CN104737901A - Method for screening mycosphaerella melonis resistance IL14 of cucumber-pickled cucumber introgression line - Google Patents
Method for screening mycosphaerella melonis resistance IL14 of cucumber-pickled cucumber introgression line Download PDFInfo
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Abstract
The invention relates to a method for screening mycosphaerella melonis-resistant IL 14 of cucumber-pickled cucumber introgression line. According to batch treatment of different concentrations, plants with good resistance are screened, and the method belongs to the field of biotechnology breeding. The method comprises the following steps: by taking wild species pickled cucumber as a female parent, cultivating cucumber 'Beijing jietou' as a male parent, and hybridizing to obtain hybrid F1; combining the hybrid F1 with a somaclone variation chromosome reduplication technology by virtue of embryo rescue, thereby obtaining a fertile inter-specific heterologous tetraploid new species which is named as Cucumis hytivus; and by taking the heterologous tetraploid new species Cucumis hytivus as a female parent, performing system backcrossing and self-cross selective breeding with the cultivated variety 'Beijing jietou', thereby obtaining 86 parts of cucumber inter-specific introgression line materials carried with wild species chromosome segments, and respectively numbering as IL1-IL86. The 86 parts of introgression line groups are screened by adopting the greenhouse seedling stage artificial inoculation identification technology, stable moderate resistance or more strains are obtained by virtue of inoculation treatment in spring and autumn, the inoculation treatment is performed by increasing the concentration of bacterium, and the mycosphaerella melonis resistance strain IL14 is screened.
Description
Technical field
The screening technique of cucumber of the present invention-pickled cucumber Introgressed line anti didymella IL14, by the batch process of variable concentrations, filters out Resistant expression good stand, belongs to biotechnology breeding field.
Background technology
Cucumber (Cucumis sativus L.) is one of large vegetables in the world ten, is mainly distributed in temperate zone and subtropical zone.According to United Nations Food and Agricultural Organization (FAO) statistics, the area under cultivation of Chinese cucumber in 2010 is 988 680hm
2, gross yield reaches 40 710 200t, is cucumber production maximum in the world and country of consumption.In recent years, along with the development of cucumber in greenhouse, cucumber blight dis-ease (Mycosphaerella melonis) becomes the second largest disease that root-knot nematode of continuing endangers cucumber production after being ill.It is multiple that this disease meets hot and humid condition in facility, and cucumber can the underproduction about 20%, serious will cause uprooting plants after their edible portions have been harvested in advance even have no harvest, this causes cucumber merchandized handling and has a strong impact on (Sun Yuanchao, 2010; Wang Peishuan and Dong Qincheng, 2010).
At present, home and abroad there is no special effective measures of carrying out preventing and treating for blight dis-ease, majority tends to general chemistry control, chemical agent not only cost is relatively high and poor in the hot and humid comparatively serious regional control efficiency of blight dis-ease morbidity that waits, effectively can not control the development of the blight dis-ease state of an illness, spray environmental pollution that chemical agent causes for a long time and the pesticide resistance that pathogen itself produces is day by day obvious, the limitation of chemical agent in actual production process becomes increasingly conspicuous; The research of biological control aspect is less and application is limited.Therefore screening has high resistance distinct variety and applies, can fundamentally effectively symptom management generation and spread.
Cucumber genetic background is narrow, aberration rate is only 3 ~ 8% (Staub et al., 1987), and Resistant germplasm is deficienter, be difficult under natural conditions obtain and have excellent Resistant germplasm or mutant, therefore cucumber anti didymella breeding process once sank into bottleneck stage.Wild species or wild relatives have accumulated degeneration-resistant hereditary factor in long-term evolution process, for crop genetic improvement provides good material.By the excellent resistant gene in distant hybridization transfer Wild related germplasm to expand cucumber hereditary basis, utilizing distant hybridization to obtain Introgressed line is widen the effective means that plant genetic background improves resistance, Introgressed line colony is excavating, is utilizing sibling species matter resource, research affect in the complex characters such as crop growth, output, resistance and plays an important role, and is that solution cucumber is disease-resistant, important method in resistance breeding process and means.
