cenospecies Anther Culture obtains regeneration plant after Raw toxin screening
Positive season plants F1 generation cenospecies, when plant enters flowering stage, chooses the plant to grow fine and carries out anther Fiber differentiation,
Operating method is identical as step 2, and the callus of acquisition is transferred to the differential medium for being added to brown spot Raw toxin screen pressing
On cultivated, differential medium formula and condition of culture are identical as step 4), obtain adventitious bud；When the adventitious bud differentiated is long
At being transferred on root media after the sprout of 2-4cm high, in 26-28 DEG C of temperature, intensity of illumination 2400-2800Lx, every daylight
According to 26-34d is cultivated under conditions of 14-16h, flower training regeneration plant is obtained；
7) acclimatization and transplants
When flower training regeneration plant root system length to 4-6 item, root long 3-5cm, moves it in greenhouse and refined using natural lighting
Seedling does not open bottle cap hardening 3d, opens bottle cap hardening 5d；Then bloom control is removed out of bottle, cleans the training for being attached to root
It is transplanted into matrix after supporting base, carries out conventional cultivation management in suitable condition；
8) brown spot Resistance Identification and offspring screen
Mature cucumber bloom control carried out the acquisition of single plant sowing do not suit to train family, further after cultivation, to its disease resistance into
Row inoculated identification, filter out brown spot highly resistance and agronomy and yield traits and breeding trunk parent similar in single plant, then through 1-2
Cucumber Target Leaf Spot resistance new lines are obtained for pedigree selection.
2. a kind of Cucumber Target Leaf Spot resistance breeding method according to claim 1, which is characterized in that small in the step 3)
The discrimination method in spore development period are as follows: identified first according to bud form size, select the closure flower that length is 0.6 ~ 1.8cm
2 ~ 4 pieces of anther picking in bud are further pulverized, release pollen by flower bud, and microscopy confirms small spore after being dyed with aceto-camine
Sub- developmental stage is mid-late uninucleate stage.
3. a kind of Cucumber Target Leaf Spot resistance breeding method according to claim 1, which is characterized in that flower in the step 3)
The method of flower bud disinfection are as follows: bud tap water is rinsed into 30min, is then transferred on superclean bench, is first soaked in 75% ethyl alcohol
Bubble disinfection 60s, with aseptic water washing 1-2 times, then is transferred to 0.1% HgCl2Middle soaking disinfection 10min, with aseptic water washing 3-5
It is secondary；Bud surface moisture is blotted with aseptic filter paper after rinsing well.
4. a kind of Cucumber Target Leaf Spot resistance breeding method according to claim 1, which is characterized in that in the step 3) more
The formula of injured tissue induced medium are as follows: N6+ cysteine hydrochloride 25mg/L+ biotin 0.8mg/L+ glutathione 0.5g/L
20 mg/L+N- methyl-N-nitrosourea 1.1mg/L+2,4-D 1.0mg/L+ tetramethyl of+polyvinyl alcohol 0.8mg/L+ glycine betaine
Glutaric acid 0.2mg/L+ polyvinylpyrrolidone 0.3g/L+mashed potatoes 33mg/L+ tea polyphenols 10mg/L, pH value are 5.6 ~ 6.0.
5. a kind of Cucumber Target Leaf Spot resistance breeding method according to claim 1, which is characterized in that special in the step 2
Fixed condition of culture refers to 23-27 DEG C of temperature, is protected from light culture.
6. a kind of Cucumber Target Leaf Spot resistance breeding method according to claim 1, which is characterized in that divide in the step 5)
Change the formula of culture medium are as follows: KNO32200mg/L, NH4NO3564mg/L, MgSO4·7H2O 280mg/L, NaH2PO4 150mg/
L, CaCl2·2H2O 255mg/L, MnSO4·4H2O 22.3mg/L, ZnSO4·7H2O 5.5mg/L, H3BO3 6.4mg/L
Na2MoO4·2H2O 0.25mg/L, CoCl2·6H2O 0.025mg/L, CuSO4·5H2O 0.25mg/L, Fe-Na2EDTA
33.6mg/L K2Cr2O7 0.019mg/L, inositol 85mg/L, niacin 1.4mg/L, glycine 2.0mg/L, tryptophan 3.4mg/L,
Beef extract 27mg/L, thiamine hydrochloride 0.3mg/L, puridoxine hydrochloride 0.5mg/L, pantothenic acid 1.2mg/L, curcumin 4.9mg/L,
L- selenium-methyl selenium substituted aminothiopropionic 0.11mg/L, L-carnitine 5.5mg/L, rosemary extracting solution 33mL/L, humic acid 40mg/
L, 6-BA2.0 mg/L, health polyphenol 0.5mg/L, brassin lactones 0.2mg/L, benzene peptide amino acid 0.2mg/L, pH value be 5.6 ~
6.0。
7. a kind of Cucumber Target Leaf Spot resistance breeding method according to claim 1, which is characterized in that raw in the step 6)
The formula of root culture medium are as follows: 1/2MS+IBA0.2mg/L+ choline chloride 0.05mg/L, pH value are 5.6 ~ 6.0.
8. a kind of Cucumber Target Leaf Spot resistance breeding method according to claim 1, which is characterized in that fitted in the step 7)
Suitable condition refers to day temperature control at 20-30 DEG C, and at 15-20 DEG C, intensity of illumination is controlled in 2500- for nocturnal temperature control
5000Lx, humid control is in 60-80%.
9. a kind of Cucumber Target Leaf Spot resistance breeding method according to claim 6, which is characterized in that the rosemary extracts
Liquid first boils 0.5h the preparation method comprises the following steps: rosemary plant is put into distilled water, then keeps 60-70 DEG C of water temperature extraction
Juice is filtered to remove solid residue by 2.5h, then filters with sterilizing filter that sterile extracting solution is made is standby in superclean bench
With.
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