CN107593626A - Utilize the method for live body plant hand-feeding propagation corn leaf aphids - Google Patents
Utilize the method for live body plant hand-feeding propagation corn leaf aphids Download PDFInfo
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Abstract
The invention discloses a kind of method using live body plant hand-feeding propagation corn leaf aphids, culture matrix of the formula soil that selection is mixed to get by loam and Nutrition Soil as live body plant, susceptible aphid corn variety seeds are first sowed in culture matrix, then plant is cultivated in growth cabinet, treat that plant was grown to the heart stage of two leaf one and be inoculated with corn leaf aphids worm sources, continue afterwards in growth cabinet on the premise of cultivating plant growth, carry out the captive breeding of corn leaf aphids.The corn leaf aphids captive breeding method of acquisition, the reproductive process of maize aphidses under access expansion state, so as to obtain having with the corn leaf aphids naturally captured the physiology such as identical biological nature, hereditary capacity, biochemical trait a large amount of corn leaf aphids samples, make that the offer of worm sources sample is more stable, more universal significance, it is detailed for aphid and accurately bion research, aphid-resistant gene be accurately positioned clone's research, the seed selection of molecular mark and excellent anti-aphid corn variety has great importance.
Description
Technical field
The present invention relates to the technical field of artificial culture of maize aphidses, more particularly to one kind are artificial using live body plant
The method of captive breeding corn leaf aphids.
Background technology
Aphid is a kind of extremely common crop pests, and its host range is extensive, can be to almost all of Main Cultivation
Crop damages.Aphid belongs to Homoptera (Homoptera) Aphidiadae (Aphididae), is commonly called as aphid or close worm.In the world about
There are nearly more than 5000 kinds of aphids, wherein that main harm is caused to corn is corn leaf aphids (Rhopalosiphum maidis), secondly
It is kaoliang aphid (Melanaphis sacchari).Corn leaf aphids are distributed widely in China northeast, northwest, North China and Central China, its
Host plant species is various, not only including the gramineous crops such as corn, sorghum, rice, wheat, in addition to barnyard grass, lady's-grass, horse hair
The weeds such as grass.The adult and nymph of corn leaf aphids inhale plant phloem juice and directly caused harm crops by piercing, and can also propagate barley
The plant viruses such as yellow dwarf virus, maize dwarf mosaic, cause the prevalence of crop virosis, cause disease.The corn leaf aphids feeding time-division
The honeydew secreted is distributed in plant leaf blade surface, not only influences the photosynthesis of plant, moreover it is possible to cause sooty mould, influence plant strain growth
Development, causes plant withered when serious.Male flower and female fringe if aphid is caused harm at corn heading stage, can cause florescence to postpone, seed
Grain is not full, the directly harm such as quality and yield declines.In recent years, as agricultural production is to scale, intensive transformation, and it is beautiful
The rice change of planting system and the increasing of planting density, and the influence of the environmental factor such as summer weather high temperature, arid, short of rain,
The harm of aphid is more and more prominent, can cause corn underproduction 10-20%, the serious time underproduction is more than 30%.And the preventing and treating of aphid
But it is always the problem in agricultural production, because aphid has high breeding potential, Single Aphids can produce 5.9 ten thousand within 6 weeks
Individual offspring, and corn leaf aphids are divided into two kinds of ecotypes of wingless aphid and alatae, have migration, have a strong impact on the effect of pesticide control
Fruit, and can also destroy the ecosystem in addition to cost is high using pesticide control and cause the pollution of environment.In addition, research table
Occurrence degree, the Harm period of bright corn leaf aphids not only have direct relation with temperature, humidity, rainfall and natural enemy quantity, and also and crop
Layout and maize sown area have close relationship.Therefore the resistant variety for cultivating high-resistance corn aphid is most economical effective anti-
The approach of corn leaf aphids is controlled, and the excavation of anti-aphides gene is the premise of resistance breeding.In order to the accuracy of corn leaf aphids Resistance Identification,
The seed selection of the screening of resistance germplasm, the finely positioning of resistant gene and further resistant variety, aphid is carried out accurately biological
Type is bred, and could be that being accurately positioned for aphid-resistant gene clones research, molecular mark and aphid resistance corn variety seed selection
Create conditions, fundamentally reach prevention effect.