Cucumber wild species C.hystrix Chakr. (2n=24), the rare wild species of an original Chinese yunnan Cucumis, through repeatedly Resistance Identification for many years, specify these wild species, on blight dis-ease, there is multiple resistance, as multiple diseases (Chen J F, 1997 such as anti didymella, downy mildew, root knot nematode diseases; Chen J F, 2003; Chen JF, 2004).The interspecific cross that Chen Jingfeng etc. (Chen J F, 2000) successfully achieve itself and susceptible cultivar ' Beijing butt ' (C.sativus L., 2n=14) obtains F
1, utilize embryo rescue and somaclone chromosome doubling technology to synthesize new allotetraploid Cucumis. × hytivus.By this allotetraploid and cultivated cucumber ' Beijing butt ' being carried out backcross, selfing, obtain a series of there is different phenotypic character gradually ooze breeding material.
The effective way creating new species and new material is not only in distant hybridization, its external source desirable genes be gradually seeped in raise crop can improve the disease-resistant resistance of crop simultaneously, by inoculated identification Introgressed line colony to the resistance of cucumber blight dis-ease, the strain to blight dis-ease high resistance can be filtered out, thus improve the quality of the merchandized handling of cucumber in raise crop
Summary of the invention
Technical problem
The present invention be directed to the above-mentioned weak point of current prior art, a kind of cultivation and screening technique of anti didymella cucumber Introgressed line material are provided
Technical scheme
The screening technique of cucumber-pickled cucumber anti didymella Introgressed line IL14, comprises following steps:
(1) acquisition of Introgressed line between seed of Fructus Cucumidis sativi: with wild species pickled cucumber for female parent, cultivated cucumber ' Beijing butt ' is that paternal hybrid obtains hybrid F
1, the comparable cultivated cucumber of wild species pickled cucumber ' Beijing butt ' sowing in 2 months in advance, and the photoperiod process it being carried out to more than 40 days, make both flower synchronizations.Hybrid F
1by embryo rescue combination cell clonal mutated chromosome doubling techniques, allotetraploid novel species between the kind that acquisition can be educated, called after Cucumis hytivus, with allotetraploid novel species Cucumis hytivus for female parent, backcrossed and selfed breeding by system with cultivar ' Beijing butt ', obtain Introgressed line material between 86 parts of seed of Fructus Cucumidis sativi carrying wild species chromosome segment.
(2) Introgressed line anti didymella inoculation just qualification (spring, Qiu Liangji) between seed of Fructus Cucumidis sativi: preparing spore concentration is 1 × 10
6the blight dis-ease pathogen spore suspension of individual/mL, utilizes greenhouse inoculation identification method in artificial seedling stage to spray the 86 portions of cucumber-pickled cucumber Introgressed line material inoculated in above-mentioned (1), inoculates 7 days and adds up the sick level of blade afterwards.Inoculated identification two season material, filter out altogether performance stable in anti-and above Introgressed line material 27 parts.
(3) cucumber-pickled cucumber anti didymella Introgressed line IL14 screens: preparing spore concentration is 1 × 10
7the blight dis-ease pathogen spore suspension of individual/mL, utilizes greenhouse inoculation identification method in artificial seedling stage to spray the 27 portions of cucumber-pickled cucumber Introgressed line material inoculated in above-mentioned (2), inoculates 7 days and adds up the sick level of blade afterwards.Filter out cucumber-pickled cucumber anti didymella Introgressed line IL14.
Between described seed of Fructus Cucumidis sativi, Introgressed line anti didymella inoculated identification concrete steps are as follows:
1) inoculate the nursery of front cucumber material and step of transplanting seedlings, concrete grammar is as follows:
Nursery: Introgressed line between each strain seed of Fructus Cucumidis sativi is soaked seed 3 ~ 5 hours in 55 DEG C of warm water, vernalization in 28 DEG C of constant incubators, the surface of the seed is rinsed every 10h, change seed soaking water, when seed base-root grows to 0.3 ~ 0.7cm, seed is sowed in the cave dish that bactericidal nurishing soil is housed, is placed in temperature 25 DEG C, relative moisture is in the greenhouse of 80%.；
Transplant seedlings: transplant seedlings in the nutritive cube that bactericidal nurishing soil is housed (bore 12cm, footpath, end 10cm, high 8cm) when cucumber seedling rough leaf flattens, the strain of every alms bowl 1.
