CN107114234A - A kind of screening technique of glossy type disease-resistant anvil pumpkin material - Google Patents
A kind of screening technique of glossy type disease-resistant anvil pumpkin material Download PDFInfo
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- CN107114234A CN107114234A CN201710365680.0A CN201710365680A CN107114234A CN 107114234 A CN107114234 A CN 107114234A CN 201710365680 A CN201710365680 A CN 201710365680A CN 107114234 A CN107114234 A CN 107114234A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The present invention relates to a kind of screening technique of glossy type disease-resistant anvil pumpkin material, step is as follows：(1) grafting：Collect different types of Pumpkin Germplasm material, whne pumpkin seedling length to two leaves wholeheartedly when Grafted Cucumber Seedling scion, before grafting by the true leaf of stock pumpkin pinch fall, keep growing point here；Grafting is colonized, and reservation two is climing, and a cucumber is climing, a pumpkin vine；(2) the glossy property screening of cucumber：Screening is compared between grafting, select cucumber dewaxing powder most substantially, the best material of brightness, removed in time by cucumber is climing after 23, cucumber knot melon, only retain pumpkin vine with screening disease resistance trait to be identified or reserve seed for planting；(3) pumpkin rootstock disease resistance is screened：Anti-blight, mildew-resistance, anti-CGMMV screening are carried out to grafting, the glossy property material selfing of disease-resistant and cucumber simultaneously retains F1 generation Pumpkin Seed.The inventive method breeding time limit is short, efficiency high, means specification and can retain pumpkin seedling and stock merit simultaneously.
Description
Technical field
The invention belongs to foliage filter screening breeding field, and in particular to a kind of screening side of the glossy disease-resistant anvil pumpkin material of type
Method.
Background technology
Pumpkin aboundresources is various, but China does not make the identification of system perfecting to many outstanding Pumpkin Germplasm resources
And evaluation, the utilization rate of material is high.
The accreditation of popularization and the public recently as graft technology, the cultivated area of Grafted Cucumber Seedling is also constantly expanding,
Grafting disease is also on the rise caused by large-scale planting and continuous cropping are aggravated simultaneously.And the Major Diseases of pumpkin such as virosis, withered
The disease-resistant Journal of Sex Research for disease, the powdery mildew etc. of withering lags behind production.
Since the eighties in last century, some domestic breeding units start anvil pumpkin in terms of breeding research, current China
Pumpkin variety seed selection mainly uses hybrid vigour, and the basic research such as relevant pumpkin hybrid vigour, Heredity is reported
It is less, therefore the heredity of many objective traits is unknown by the people with stable law, causes current anvil pumpkin seed selection to be in relatively more mixed
Random state, using the most frequently used screening technique of traditional breeding for heterosis is searched by modes such as farmers', market and purchases
Collect pumpkin material and kind, carry out it is many turn into the stable self-mating system of character for directive breeding, strain F5 for character it is stable after enter again
Row Grafting Experiment, carries out a series of screening of anvil objective traits such as affinity, symbiosis power, glossy property, disease resistance.
Wherein the screening technique of disease resistance has two kinds at present：(1) to take sick nursery plot field plot test as Main Means, draw
Permanent year sample plot, the serious soil sample of disease is gathered in Grafted Variety Zhu Zai areas, is spreaded manuer in holes with cultivation of per season orientation and is puddled disease
Soil field planting, creates the condition (high temperature, high humidity etc.) beneficial to morbidity, the incidence of object observing material in the course of cultivation.(2)
It is micro- that the environmental quality of disease time and requirement since Germination period to foundation disease of whole growth period is inoculated with cause of disease in real time
Biology, according to its disease index screening material.
Affinity and glossy property screening technique are：By homozygosis material and scion grafting, its survival rate is observed, can be complete after field planting
Into normally surviving, stretch it is climing, bloom, the growth course of result and more than professional standard, as affinity is qualified；Grafting green cucumber
Afterwards, with cucumber seedling (kind do not dewax powder) for blank control, cucumber sugar-preserved gourd sloughs wax powder, compares sieve according to its glossy degree
Choosing.