In the research to aphid, obtaining aphid sample can be by way of field collection or artificial feeding.Corn leaf aphids exist
In more than 10 generations, can occur every year for northern China, be survived the winter with aptery viviparous female on wheat seedling and grassy weed；In May by the end of April
First alatae migrates to spring maize, sorghum field to be endangered；In Summer Maize reproductive stage, spread to corn summer seeding, this
When worm amount be doubled and redoubled, into seriously endanger the stage；On October, the middle ten days, the gradual aging of corn, corn leaf aphids finally produce once have
Wing aphid, move into wheatland and endangered and survived the winter；Therefore field collect corn leaf aphids by studies of migration of aphids, season, weather etc. it is a variety of because
The influence of element, makes that identification for resistance to aphis efficiency is low and result is unreliable.And the artificial feeding of aphid, exist at present frequently with solid medium
Raised in fixed container, such as Chinese patent application CN201710099350.1, although the method for breeding can successfully obtain aphid
Breeding, but obtained aphid sample is because of the directional induction of artificial feeding mode, with the aphid sample of natural environment trapping biology,
Had differences in terms of heredity, genetic characteristics, cause result of study to produce larger difference with natural control effect, being unfavorable for reality should
With.
The content of the invention
To solve the shortcomings of the prior art, the invention provides one kind to utilize live body plant hand-feeding propagation
The method of corn leaf aphids, the corn leaf aphids for breeding to obtain by this method have the corn leaf aphids identical biological nature and something lost with capturing naturally
Characteristic is passed, the preventing and treating of bion difference, aphid to research corn leaf aphids, the anti-aphid mechanism of research corn, heredity, authentication method have
Significance.
To achieve the above object, the method provided by the invention using live body plant hand-feeding propagation corn leaf aphids,
Culture matrix of the formula soil that selection is mixed to get by loam and Nutrition Soil as live body plant, first broadcasts in culture matrix
The susceptible aphid corn variety seeds of kind, plant is then cultivated in growth cabinet, treats that plant was grown to the heart stage of two leaf one
Corn leaf aphids worm sources are inoculated with, continues afterwards in growth cabinet on the premise of cultivating plant growth, carries out the feeding of corn leaf aphids
Support breeding.
The corn leaf aphids captive breeding method of the present invention, is by the artificial culture in susceptible aphid plant, growth course
The middle artificial mode for connecing worm, raising and this bion aphid of propagating corn aphid of progress, is obtained closer to corn under nature
The reproductive process of aphid, so as to which the corn leaf aphids for obtaining with capturing naturally have a large amount of corn leaf aphids of identical biological nature and hereditary capacity
Sample, effective sample is provided for the research of corn leaf aphids, being advantageous to aphid, accurately bion is studied, and accurately corn aphid resistance
Identification, the screening of resistance germplasm, the seed selection of the finely positioning of resistant gene and further resistant variety；And the method compared to
Natural Trapping ways, offer to aphid sample is more stable, more universal significance；, can compared to other artificial feeding manners
Reduce caused by human intervention factor on the ecotypic influence of aphid sample, so as to obtain more accurately result of study and more effective
Means of prevention.In the captive breeding method of the present invention, the culture of plant and the captive breeding of corn leaf aphids are to close closely
Join and interactional, plant should be enable to enter reproductive stage as early as possible, excellent host's ring is provided for corn leaf aphids
Border, it can not be restricted the breeding of corn leaf aphids because of the growth of promotion plant again, therefore selection, the corn of culture matrix
The culture of plant and the determination of corn leaf aphids Inoculating date, the factor such as captive breeding condition of corn leaf aphids be interrelated, mutual association
Adjust, to obtain the corn leaf aphids sample of mass efficient.