Described bactericidal nurishing soil is that the peat composed of rotten mosses and vermiculite 1: 1 mix, and through 121 DEG C of sterilizing gained.
2) acquisition of pathogen and cultivation
Climing 1 ~ the 2cm of cucumber blight dis-ease that fruit body gives prominence to epidermis is got in the acquisition of pathogen, tap water 2 ~ 3min, 75% alcohol surface sterilization 30s, sterile water wash 2 ~ 3min, by the single fruit body of sterilizing dissecting needle picking, be placed in the sterilizing culture dish filling sterile water and soak 2min.Fruit body is placed on the potato dextrose medium (PDA) containing rifampin and carries out cultivating (PH=5).Culture dish cultivates 3d at being placed on 28 DEG C, is cultivated by separating for several times, finally obtains isozygoty cultivation (Li Ying etc., 2007; Ellis and Holliday, 1971).
The pathogen of purified cultivation is adopted the mode of picking mycelia or spore by the cultivation of pathogen, being inoculated in containing rifampin, PH is in the PDA medium of 5.0, the method induced meristem spore adopting light culture and interval uviol lamp process (12h dark/12h uviol lamp) to combine after light culture 4d produces, 25 DEG C of preservations.
Pathogen rejuvenation: the pathogen cultivated through purifying is inoculated in the fresh cucumber epidermis after sterilization treatment, after 4d, picking black spore inoculating is in the PDA medium containing rifampin (PH=5.0).
3) pathogen spore suspension configuration
Add 5 ~ 10mL aseptic water washing Bacteria culturing primary surface, scrape medium top layer gently with L-type glass bar, the spore suspension of acquisition is through four layers of filtered through gauze, and the spore crude extract of impurity such as removing mycelia, agar etc. is as mother liquor.With pH value to 3.5 ~ 4.0 of lactic acid adjustment spore suspension, use the spore concentration of blood counting chamber statistics suspension under the microscope, by the preparation of inoculating spores concentration to 1 × 106mL
-1, before inoculation, add 2.5gL
-1gelatin as surfactant.
4) inoculate
Carry out artificial spray inoculation during slice true leaf of cucumber seedling growth to 3 ~ 4, inoculating spores suspension concentration is 1 × 10
6mL
-1, adjoining tree inoculation sterile water.Cover canopy film 3d after inoculation, the airtight hut of shading, and keep relative moisture 92% ~ 95% in canopy, temperature 21 ~ 25 DEG C.
Described inoculation step concrete grammar, for inoculating when growing to 3 ~ 4 true leaves until seedling, uses the spraying of sprayer complete stool, and control water droplet wish in the amount of spraying to plant leaf and drip, spore suspension concentration is 1 × 10
6mL
-1, in airtight canopy, place humidifier after inoculation carry out adding wet process to ensure humidity, control group inoculation liquid does not add pathogen conidium.Keep relative moisture 92% ~ 95% in canopy, temperature 21 ~ 25 DEG C, carries out normal rich water quality management.
5) identify
Add up the sick level in leaf portion after inoculation after 7d, statistics position is 4 true leaves from basal part of stem, and the sick level of the method statistic with reference to Wehner and Zhang, according to following formulae discovery disease index.
Disease index (DI)=∑ (sick level × this sick level strain number)/(the highest sick level × investigate total strain number) × 100.With reference to the method for Fang Zhongda (1998).Leaf, stem's disease refers to that DI≤15 are for high resistance (HR); 15 < DI≤45 are anti-(R); 45 < DI≤75 are sense (S); 75 < DI are high sense (HS).
Leaf part grade standard: 0 ~ 9 grade: 0) asymptomatic; 1 ~ 2) sporadicly infect; 3 ~ 4) a small amount of fleck; 5 ~ 6) blade of 20 ~ 50% has fleck; 7 ~ 8) plant wither and spot are more than 50%; 9) plant death (Wehner & Shetty, 2000).