There is following four in existing screening technique：
One is that the seed selection time is long, and management cost is high.Traditional material purifying and creation are whole by type after resource is collected
Reason is sorted out, and carries out plantation evaluation, and selfing is reserved seed for planting, and superior and inferior evaluating screening is carried out with reference to breeding objective, and objective trait stably obtains kind
The screening for tapping into a series of anvil objective traits such as row affinity, symbiosis power, glossy property, disease resistance is remarried after son.Whole flow process
Get off to take around time of 5 years or so.
Two be that Breeding objective is clear and definite, but is easily lost in screening process.Existing material purifying is to provide with creation
Source is arranged by type after collecting to be sorted out, and carries out plantation evaluation, selfing is reserved seed for planting, superior and inferior evaluating screening, mesh are carried out with reference to breeding objective
The screening for tapping into a series of anvil objective traits such as row affinity, symbiosis power, glossy property, disease resistance is remarried after mark character is stable.
This screening mode can only be screened pumpkin seedling trait expression is good, may choosing some characters when losing as stock
The good material having outstanding performance.
During three are stock material selfing homozygosis screening process, each generation planting number, selected quantity, selection approach are not advised
Model.Prior art is in stock material screening process, and screening direction (1) requires that seed meat is thin, fruit caving is big, seed is more and honest and kindhearted,
(2) resistance, high temperature resistant, high humidity or low temperature resistant, (3) growing way, well developed root system, plant are vigorous, (4) disease resistance.According to this several Xiang Xuan
Educate object filtering and go out a number of material and add generation stable to strain, workload is big in screening process, is selected in quantity many (typically
Need to select nearly 100 plants or so), Grafting Experiment is carried out after the completion of screening, it is necessary to each strain when affinity and symbiosis power are determined
Every part of material all carry out substantial amounts of experiment, data could have certain convincingness, and this nonstandard screening mode can be with
Adding for self-mating system more and more for drawback.
Four be that breed cucumber comprehensive resistance level has much room for improvement.Threatening the disease of cucumber furnished equipments at present mainly has on the ground
Disease and underground disease two parts, underground disease refer mainly to various soil-borne diseases, such as droop, epidemic disease, root rot, on the ground
Some Disease refers mainly to the leaf diseases such as powdery mildew, virosis of cucumber, downy mildew, gray mold etc., recently as grafting
The popularization of seedling application, the various soil-borne diseases in underground are paid attention to, but the anti-leaf diseases of stock are ignored, and relevant research shows to transfer
Certain inducing action is connected to, the genetic signal material from stock can be transferred in scion and be adjusted by plasmodesmus and vascular bundle
The pattern of growing of scion is controlled, the anti-leaf diseases ability of stock can be transmitted among scion, and seed selection is disease-resistant to scion leaf portion
Property the strong stock material of influence, the disease-resistant level of synthesis of breed cucumber can be effectively improved.It is common in pumpkin material and grafting green cucumber
In one period of growth, while identify the leaf portion resistance against diseases of pumpkin leaf diseases resistance against diseases and cucumber plant, select pair
Cucumber scion leaf portion resistance against diseases influences big pumpkin material, and the synthesis that can not only improve peasant household's cultivating and growing cucumber plant resists
Sick level, and it is substantially shorter breeding time.
The content of the invention
For the problem of pumpkin rootstock is screened in the prior art, the invention aims to provide a kind of breeding time limit it is short,
Efficiency high, means specification and the disease-resistant anvil pumpkin material of the glossy type of pumpkin seedling and stock merit can be retained simultaneously
Screening technique.