As the restriction to above-mentioned technical proposal, the method using live body plant hand-feeding propagation corn leaf aphids
Comprise the following steps：
A, the culture matrix of live body plant is prepared：By loam and Nutrition Soil by volume (1~3):1 is well mixed,
Obtained formula soil is culture matrix；
B, plant is cultivated：Susceptible aphid corn variety seeds are sowed in culture matrix, then in growth cabinet
In 7~12 hours 25~31 DEG C of daytime temperature, 18~20 DEG C of night temperature, humidity 55~70%, intensity of illumination 9600Lx, light application time conditions
The growth of lower culture plant；
C, captive breeding aphid：Treat that plant was grown to the heart stage of two leaf one and be inoculated with corn leaf aphids worm sources, afterwards in artificial climate
In 7~12 hours 25~31 DEG C of daytime temperature, 18~20 DEG C of night temperature, humidity 55~70%, intensity of illumination 9600Lx, illumination bars in case
Part, on the premise of continuing to cultivate plant growth, carry out the captive breeding of corn leaf aphids.
Further limit the culture matrix of live body plant in captive breeding method, and suitable plant and aphid
Under the temperature of syntrophism, humidity, illumination condition, wherein the condition of control corn culture and aphid captive breeding is consistent,
Corn and aphid is set to access good growth, to reach to the hand-feeding propagations of corn leaf aphids closer to aphid under natural environment
The breeding of worm, obtain a large amount of, reliable corn leaf aphids sample.
As the restriction to above-mentioned technical proposal, the volume ratio of the formula soil medium loam and Nutrition Soil is 3:1.
As the restriction to above-mentioned technical proposal, the susceptible aphid corn variety include the entitled S221, PH6WC of kind,
JS06330 corn material.
As the restriction to above-mentioned technical proposal, step b, the condition of culture described in c in growth cabinet be 28 DEG C of daytime temperature,
20 DEG C of night temperature, humidity 70%, illumination 10 hours.
As the restriction to above-mentioned technical proposal, the inoculum concentration of the corn leaf aphids worm sources is inoculated with least for every plant of plant
3 corn leaf aphids.
Further refine formula soil proportion optimizing, susceptible aphid corn variety, optimal plant in captive breeding method
Condition of culture and corn leaf aphids captive breeding condition, the condition elements such as worm amount are connect, enable the method to the implementation smoothly and optimized.
In summary, using technical scheme, the corn leaf aphids captive breeding method of acquisition, is by susceptible aphid
The artificial mode for connecing worm, raising and propagating corn aphid is carried out in the artificial culture of plant, growth course, is obtained closer
The growth and breeding process of corn leaf aphids under nature, so as to which the corn leaf aphids for obtaining with capturing naturally have identical biological nature, something lost
A large amount of corn leaf aphids samples of characteristic are passed, are that corn leaf aphids accurately study by bion, and the accurately identification of corn aphid resistance, resistance kind
The seed selection of the screening of matter, the finely positioning of resistant gene and further resistant variety provides effective sample, and the aphid sample provided
This is more stable, more universal significance, is not limited by seasonal variations and other natural environments, and greatly reduce because human intervention because
Element makes the influence of paired samples performance, so as to obtain more accurately result of study and more effective means of prevention.
Brief description of the drawings
The present invention is further detailed explanation with reference to the accompanying drawings and detailed description.
Fig. 1 is that the different culture media matter cropping pattern of the embodiment of the present invention compares photo；
Fig. 2 is that corn different times aphid breeds photo in the embodiment of the present invention；
Plant highly compares photo under the different photoperiods that Fig. 3 is the embodiment of the present invention；
Fig. 4 is corn growth number of days and Aphed population changing trend diagram in the embodiment of the present invention；
Fig. 5 is to cultivate live body plant captive breeding corn leaf aphids photo using growth cabinet；
Fig. 6 is the corn leaf aphids different development stage microscopic photo of captive breeding of the embodiment of the present invention；
Fig. 7 is the DNA sequence dna result figure of COI gene sequencing on the corn leaf aphids mitochondria of captive breeding of the embodiment of the present invention.
Embodiment
Below in conjunction with embodiment, technical scheme is clearly and completely described, it is clear that described
Embodiment is only the part of the embodiment of the present invention, rather than whole embodiments.Based on the embodiment in the present invention, this area
The every other embodiment that those of ordinary skill is obtained under the premise of creative work is not made, belongs to protection of the present invention
Scope.