The screening of cucumber-pickled cucumber anti didymella Introgressed line IL14 obtains by the following method:
(1) cucumber-pickled cucumber Introgressed line colony anti didymella spring and autumn just screens:
With cucumber-pickled cucumber Introgressed line colony (86 parts), common cultivation cucumber ' Beijing butt ' (susceptible contrast) for material, adopt the method for artificial inoculation on seeding in greenhouse qualification to inoculate, preparing spore suspension concentration is 1 × 10
6individual/mL.Cover canopy film 3d after inoculation, shading is airtight, ensures relative moisture 92% ~ 95% in canopy, temperature 21 ~ 25 DEG C.The sick level in statistics leaf portion after inoculating 1 week, statistics position is 4 true leaves from basal part of stem.According to formula draw disease index in anti-above material, two seasons sifted out 27 parts altogether.
(2) screening of cucumber-pickled cucumber anti didymella Introgressed line IL14:
With gained 27 portions of cucumber-pickled cucumber Introgressed line in (1) for material, common cultivation cucumber ' Beijing butt ' is susceptible contrast, and the same method of artificial inoculation on seeding in greenhouse qualification that adopts is inoculated, and preparing spore suspension concentration is 1 × 10
7individual/mL, can produce obvious resistance class difference after making to have the different strain inoculation of resistance, cover canopy film 3d after inoculation, shading is airtight, ensures relative moisture 92% ~ 95% in canopy, temperature 21 ~ 25 DEG C.The sick level in statistics leaf portion after inoculating 1 week, statistics position is 4 true leaves from basal part of stem.Filter out and there is high resistance and the cucumber of resistance-pickled cucumber anti didymella Introgressed line IL14.
Beneficial effect:
The cultivation of cucumber of the present invention-pickled cucumber anti didymella Introgressed line IL14 and screening and application method thereof, compared with existing cucumber cultivation present situation, tool has the following advantages and good effect:
Cucumber is a kind of important worldwide vegetables, because its background is narrow, diversity is lower, traditional breeding method cannot meet breeding for disease resistance demand, the present invention be utilize interspecific cross, many generations backcross and the mode of selfing by favorable genes transformation in wild pickled cucumber in 86 parts of Introgressed line cucumber, the disease-resistant resistance of common cultivation kind can be improved by useful resistant gene in transfer wild species.The present invention adopts the method for greenhouse artificial infection idenfication in seedling stage, filters out anti didymella Introgressed line IL14 in Introgressed line colony, for anti didymella breeding seed selection provides useful material.The cucumber anti didymella Introgressed line material IL14 screened by inoculated identification has obvious application prospect, not only solve the narrow problem of cucumber background, also reliable guarantee is provided for cucumber anti didymella lacks stable antigen problem, germ plasm resource excellent in cucumber-pickled cucumber Introgressed line is used, this is for breaking the narrow bottleneck of cucumber genetic background in cucumber anti didymella breeding research from now on and excavating wild resource favorable genes, Effect of Anti blight dis-ease hereditary capacity, meticulous location, the map based cloning of gene and the genetic improvement etc. of kind are with a wide range of applications.
By field observation, cucumber-pickled cucumber Introgressed line IL14 has stable resistance to blight dis-ease, and plant growing way is strong, the long 14.2cm of average main internode, the climing thick 0.3cm of average master, average branch number is 4, leaf green, the long 22.5cm of average leaf, mean blade width 23.1cm, the long 14.4cm of average petiole, average first female flower joint position is at 8 joints, and average single-unit female flower number is 3, and commodity melon is corynebacterium, light green color, melon thorn white, quantity is medium, the long 14.5cm of average melon, thick 4.2cm, master is climing all can tie melon.Therefore, cucumber-pickled cucumber Introgressed line IL14 can be used as excellent disease-resistant materials application in new breeds of cucumbers breeding practice.
Accompanying drawing explanation
Fig. 1: the cultivation of anti didymella cucumber Introgressed line material IL14 and triage techniques route map;
Fig. 2: blight dis-ease pathogen mycelia and spore;
Fig. 3: cucumber seedling to be seeded;
Fig. 4: different brackets cucumber leaves after inoculation;
Fig. 5: different brackets cucumber plant after inoculation;
Fig. 6: anti-after inoculation blight dis-ease, to feel plant variable rate technology.