A kind of screening technique of glossy type disease-resistant anvil pumpkin material, step is as follows：
(1) grafting：Different types of Pumpkin Germplasm material is collected, and Pumpkin Seed is sowed, each kind sows 15-25
Strain, whne pumpkin seedling length to two leaves wholeheartedly when Grafted Cucumber Seedling scion, before grafting by the true leaf of stock pumpkin pinch fall, keep growing point here；
Grafting is colonized 10-15 plants, and reservation two is climing, and a cucumber is climing, a pumpkin vine；
(2) the glossy property screening of cucumber：Cucumber seedling using the powder that do not dewax is compared as blank control between grafting
Screen, select the cucumber dewaxing powder most best material of obvious, brightness, in time by the climing removal of cucumber after cucumber knot melon 2-3 bars,
Retain pumpkin vine with screening disease resistance trait to be identified or reserve seed for planting；
(3) pumpkin rootstock disease resistance is screened：Anti-blight, mildew-resistance, anti-CGMMV screening are carried out to grafting, is resisted
Disease and the glossy property material selfing of cucumber simultaneously retain F1 generation Pumpkin Seed；The screening of pumpkin rootstock anti-blight is opened when grafting is colonized
Begin, mildew-resistance and CGMMV screening grafting be colonized after start, or remove cucumber it is climing after start；
(4) screening of stable character：The sowing of F1 generation Pumpkin Seed is obtained into pumpkin seedling, repeat step (1)-(3) are filtered out
F2 is for Pumpkin Seed；F2 is obtained into pumpkin seedling for Pumpkin Seed sowing, repeat step (1)-(3) filter out F3 for Pumpkin Seed；
F3 is obtained into pumpkin seedling for Pumpkin Seed sowing, repeat step (1)-(3) filter out F4 for Pumpkin Seed；By F4 for Pumpkin Seed
Sowing obtains pumpkin seedling, and repeat step (1)-(3) filter out F5 for Pumpkin Seed, as stablize genetic stocks.
Further, in step (1) before grafting, by stock, scion, grafting pin, grafting knife and hand all with it is general make every effort to overcome it is molten
Liquid is sterilized, and prevents grafting wound infection.
Further, insertion method grafting is used in step (1), easy to operate, survival rate is high, and interface position is high, diseases prevention
Effect is good.
Further, sheltered from heat or light moisturizing 3-4 days with mulch film after grafting is complete in step (1), mulch film is raised afterwards, is typically just raised
Mulch film, young plant is more fragile, and to prevent from dodging seedling, mulch film should not be raised for a long time, premised on grafting is not wilted, rationally covering
Grafting substantially completely heals after mulch film, grafting 5-7 days, can remove mulch film, if illumination is not strong, shade net can be removed after 10 days,
Premised on grafting is not wilted.
Further, in step (1) during grafting healing, pumpkin is faster than the speed of growth of scion, raises after mulch film
Remove 1-2 piece pumpkin true leaves, prevent shading from causing scion healing is slow to fight for nutrient with scion simultaneously.
Further, the screening of anti-blight uses leaching root inocalation method in step (3)：It is 10 to prepare concentration6-107Individual spore
Son/ml Fusarium oxysporum spore suspension, the grafting of 2 leaf periods is taken out from Nutrition Soil, and root system table is washed out in washing
The Nutrition Soil in face, the root system for cutting grafting 1/5-1/3 causes to infect wound, by remaining root system all not in spore suspension
In, taken out after 8-12 minutes, in being then colonized in experimental field；Every part of material must be inoculated with least 10 plants graftings as repetition；It is fixed
The condition (21-30 DEG C of temperature) that pathogenic bacteria fall ill that is beneficial to, the incidence of object observing material, 18-25 after inoculation are created after plant
My god, plant incidence is stable, you can evaluate the disease resistance of kind.
Further, the preparation method of the Fusarium oxysporum spore suspension is：Take droop morbidity serious (4 grades)
Pumpkin plant, from the withered stalk of site of pathological change collection, aseptically, be put into PDA (potato dextrose agar cultures
Base) cultivated 2 days in 28 DEG C of incubator on flat board after observe bacterium colony situation, picking mycelia is purified and cultivated, culture 8-
10 days, mycelia to be purified covered with flat board and then beats bacteria cake in PDB (potato fluid nutrient medium), 25 DEG C, 180rpm, in perseverance
Bacterium is shaken in warm shaking table 24-36 hours, microscopy conidium, and determine spore concentration with blood counting chamber.Then aqua sterilisa system is used
Into the suspension of required concentration.