Embodiment one
The present embodiment relates to the use of the method for live body plant hand-feeding propagation corn leaf aphids, carries out as follows：
A, the preparation of culture matrix：By loam and Nutrition Soil by volume (1~3):1 is well mixed, obtained formula soil
As culture matrix；
Loam, it is derived from Di Shang experiment stations of Food and Oil Crops Inst., Hebei Agriculture and Forestry Academy, the farmland for maize planting
Light loam, the content of organic matter 13.0~14.6g/kg, complete 1.14~1.20g/kg of nitrogen, complete 1.5~1.6g/kg of phosphorus, alkali-hydrolyzable nitrogen
76.5~81.4mg/kg, available phosphorus 23.12~26.22mg/kg, effective 103.5~106.0mg/kg of potassium.Soil sample is through weeding out stone
It is standby after the larger particles such as block；
Nutrition Soil, it is from the general Nutrition Soil of plant (Changbai Mountain board) of market purchase, is that scientifically is formulated
, containing abundant organic fertilizer, sustained slow-release fertilizer and multiple beneficial microorganism, pH value is 5.5~6.5, main component：Peat,
Vermiculite, perlite, wetting agent, humic acid, sustained slow-release fertilizer and multiple beneficial microorganism；
B, plant is cultivated：To at from 1~2cm of mouth, it will be then placed in easily in culture matrix loading culturing pot or flowerpot
Feel aphid corn variety (such as corn material S221, PH6WC, JS06330) seed, then add culture matrix to expiring, in people after watering
In 25~31 DEG C of daytime temperature, intensity of illumination 9600Lx, light application time 7~12 hours in work climate box, 18~20 DEG C of night temperature, illumination are strong
0Lx is spent, the growth of plant is cultivated under the conditions of humidity 55~70%；
C, captive breeding aphid：Treat that plant was grown to the heart stage of two leaf one and be inoculated with corn leaf aphids worm sources, every plant of inoculation at least 3
Head, afterwards in growth cabinet in 25~31 DEG C of daytime temperature, intensity of illumination 9600Lx, light application time 7~12 hours, night temperature 18~
20 DEG C, intensity of illumination 0Lx, the condition of humidity 55~70%, on the premise of continuing to cultivate plant growth, carry out corn leaf aphids
Captive breeding, obtain the corn leaf aphids of hand-feeding propagation, can be used as all kinds of research samples.
Embodiment two
The present embodiment is related to the selection and its influence of each condition element in captive breeding corn leaf aphids method.
Embodiment 2.1
The selection of culture matrix.
Test method：By tri- parts of corn materials of S221, PH6WC, JS06330, nutrient solution, Nutrition Soil, earth are planted in respectively
In native three kinds of matrix, every part of corn material plants 3 basins, altogether 9 test specimens, and each sample plants two plants of plants,
To be cultivated in growth cabinet, condition of culture is humidity 70%, 25 DEG C of day temperature, intensity of illumination 9600Lx, 18 DEG C of nocturnal temperature,
Intensity of illumination 0Lx, periodicity of illumination day night are 14 hours/10 hours.
Wherein, Solution culture method mode is that corn seed is placed in distilled water to soak 24h, and paving is transferred to after expansion water suction
Have in the filter paper culture dish of water saturation and cultivate, until germination (24-48h, centre keep the skin wet), then the seed by germination
Fixed, be placed in small flower with foam, poured into appropriate nutrient solution and cultivated.The preparation of nutrient solution：Using for plant group
Knit the MS culture medium (production code members without agar and sucrose of culture：HB8469-5), it is molten to weigh 4.74 grams of MS culture mediums heating
For solution in 1000 milliliters of distilled water, 115 DEG C of autoclavings 30 minutes are standby, are transferred to pH value with 3M hydrochloric acid as 7.0 during use.