Embodiment
Cucumber blight dis-ease is a kind of serious worldwide disease, makes a big impact to the quality and yield of cucumber.The present invention for material, utilizes seedling stage greenhouse Inoculation Method to carry out Resistance Identification to Introgressed line material with cucumber-pickled cucumber Introgressed line, through two season inoculated identification, filter out cucumber-pickled cucumber anti didymella Introgressed line IL14.The cultivation of anti didymella cucumber Introgressed line material IL14 and screening technique, comprising:
The acquisition of 1, cucumber-pickled cucumber Introgressed line
Cucumber wild species C.hystrix Chakr. (2n=24) is the rare wild species of an original Chinese yunnan Cucumis, for short-day plant, the growing way time at initial stage is longer, ' Beijing butt ' is common cultivation cucumber, therefore wild species pickled cucumber shifts to an earlier date sowing in 2 months than common cultivation cucumber, and it is carried out to the photoperiod process of more than 40d, make both flower synchronizations.With wild species pickled cucumber for female parent, cultivated cucumber ' Beijing butt ' is paternal hybrid acquisition Hybrids F1, hybrid F
1by embryo rescue combination cell clonal mutated chromosome doubling techniques, allotetraploid novel species between the kind that acquisition can be educated, called after Cucumis hytivus, with allotetraploid novel species Cucumis hytivus for female parent, to be backcrossed 1 ~ 2 generation, selfing 5 ~ 7 generation seed selection by system with cultivar ' Beijing butt ', obtain Introgressed line material between 86 parts of seed of Fructus Cucumidis sativi carrying wild species chromosome segment, its somatic chromosome qualification result shows that cucumber-pickled cucumber Introgressed line colony is identical with common cultivation cucumber chromosomal number, 2n=14.Concrete Cultivating techniques route map is shown in Fig. 1.
2, cucumber 86 parts of Introgressed line anti didymella inoculated identifications
1) pathogen gathers: blight dis-ease pathogen picks up from the sick stem of field cucumber natural occurrence (blight dis-ease) diseased plant, carries out separation and purification, and collect preservation by cucumber seminar of College of Horticulture of Agricultural University Of Nanjing with the inoculation method of the single fruit body of picking.
2) cultivation of pathogen: the mode pathogen of purified cultivation being adopted picking mycelia or spore, being inoculated in containing rifampin, PH is in the PDA medium of 5.0, the method induced meristem spore adopting light culture and interval uviol lamp process (12h dark/12h uviol lamp) to combine after light culture 4d produces, 25 DEG C of preservations, are shown in Fig. 2.
3) proportioning of matrix and sterilizing: vermiculite and the peat composed of rotten mosses are mixed obtained Nutrition Soil in 1: 1 ratio, autoclaving at 121 DEG C.
4) material plantation:
Nursery: by 86 parts of seed of Fructus Cucumidis sativi intermolecular hybrid Introgressed line materials, disease-resistant contrast wild species (C.hystrix), susceptible contrast ' Beijing butt ', the strain of every part of material 10, soak seed 4 hours in 55 DEG C of warm water, be placed in 28 DEG C of constant incubators and carry out vernalization, observe and rinse the surface of the seed, supplement a small amount of moisture in time when the surface of the seed moisture is few, when seed base-root grows to 0.5cm, seed is sowed in the cave dish that bactericidal nurishing soil is housed.
Transplant seedlings: transplant seedlings in the nutritive cube that bactericidal nurishing soil is housed (bore 12cm, footpath, end 10cm, high 8cm) when cucumber seedling rough leaf flattens i.e. " two leaves wholeheartedly ", the strain of every alms bowl 1.
5) material inoculation:
Pathogen spore suspension configures: on Bacteria culturing ware, add 5 ~ 10mL sterile water to rinse Bacteria culturing primary surface, medium top layer is scraped gently with L-type glass bar, the spore suspension obtained is through four layers of filtered through gauze, and the spore crude extract of impurity such as removing mycelia, agar etc. is as mother liquor.With pH value to 3.5 ~ 4.0 of lactic acid adjustment spore suspension, use the spore concentration of blood counting chamber statistics suspension under the microscope, by the preparation of inoculating spores concentration to 1 × 10
6mL
-1, before inoculation, add 2.5gL
-1gelatin as surfactant.