Further, the screening of mildew-resistance is inoculated with using spray inoculation method in step (3), is by concentration with sprayer
105-106Individual spore/ml Powdery Mildew spore suspension uniformly sprays the tow sides in pumpkin old leaf, sprays to bacterium solution
It is covered with blade but not lower drop, 20~25 DEG C of keeping temperature after inoculation, humidity is 70-80%, dark culturing 20-30 hours, then
Normal management in 22-28 DEG C of daytime temperature, the greenhouse of 15-20 DEG C of night temperature is transferred to, it is substantially steady that inoculation starts the state of an illness in observation, 20 days for 7 days
It is fixed.Genus erysiphe is not suitable for the morbidity of its Spores in high temperature low humidity type in environment too high or too low for temperature, therefore in resistance
Temperature and humidity all needs strict control during screening.
Further, the preparation method of the Powdery Mildew spore suspension is：Directly Fresh leaves are gathered in field
Upper powdered conidium, adds distilled water, centrifuges 3~5 minutes, microscopy is simultaneously counted with blood counting chamber, is prepared with sterilized water
It is 10 into concentration5-106Individual spore/ml suspension.It is good to inoculation no more than 2 hours from suspension preparation.
Further, the screening of anti-cucumber green mottle mosaic virus (CGMMV) is inoculated with using frictional inoculation method, growth selection
The cushaw leaf of health, a small amount of diamond dust is equably spread on blade, virus inoculation liquid is dipped with gauze, on blade face in one direction
Gently rub 3-5 times；Start to observe the symptoms after 7 days, investigated 1 time every 7 days, disease symptom can be stablized within one month and be identified
As a result.
Further, the preparation method of the virus inoculation liquid is：CGMMV is expanded numerous on cushaw leaf, takes morbidity bright
The fresh blade of aobvious (more than 7 grades), according to mass volume ratio 1:10 add phosphate buffer, and are fully ground in mortar, yarn
Cloth is filtered, now with the current.
Further, material strain screen the 5th generation character it is basicly stable after, start to be identified with potted plant checking test
As a result, carried out under conditions of ensureing not carrying any pathogenic bacteria comprising flowerpot, the potted plant experiment articles for use with including soil, grafting
The inoculation of pathogenic bacteria, so as to verify the accuracy of screening.
The present invention solves the main contents of breeding objective seed selection and premunition screening, bag in a grafting cultivation generation
Include：Anti- CGMMV, powdery mildew, the droop evaluation and screening of pumpkin rootstock；The grafting affinity of stock pumpkin, grain plumpness etc.
The evaluation and screening of index；The evaluation and screening of the glossy character of sugar-preserved gourd after grafting green cucumber.Grafting Experiment is all carried out per generation self-mating system,
A part of affinity can be all eliminated in every generation or symbiosis power is poor, the material of disease resistance difference, selects excellent during material is excellent, so that thing half
Work(times.Compared to the method for existing screening pumpkin rootstock material, method of the invention possesses following beneficial effect：
(1) shorten the seed selection time limit, improve screening accuracy.Conventional screening methods are that anvil pumpkin material disease resistance is screened
And obtain after seed, then Grafting Experiment is carried out, identify the anvil performance of this material, such as grafting affinity and growing way, the present invention
Method is screened or added with regard to carrying out Grafting Experiment for during, using graft technology, directly with anvil objective trait in stock material
For starting point, the material grafting in Fn generations will be in, while keeping the pumpkin growing point of itself here, can so be seen in whole growth period
Glossy sex expression after grafting green cucumber is examined, a series of Evaluating and screening of objective traits such as seedling disease resistance can be carried out again, according to material
The variable rate technology combining target character of material, the material that advantage is protruded keeps pumpkin vine selfing here and carries out next step seed selection work, so that
Shorten the seed selection time of half.
(2) each generation planting number, is selected in quantity, selection approach specification and evaluation method science, per generation self-mating system
Grafting Experiment is all carried out, the material of a part of affinity or symbiosis power difference can be all eliminated in every generation, selects excellent during material is excellent.
(3) material filtered out is more accurate, effectively.In anti-blight screening process, with the cause of disease of medium culture
Microorganism is made spore suspension and is inoculated into the mode that the method for stock root replaces spreading sick soil in root after tradition directly field planting.