Result of the test：As shown in figure 1, three parts of corn materials grow preferably in loam and Nutrition Soil, and it is grown in battalion
Corn seedling in nutrient solution, because nutrient solution can not meet nutrition needed for growth of maize development, bottom root growth is good, on
Portion's yellow leaf is wilted.Corn seedling is carried out into investigation according to the growing way of blade to collect, is shown in Table 1.
The plant seedling growth situation of table 1
This result of the test shows：(1) the easily raw bacterium of nutrient solution, it can not meet that normal plants were developed needed for each period
Nutrition, and financial cost is higher；(2) financial cost of loam is low, and convenient material drawing, plant strain growth is normal, but poor air permeability；(3)
Nutrition Soil financial cost is moderate, good permeability, plant energy normal growth, but poor water retention property.Therefore by further using loam
Volume ratio with Nutrition Soil is respectively 1:1、2:1、3:1 formula soil is as corn growth matrix, according to the growing way of corn seedling
It was found that 3:1 formula soil optimum corn growth, the volume ratio for determining loam and Nutrition Soil is 3:1 formula soil is given birth to as corn
Long matrix, the culture of the plant for raising aphid.
Embodiment 2.2
Plant condition of culture and the determination in the corn growth period bred suitable for corn leaf aphids.
Test method：9 basins sow susceptible aphid corn variety PH6WC test specimen, are divided into 3 groups, every group of 3 basins, per 2 plants of basin
Corn.In growth cabinet, humidity 70%, nocturnal temperature is 18 DEG C, and day temperature is respectively 25 DEG C, 28 DEG C and 31 DEG C,
Periodicity of illumination is corresponding be respectively day night be 8 hours/16 hours, day night is 10 hours/14 hours and day night is 14 hours/10
Under 3 environment of hour, corn growth is first cultivated, then connects corn leaf aphids in the different times of corn growth, carries out the feeding of aphid
Breeding is supported, every plant connects aphid 3, next day, worm is connect again to the plant of no aphid, institutes an inquiry aphid within the 3rd day after worm is connect
Decortication and breeding situation, death toll, aphid number, every investigation in 3 days once, the strain of 40 days investigation plants after plant takes out hero
Height, for primarily determining that the corn growth period that aphid most preferably breeds grows bar with the plant for determining suitable corn leaf aphids breeding
Part.
Result of the test：The plant height investigation result of plant is shown in Table 2 (by taking wherein 6 plants of plants as an example)：
Plant height of the corn of table 2 under the different photoperiods
Corn is short-day plant, can be blossomed and beared fruit under the conditions of field nature short-day (8~10 hours).By upper table
As a result visible, the different photoperiods have an impact to growing for corn, and with the growth of light application time, corn growth prolongs
Late, plant significantly increases.
Aphid is bred shown in condition survey result as Fig. 2 and table 3：
The aphid of table 3 breeds condition survey
Connect worm in the different growth period of corn, as a result find, Seedling Stage corn leaf aphids nymph have breeding but it is poor；Big loudspeaker
Mouth phase aphid starts to breed vigorous.Corn leaf aphids are divided to lonely female adult aphid, two kinds of larvae aphid, and in the case of the nature of field, Maize at Seedling Stage is put down
Equal temperature is less than 23 DEG C, and corn leaf aphids are equal in the corn growth later stage (reproductive stage) mainly to there is the lonely female adult aphid of wing to be caused harm
Temperature is less than 28 DEG C, and suitable corn leaf aphids breeding, corn leaf aphids are mainly caused harm with the aptery female larvae aphid of orphan.The study find that artificial
In climate box, nocturnal temperature is 18 DEG C, and day temperature is at 25-31 DEG C, in the larvae aphid that Maize at Seedling Stage connects (before trumpet period), because
It is not suitable for nymph breeding, has degenerated into the lonely female adult aphid of wing, in the corn typhon mouth phase to be followed by larvae aphid, larvae aphid starts to breed, special
It it is not the phase of reeling off raw silk from cocoons, population number increases sharply.Therefore, photoperiod and temperature are adjusted, shortens corn nourishment growth period, it is relative to extend
Reproductive stage, suitable corn leaf aphids nymph Reproduction Conditions are created, be advantageous to improve hand-feeding propagation's corn leaf aphids group's mouth amount.