Inoculation: carry out artificial spray inoculation during slice true leaf of cucumber seedling growth to 3 ~ 4, see Fig. 3.Inoculating spores suspension concentration is 1 × 10
6mL
-1, adjoining tree inoculation sterile water.Use the spraying of sprayer complete stool, control water droplet wish in the amount of spraying to plant leaf and drip, in airtight canopy, place humidifier after inoculation carry out adding wet process to ensure humidity, control group inoculation liquid does not add pathogen conidium.Cover canopy film 3d after inoculation, the airtight hut of shading, keep relative moisture 92% ~ 95% in canopy, temperature 21 ~ 25 DEG C, carries out normal rich water quality management.
6) identify
Add up the sick level in leaf portion after inoculation after 7d, statistics position is 4 true leaves from basal part of stem, and the sick level of the method statistic with reference to Wehner and Zhang, according to following formulae discovery disease index.
Disease index (DI)=∑ (sick level × this sick level strain number)/(the highest sick level × investigate total strain number) × 100.With reference to the method for Fang Zhongda (1998).Leaf, stem's disease refers to that DI≤15 are for high resistance (HR); 15 < DI≤45 are anti-(R); 45 < DI≤75 are sense (S); 75 < DI are high sense (HS).
Leaf part grade standard: 0 ~ 9 grade: 0) asymptomatic; 1 ~ 2) sporadicly infect; 3 ~ 4) a small amount of fleck; 5 ~ 6) blade of 20 ~ 50% has fleck; 7 ~ 8) plant wither and spot are more than 50%; 9) plant death (Wehner & Shetty, 2000).
7) add up according to above-mentioned inoculation identification method, in spring and autumn experiment, filter out Introgressed line material 27 parts anti-and above in performance altogether, see Fig. 4 and Fig. 5.
8) material of resistance is had to carry out third quarter inoculated identification with filter out 27 parts of performances, with common cultivation cucumber ' Beijing butt ' for susceptible contrast, the method of same employing artificial inoculation on seeding in greenhouse qualification is inoculated, material plantation and Spawn incubation method the same.During inoculation, pathogen spore suspension concentration is risen to 1 × 10
7individual mL
-1, can produce obvious resistance class difference after making to have the different strain inoculation of resistance, cover canopy film 3d after inoculation, shading is airtight, ensures relative moisture 92% ~ 95% in canopy, temperature 21 ~ 25 DEG C.Inoculate 7 days and add up the sick level in leaf portion afterwards, statistics position is 4 true leaves from basal part of stem.Filter out and there is high resistance and the cucumber of resistance-pickled cucumber anti didymella Introgressed line IL14, after qualification, find that the Introgressed line material being numbered IL14 shows resistance to blight dis-ease, and resistance is comparatively stable in three seasons observed, and sees Fig. 6.
The cucumber anti didymella Introgressed line material IL14 that application the present invention obtains, can directly as breeding material, this Material growth gesture is strong, the long 14.2cm of average main internode, average main climing thick 0.3cm, average branch number is 4, leaf green, the long 22.5cm of average leaf, mean blade width 23.1cm, the long 14.4cm of average petiole, average first female flower joint position is at 8 joints, and average single-unit female flower number is 3, and commodity melon is corynebacterium, light green color, melon thorn white, quantity is medium, the long 14.5cm of average melon, thick 4.2cm, master is climing all can tie melon.The acquisition of this disease-resistant material can be used as excellent disease-resistant materials application in new breeds of cucumbers breeding practice, has also established solid foundation for studying the location of cucumber disease-resistant gene molecule marker, the structure of collection of illustrative plates and clone etc. from now on simultaneously.