By manually regulating and controlling to cause of disease bacteria concentration, the disease cycle of material can be shortened, screening efficiency is improved, to superseded material and
When pull out, reduce management cost；It also ensure that the pathogen quantity of every plant of grafting inoculation is identical, control variable, it is ensured that its
The accuracy of the selection result.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that specific embodiment described herein is not used to limit only to explain the present invention
The fixed present invention.
Used material is as follows in the embodiment of the present invention：
PDA (potato dextrose agar)：200g potatoes, 20g glucose, 10g agar, 1L water, 121 DEG C of high pressures
Sterilize 20min.
PDB (potato fluid nutrient medium)：200g potatoes, 20g glucose, 1L water, 121 DEG C of autoclaving 20min.
Droop severity Scaling standard：0 grade：Basal part of stem is without brown；1 grade：Basal part of stem brown stain；2 grades：The brown stain of basal part of stem 1/2；
3 grades：The brown stain of basal part of stem more than 2/3；4 grades：Basal part of stem is to all vascular bundle brown stains in top, root necrosis.
Powdery mildew severity Scaling standard：0 grade：Without illness；1 grade：The positive lesion area of leaf is less than 30%, and leaf back is disease-free
Spot；3 grades：The positive lesion area of leaf is more than 30%, and the lesion area of leaf back is less than 10%；5 grades：The positive lesion area of leaf
More than 30%, the lesion area of leaf back, which is more than on 10%, petiole, there is a small amount of scab；7 grades：The mould layer of leaf front covering, blade back
The lesion area in face, which is more than to have on 10%, petiole, there is a small amount of scab on more scab, stem；9 grades：The mould layer of leaf front covering,
The lesion area of leaf back, which is more than to have on 10%, petiole, covers mould layer on more scab, stem.
Cucumber green mottle mosaic virus (CGMMV) severity Scaling standard：0 grade：Without any symptom；1 grade：The bright arteries and veins of lobus cardiacus, gently
Micro- floral leaf；3 grades：Lobus cardiacus and a small number of blade floral leaves；5 grades：Lobus cardiacus and most blade floral leaves, a small number of leaf-shrinkages, deformity, or it is bright
Arteries and veins, petiole have brown necrotic spot, and plant is slightly downgraded；7 grades：Weight floral leaf, most leaf-shrinkages, attenuate has in deformity or stem
Brown long streak, plant is downgraded；9 grades：Weight floral leaf, sick leaf severe deformities, plant seriously downgrades, or into Cong Zhizhuan, or even it is withered.
Incidence of disease %=(morbidity strain number/investigation total strain number) * 100%.
Glossy type disease-resistant anvil pumpkin material is screened according to the inventive method, is comprised the following steps that：
The first step, grafting.
Different types of Pumpkin Germplasm material is collected, Pumpkin Seed is sowed, every kind of germplasm materials plant 20 plants, treat pumpkin
Seedling length to two leaves wholeheartedly when Grafted Cucumber Seedling scion, before grafting by the true leaf of stock pumpkin pinch fall, keep growing point here.Stock, scion,
Grafting pin, grafting knife and hand are all sterilized before grafting with general solution of making every effort to overcome, and prevent grafting wound infection.Transferred using insertion method
Connect, easy to operate, survival rate is high, and interface position is high, and protection effect is good.
Sheltered from heat or light moisturizing 3 days with mulch film after grafting is complete, raise mulch film afterwards, grafting substantially completely heals after grafting 6 days, removes
Remove mulch film.Raise and suitably remove 1-2 piece pumpkin true leaves after mulch film.
Second step, the screening of grafting anti-blight.
The preparation method of Fusarium oxysporum spore suspension：The pumpkin plant for taking droop morbidity serious, from site of pathological change
The withered stalk of collection, aseptically, is put on PDA plate after being cultivated 2 days in 28 DEG C of incubator bacterium colony situation of observing,
Picking mycelia is purified and cultivated, and is cultivated 9 days, and mycelia to be purified covers with flat board and then beats bacteria cake in PDB, 25 DEG C,
180rpm, shakes bacterium 30 hours in constant-temperature table, microscopy conidium, and determines spore concentration with blood counting chamber.Then use
It is 10 that concentration, which is made, in aqua sterilisa7Individual spore/ml suspension.