In addition, there is aphid growth under 3 kinds of environment, illustrate that 25~31 DEG C of temperature range is adapted to aphid growth；Illumination
Duration had aphid growth, wherein 10 hours periodicity of illuminations for the breeding of optimum aphid of illumination at 8~14 hours.
Embodiment 2.3
The determination of corn leaf aphids captive breeding condition, primarily determining that corn growth condition and corn different growing stage to jade
On the basis of rice aphid breeding influences, continue the corn growth most suitable photoperiod of suitable corn leaf aphids breeding.
Test method：Susceptible aphid corn variety PH6WC 9 basin test specimens will be sowed, be put in day temperature respectively as 28
DEG C, nocturnal temperature be 20 DEG C, humidity 70%, periodicity of illumination is respectively day night 7 hours/17 hours, day night 10 hours/14 is small
When and day night 12 hours/12 hours, under 3 environment, first cultivate corn growth, then in corn seedling length to the heart stage of 2 leaf 1, often
Strain seedling connects aphid 5, carries out the captive breeding of aphid.In plant strain growth different times, quantity and the plant strain of aphid are investigated
The high and number of blade, compared with the growing state for the PH6WC corn varieties that field grows naturally, for determining growth cabinet condition
The corn growth most suitable photoperiod of suitable aphid breeding down.
Result of the test：Shown in influence result as Fig. 3 and table 4 of the periodicity of illumination to corn growth stage：
Influence of the photoperiod of table 4 to corn growth stage
Compared with the plant that field grows naturally, the corn in growth cabinet is grown in by different periodicity of illuminations
Influenceing its breeding time has significant change, grows substantially in advance, reduction in the life period, plant becomes short, total leaf number and fringe position
Reduce.The reproductive growth of plant shifts to an earlier date, and more favorable condition is provided for the breeding of aphid.
The variation tendency of aphid group mouth quantity as shown in Figure 4, is cultivated live body corn using growth cabinet and planted under 3 environment
The corn leaf aphids experiment of strain captive breeding is as shown in Figure 5, the results showed that corn is 10 hours/14 hours conditions in periodicity of illumination day night
Under most beneficial for aphid breeding.
Corn leaf aphids hand-feeding propagation's method of the present invention, most suitable aphid Reproduction Conditions are:Using loam and Nutrition Soil
Mass ratio is 3:1 formula soil is as plant culture matrix, plant condition of culture and corn leaf aphids captive breeding condition
It is 28 DEG C of daytime temperature, 20 DEG C of night temperature, humidity 70%, intensity of illumination 9600Lx, illumination 10 hours, corn leaf aphids seed stage is that corn is planted
Strain is long to the heart stage of two leaf one.
Embodiment three
The present embodiment is related to the bion DNA level and Morphological Identification of hand-feeding propagation corn leaf aphids, as follows
Carry out：
(1) aphid, is collected respectively from different growth cabinets and corn field, puts and observes under an optical microscope, from shape
Identified in state feature.
(2), the aphid to above-mentioned collection is separately immersed in absolute ethyl alcohol 2 days (changing once fresh absolute ethyl alcohol daily),
It is placed in -20 DEG C of refrigerators and stores for future use.
(3) aphid DNA, is extracted using CTAB methods：
The aphid that the fresh aphid of single head or absolute ethyl alcohol preserve is placed on blotting paper, ethanol is blotted, is placed in 1.5mL's
In centrifuge tube.50 μ L CTAB extracts are added in centrifuge tube, the pipette tips (1000 μ L) liquid-transfering gun are sophisticated on alcolhol burner
The empty centrifugation bottom of the tube for being rapidly inserted into a 1.5mL afterwards is burnt after its cooling, and aphid of each pipe is chosen with it and left
On the tube wall of liquid level, aphid is then ground to complete crushing with this instrument on tube wall, adds 150 μ L CTAB bufferings
Liquid.Centrifuge tube is put into water-bath, 65 DEG C of warm bath 1h.Centrifuge tube is taken out, adds 1U RNase, is placed in 1h in 37 DEG C of incubators
After taking-up, 500 μ L chloroforms/isoamyl alcohol (volume ratio 24 is added:1), slowly shake and mix, 13000~15000r/min centrifugations
15min.Supernatant (about 150 μ L) is taken to be placed in new 1.5mL centrifuge tubes.The absolute ethyl alcohol of the ice of 2 times of volumes of addition, about 1/10
The sodium acetate (pH5.2) of volume, -20 DEG C are placed in after mixing, overnight (more than 8h).13000r/min centrifuges 15min, abandons supernatant,
Cleaned 2 times with 70% ethanol of -20 DEG C of preservations.After drying at room temperature add 1 × TE of 20mL (pH 8.0) dissolving, be placed in 4 DEG C it is stand-by
Or preserved in -20 DEG C of refrigerators.