Bibliography
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Chen J F，Staub J E，Qian C T，et al.Reproduction and cytogenetic characterization of interspecific hybridsderived from Cucumis hystrix Chakr.×Cucumis sativus L.[J].Theoretical and Applied Genetics，2003，106(4)：688-695
Chen J F，Staub J E，Tashiro Y，et al.Successful interspecific hybridization between Cucumis sativus L.and C.hystrix Chakr[J].Euphytica，1997，96(3)：413-419
Chen JF，MoriatyG，Jahn M.2004.Some disease resistance tests in Cucumis hystrix and its progenies frominterspecific hybridization with cucumber[J].Proceedings of Cucurbitaceae：189-196
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Staub JE，Fredric L，Marty TL.1987.Electrophoretic variation in cross compatible wild diploid species ofCucunis[J].Can.J.Bot，65：792-798
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Claims (4)
1. the acquisition of cucumber-pickled cucumber Introgressed line:
Cucumber wild species C.hystrix Chakr. (2n=24) is the rare wild species of an original Chinese yunnan Cucumis, for short-day plant, the growing way time at initial stage is longer, ' Beijing butt ' is common cultivation cucumber, therefore wild species pickled cucumber shifts to an earlier date sowing in 2 months than common cultivation cucumber, and it is carried out to the photoperiod process of more than 40d, make both flower synchronizations.With wild species pickled cucumber for female parent, cultivated cucumber ' Beijing butt ' is paternal hybrid acquisition hybrid F
1, hybrid F
1by embryo rescue combination cell clonal mutated chromosome doubling techniques, allotetraploid novel species between the kind that acquisition can be educated, called after Cucumis hytivus, with allotetraploid novel species Cucumis hytivus for female parent, to be backcrossed 1 ~ 2 generation, selfing 5 ~ 7 generation seed selection by system with cultivar ' Beijing butt ', obtain Introgressed line material between 86 parts of seed of Fructus Cucumidis sativi carrying wild species chromosome segment, its somatic chromosome qualification result shows that cucumber-pickled cucumber Introgressed line colony is identical with common cultivation cucumber chromosomal number, 2n=14.
2. cucumber 86 parts of Introgressed line anti didymella inoculated identifications filter out anti-material in 27 parts:
1) pathogen gathers: blight dis-ease pathogen picks up from the sick stem of field cucumber natural occurrence (blight dis-ease) diseased plant, carries out separation and purification, and collect preservation by cucumber seminar of College of Horticulture of Agricultural University Of Nanjing with the inoculation method of the single fruit body of picking.
2) cultivation of pathogen: the mode pathogen of purified cultivation being adopted picking mycelia or spore, being inoculated in containing rifampin, PH is in the PDA medium of 5.0, the method induced meristem spore adopting light culture and interval uviol lamp process (12h dark/12h uviol lamp) to combine after light culture 4d produces, 25 DEG C of preservations, are shown in Fig. 2.
3) proportioning of matrix and sterilizing: vermiculite and the peat composed of rotten mosses are mixed obtained Nutrition Soil in 1: 1 ratio, autoclaving at 121 DEG C.
4) material plantation:
Nursery: by 86 parts of seed of Fructus Cucumidis sativi intermolecular hybrid Introgressed line materials, disease-resistant contrast wild species (C.hystrix), susceptible contrast ' Beijing butt ', the strain of every part of material 10, soak seed 4 hours in 55 DEG C of warm water, be placed in 28 DEG C of constant incubators and carry out vernalization, observe and rinse the surface of the seed, supplement a small amount of moisture in time when the surface of the seed moisture is few, when seed base-root grows to 0.5cm, seed is sowed in the cave dish that bactericidal nurishing soil is housed.
Transplant seedlings: transplant seedlings in the nutritive cube that bactericidal nurishing soil is housed (bore 12cm, footpath, end 10cm, high 8cm) when cucumber seedling rough leaf flattens i.e. " two leaves wholeheartedly ", the strain of every alms bowl 1.
5) material inoculation:
Pathogen spore suspension configures: on Bacteria culturing ware, add 5 ~ 10mL sterile water to rinse Bacteria culturing primary surface, medium top layer is scraped gently with L-type glass bar, the spore suspension obtained is through four layers of filtered through gauze, and the spore crude extract of impurity such as removing mycelia, agar etc. is as mother liquor.With pH value to 3.5 ~ 4.0 of lactic acid adjustment spore suspension, use the spore concentration of blood counting chamber statistics suspension under the microscope, by the preparation of inoculating spores concentration to 1 × 10
6mL
-1, before inoculation, add 2.5gL
-1gelatin as surfactant.