Using leaching root inocalation method screening：The grafting of 2 leaf periods is taken out from Nutrition Soil, root system surface is washed out in washing
Nutrition Soil, the root system for cutting grafting 1/4 causes to infect wound, by remaining root system all not in spore suspension, 10
Taken out after minute, in being then colonized in experimental field；Every part of material is inoculated with 10 plants of graftings as repetition；Created after field planting and be beneficial to cause
The condition (25 DEG C of temperature) of germ morbidity, the incidence of object observing material, during which by disease plant (2 grades and more than) in time
Pull out, 20 days after inoculation, plant incidence is stable, you can evaluate the disease resistance of kind.Remaining anti-blight grafting
Continue on for the screening of other characters.
3rd step, the screening of grafting mildew-resistance.
The preparation of Powdery Mildew spore suspension：The powdered conidium directly on field collection Fresh leaves, adds
Distilled water, is centrifuged 3~5 minutes, microscopy is simultaneously counted with blood counting chamber, and it is 10 that concentration is configured to distilled water6Individual spore/ml's
Suspension.It is good to inoculation no more than 2 hours from suspension preparation.
Screened using brush leaf inocalation method：Concentration is 10 by second step residue grafting sprayer6Individual spore/ml white powder
Germ spore suspension uniformly sprays the tow sides in pumpkin old leaf, sprays to bacterium solution and is covered with blade but not lower drop, inoculation
22 DEG C of keeping temperature afterwards, humidity is 75%, dark culturing 24 hours, is then transferred to 25 DEG C of daytime temperature, the greenhouse that 16 DEG C or so of night temperature
Interior normal management, inoculation starts observation for 7 days, during which pulls out disease plant (3 grades and more than) in time, the state of an illness is substantially steady in 20 days
It is fixed, make disease resistance evaluation.
4th step, the glossy property screening of cucumber.
Cucumber sugar-preserved gourd grows up to when being colonized 30 days or so, is now in mildew-resistance character screening, two kinds of character screenings
Carry out simultaneously.Using cucumber seedling (kind is the powder that do not dewax) for blank control, screening is compared between grafting, is selected de-
The wax powder most best material of obvious, brightness.In time by the climing removal of cucumber after cucumber knot melon 2-3 bars can identify glossy property,
Retain pumpkin vine with the anti-CGMMV characters of screening to be identified and reserve seed for planting.Period pulls out the plant for the powder difference that dewaxes in time, remaining anti-
Droop, mildew-resistance, glossy property grafting continue on for the screening of other characters.
5th step, the anti-CGMMV screenings of pumpkin rootstock.
The CGMMV bought from commercial channel viruses are expanded numerous on cushaw leaf, the obvious fresh blade of morbidity are taken, according to matter
Measure volume ratio 1:10 add phosphate buffer, and are fully ground in mortar, and filtered through gauze is now with the current.
Screened using frictional inoculation method：The cushaw leaf of growth selection health, equably spreads a small amount of diamond dust on blade, uses
Gauze dips virus inoculation liquid, is gently rubbed 4 times in one direction on blade face；Start to observe the symptoms after 7 days, during which plant morbidity
Strain (3 grades and more than) is pulled out in time, every investigation in 7 days 1 time, can be stablized disease symptom within one month and be obtained qualification result.It is remaining
Anti- CGMMV, anti-blight, mildew-resistance, glossy property grafting, individual plant selfing obtains F1 generation Pumpkin Seed.
6th step, the screening of stable character.
The sowing of F1 generation Pumpkin Seed is obtained into pumpkin seedling, the first to five step is repeated and obtains F2 for Pumpkin Seed；By F2 generation south
The sowing of melon seed obtains pumpkin seedling, and the step of repeat step the first to five obtains F3 for Pumpkin Seed；F3 is sowed for Pumpkin Seed
To pumpkin seedling, the step of repeat step the first to five obtains F4 for Pumpkin Seed；F4 is obtained into pumpkin seedling for Pumpkin Seed sowing, repeated
Step the first to five steps obtain F5 for Pumpkin Seed, to stablize genetic stocks.
7th step, screens Accuracy Verification.
Material strain screen the 5th generation character it is basicly stable after, start, with potted plant checking test ground qualification result, protecting
Card carries out pathogenic bacteria under conditions of not carrying any pathogenic bacteria comprising flowerpot, the potted plant experiment articles for use with including soil, grafting
Inoculation, so as to verify the accuracy of screening.
The pumpkin rootstock character that the present invention of table 1 is screened
The present invention can select the disease-resistant anvil pumpkin of glossy type with five growth cycles, and breeding time is compared to conventional method
Shorten half.
It should be appreciated that for those of ordinary skills, can according to the above description be improved or converted,
And all these modifications and variations should all belong to the protection domain of appended claims of the present invention.
Claims (10)
1. a kind of screening technique of glossy type disease-resistant anvil pumpkin material, it is characterised in that step is as follows：
(1) grafting：Different types of Pumpkin Germplasm material is collected, and Pumpkin Seed is sowed, each kind sows 15-25 plants,
Whne pumpkin seedling length to two leaves wholeheartedly when Grafted Cucumber Seedling scion, before grafting by the true leaf of stock pumpkin pinch fall, keep growing point here；Grafting
Seedling is colonized 10-15 plants, and reservation two is climing, and a cucumber is climing, a pumpkin vine；
(2) the glossy property screening of cucumber：Cucumber seedling using the powder that do not dewax is compared sieve as blank control between grafting
Choosing, selects the cucumber dewaxing powder most best material of obvious, brightness, in time only protects the climing removal of cucumber after cucumber knot melon 2-3 bars
Pumpkin vine is stayed with screening disease resistance trait to be identified or is reserved seed for planting；
(3) pumpkin rootstock disease resistance is screened：To grafting carry out anti-blight, mildew-resistance, anti-CGMMV screening, it is disease-resistant and
The glossy property material selfing of cucumber simultaneously retains F1 generation Pumpkin Seed；Pumpkin rootstock anti-blight is screened when grafting is colonized,
Mildew-resistance and CGMMV screening grafting be colonized after start, or remove cucumber it is climing after start；
(4) screening of stable character：The sowing of F1 generation Pumpkin Seed is obtained into pumpkin seedling, repeat step (1)-(3) filter out F2 generations
Pumpkin Seed；F2 is obtained into pumpkin seedling for Pumpkin Seed sowing, repeat step (1)-(3) filter out F3 for Pumpkin Seed；By F3
Pumpkin seedling is obtained for Pumpkin Seed sowing, repeat step (1)-(3) filter out F4 for Pumpkin Seed；F4 is sowed for Pumpkin Seed
Pumpkin seedling is obtained, repeat step (1)-(3) filter out F5 for Pumpkin Seed, as stablize genetic stocks.
2. the screening technique of glossy type disease-resistant anvil pumpkin material according to claim 1, it is characterised in that step (1)
Middle use insertion method grafting.
3. the screening technique of glossy type disease-resistant anvil pumpkin material according to claim 1, it is characterised in that step (1)
Sheltered from heat or light moisturizing 3-4 days with mulch film after middle grafting is complete, mulch film is raised afterwards, grafting removes mulch film after 5-7 days.
4. the screening technique of glossy type disease-resistant anvil pumpkin material according to claim 3, it is characterised in that step (1)
In raise and remove 1-2 piece pumpkin true leaves after mulch film.
5. the screening technique of glossy type disease-resistant anvil pumpkin material according to claim 1, it is characterised in that step (3)
The screening of middle anti-blight is using leaching root inocalation method：It is 10 to prepare concentration6-107Individual spore/ml Fusarium oxysporum spore suspension
Liquid, the grafting of 2 leaf periods is taken out from Nutrition Soil, and the Nutrition Soil on root system surface is washed out in washing, cuts grafting 1/5-
1/3 root system causes to infect wound, and remaining root system is whole not in spore suspension, is taken out after 8-12 minutes, Ran Houding
Plant in experimental field；Every part of material must be inoculated with least 10 plants graftings as repetition；21-30 DEG C of keeping temperature after field planting, observation
18-25 days after the incidence of target material, inoculation, plant incidence is stable, you can evaluate the disease resistance of kind.