Inoculation: carry out artificial spray inoculation during slice true leaf of cucumber seedling growth to 3 ~ 4, see Fig. 3.Inoculating spores suspension concentration is 1 × 10
6mL
-1, adjoining tree inoculation sterile water.Use the spraying of sprayer complete stool, control water droplet wish in the amount of spraying to plant leaf and drip, in airtight canopy, place humidifier after inoculation carry out adding wet process to ensure humidity, control group inoculation liquid does not add pathogen conidium.Cover canopy film 3d after inoculation, the airtight hut of shading, keep relative moisture 92% ~ 95% in canopy, temperature 21 ~ 25 DEG C, carries out normal rich water quality management.
6) identify
Add up the sick level in leaf portion after inoculation after 7d, statistics position is 4 true leaves from basal part of stem, and the sick level of the method statistic with reference to Wehner and Zhang, according to following formulae discovery disease index.
Disease index (DI)=∑ (sick level × this sick level strain number)/(the highest sick level × investigate total strain number) × 100.With reference to the method for Fang Zhongda (1998).Leaf, stem's disease refers to that DI≤15 are for high resistance (HR); 15<DI≤45 are anti-(R); 45<DI≤75 are sense (S); 75<DI is high sense (HS).
Leaf part grade standard: 0 ~ 9 grade: 0) asymptomatic; 1 ~ 2) sporadicly infect; 3 ~ 4) a small amount of fleck; 5 ~ 6) blade of 20 ~ 50% has fleck; 7 ~ 8) plant wither and spot are more than 50%; 9) plant death (Wehner & Shetty, 2000).
7) add up according to above-mentioned inoculation identification method, in spring and autumn experiment, filter out Introgressed line material 27 parts anti-and above in performance altogether.
3. the screening of cucumber-pickled cucumber Introgressed line anti didymella IL14:
Have the material of resistance to carry out third quarter inoculated identification with filter out 27 parts of performances, with common cultivation cucumber ' Beijing butt ' for susceptible contrast, the same method of artificial inoculation on seeding in greenhouse qualification that adopts is inoculated, material plantation and Spawn incubation method the same.During inoculation, pathogen spore suspension concentration is risen to 1 × 10
7individual mL
-1, can produce obvious resistance class difference after making to have the different strain inoculation of resistance, cover canopy film 3d after inoculation, shading is airtight, ensures relative moisture 92% ~ 95% in canopy, temperature 21 ~ 25 DEG C.Inoculate 7 days and add up the sick level in leaf portion afterwards, statistics position is 4 true leaves from basal part of stem.Filter out and there is high resistance and the cucumber of resistance-pickled cucumber anti didymella Introgressed line IL14, after qualification, find that the Introgressed line material being numbered IL14 shows resistance to blight dis-ease, and resistance is comparatively stable in three seasons observed.
4. the cucumber anti didymella Introgressed line material IL14 obtained, can directly as breeding material, and this Material growth gesture is strong, the long 14.2cm of average main internode, the climing thick 0.3cm of average master, average branch number is 4, leaf green, the long 22.5cm of average leaf, mean blade width 23.1cm, the long 14.4cm of average petiole, average first female flower joint position is at 8 joints, and average single-unit female flower number is 3, and commodity melon is corynebacterium, light green color, melon thorn white, quantity is medium, the long 14.5cm of average melon, thick 4.2cm, master is climing all can tie melon.The acquisition of this disease-resistant material can be used as excellent disease-resistant materials application in new breeds of cucumbers breeding practice, has also established solid foundation for studying the location of cucumber disease-resistant gene molecule marker, the structure of collection of illustrative plates and clone etc. from now on simultaneously.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106171963A (en) * | 2016-08-25 | 2016-12-07 | 南京农业大学 | The breeding method of Fructus Cucumidis sativi anti didymella Introgressed line HH1 812 and resistant gene site molecular marker |
| CN117586931A (en) * | 2024-01-19 | 2024-02-23 | 中国农业科学院蔬菜花卉研究所 | Achromobacter xylosoxidans IVF-WK240 and application thereof |
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| CN103355160A (en) * | 2013-07-31 | 2013-10-23 | 南京农业大学 | Gradually oozing between the Cucumis kind is the method that colony made up and be used for breed cucumber |
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| CN117586931A (en) * | 2024-01-19 | 2024-02-23 | 中国农业科学院蔬菜花卉研究所 | Achromobacter xylosoxidans IVF-WK240 and application thereof |
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