6. the screening technique of glossy type disease-resistant anvil pumpkin material according to claim 5, it is characterised in that the sharp spore
The preparation method of sickle-like bacteria spore suspension is：The pumpkin plant for taking droop morbidity serious, from the withered stem of site of pathological change collection
Stalk, aseptically, is put on PDA plate after being cultivated 2 days in 28 DEG C of incubator bacterium colony situation of observing, picking mycelia enters
Row purifying and culture, are cultivated 8-10 days, and mycelia to be purified covers with flat board and then beats bacteria cake in PDB, 25 DEG C, 180rpm,
Bacterium is shaken in constant-temperature table 24-36 hours, microscopy is counted, the suspension of concentration needed for being then made of aqua sterilisa.
7. the screening technique of glossy type disease-resistant anvil pumpkin material according to claim 1, it is characterised in that step (3)
The screening of middle mildew-resistance using spray inoculation method be inoculated with, with sprayer by concentration be 105-106Individual spore/ml Powdery Mildew
Spore suspension uniformly sprays the tow sides in pumpkin old leaf, sprays to bacterium solution and is covered with blade but not lower drop, is protected after inoculation
20~25 DEG C of temperature is held, humidity is 70-80%, and then dark culturing 20-30 hours is transferred to 22-28 DEG C of daytime temperature, night temperature 15-20
DEG C greenhouse in normal management, inoculation starts observation for 7 days, and the state of an illness is basicly stable in 20 days.
8. the screening technique of glossy type disease-resistant anvil pumpkin material according to claim 7, it is characterised in that the white powder
The preparation method of germ spore suspension is：The powdered conidium directly on field collection Fresh leaves, adds distilled water,
Centrifugation 3~5 minutes, microscopy is counted, and it is 10 that concentration is configured to sterilized water5-106Individual spore/ml suspension.
9. the screening technique of glossy type disease-resistant anvil pumpkin material according to claim 1, it is characterised in that anti-CGMMV
Screening using frictional inoculation method be inoculated with, growth selection health cushaw leaf, a small amount of diamond dust is equably spread on blade, yarn is used
Cloth dips virus inoculation liquid, is gently rubbed 3-5 times in one direction on blade face；Start to observe the symptoms after 7 days, 1 was investigated every 7 days
It is secondary, disease symptom can be stablized within one month and obtain qualification result.
10. the screening technique of glossy type disease-resistant anvil pumpkin material according to claim 9, it is characterised in that the disease
The preparation method of malicious inoculation liquid is：CGMMV is expanded numerous on cushaw leaf, the obvious fresh blade of morbidity is taken, according to quality volume
Than 1:10 add phosphate buffer, and are fully ground in mortar, and filtered through gauze is now with the current.
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| CN108450169A (en) * | 2018-04-23 | 2018-08-28 | 潘文鋆 | A method of improving camellia graft survival rate |
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| CN114107535A (en) * | 2020-08-31 | 2022-03-01 | 北京市农林科学院 | Molecular marker for identifying character of removing wax powder of grafted cucumber fruit from Chinese pumpkin rootstock and application of molecular marker |
| CN114107535B (en) * | 2020-08-31 | 2023-08-18 | 北京市农林科学院 | Molecular marker for identifying properties of removing wax powder of grafted cucumber fruits of pumpkin stock and application of molecular marker |
| CN114885693A (en) * | 2022-06-07 | 2022-08-12 | 青岛金妈妈农业科技有限公司 | Method for obtaining excellent seeds of pumpkin rootstock for powdery mildew-resistant cucumbers |
| CN114885694A (en) * | 2022-06-07 | 2022-08-12 | 青岛金妈妈农业科技有限公司 | Method for obtaining root rot resistant cucumber stock material seeds |
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