(4), DNA concentration and purity detecting：
The quantitative determination of DNA concentration and purity is carried out from NanoDrop ND-1000 spectrophotometers.
(5), PCR amplifications and detection：
From LepF (5 '-ATTCAACCAATCATAAAGATATTGG-3 '), LepR (5 '-TAAACTTCTG
GATGTCCAAAAAATCA-3 ') special primer (Lep primers are the specific amplification primer of the COI genes on aphid mitochondria), make
Augmentation detection is carried out to the DNA extracted with Thermal Cycler PCR instruments.PCR reactants are：10×TransStart
The μ L of 3 2.4 0.6 0.6 μ L, Taq archaeal dna polymerases of μ L, LepR of μ L, LepF of μ L, dNTPs of Taq Buffer 0.4, sterilizing are ultrapure
The μ L of water 20, the μ L of DNA profiling 3.Response procedures are：95℃7min；94 DEG C of 1min, 50 DEG C of 1min, 72 DEG C of 1min, 15 circulations；94℃
1min, 52 DEG C of 1min, 72 DEG C of 1min, 20 circulations；72 DEG C of 10min, 4 DEG C of 30min.
(6), it is sequenced：
PCR primer is sent BeiJing ZhongKe to wish woods sequencing portion and is sequenced, and sequencing instrument used is ABI PRISM3730xl.
Result of the test：The aphid collected in growth cabinet observes result as shown in Figure 6 under an optical microscope.To artificial
The aphid of climate box breeding carries out morphologic observation under the microscope, the results showed that nymph and has wing female insect to meet corn leaf aphids
Morphological feature.
DNA level sequencing is carried out to the aphid collected from the growth cabinet of 3 varying environment conditions respectively, as a result sees figure
Shown in 7.As a result show, compared with the abiogenous corn leaf aphids of the corn field of collection, aphid and the field nature ring of artificial propagation
The aphid DNA sequence dna of border breeding is identical, and genetic constitution is consistent.
In summary, corn leaf aphids hand-feeding propagation's method of the invention, closer to aphid infection corn under nature
Growth and breeding process, so as to obtain having with the corn leaf aphids naturally captured the physiology such as identical biomorph characteristic, hereditary capacity,
A large amount of corn leaf aphids samples of biochemical trait, it is research and preventing and treating and the standard of the anti-corn leaf aphids identification of corn and resistant gene of corn leaf aphids
True positional cloning provides effective sample, and sample has more stable, more universal significance, not by seasonal variations and other natural rings
The limitation in border, and the influence caused by human intervention factor to aphid sample bion is greatly reduced, it is deeply and smart for aphid
Accurate biological study, the seed selection of excellent anti-aphid corn variety are extremely important.
Claims (6)
- A kind of 1. method using live body plant hand-feeding propagation corn leaf aphids, it is characterised in that this method is selected by earth Culture matrix of the formula soil that soil is mixed to get with Nutrition Soil as live body plant, first sows susceptible aphid in culture matrix Corn variety seeds, plant is then cultivated in growth cabinet, treat that plant was grown to the heart stage of two leaf one and be inoculated with corn Aphid source, continue afterwards in growth cabinet on the premise of cultivating plant growth, carry out the captive breeding of corn leaf aphids.
- 2. the method according to claim 1 using live body plant hand-feeding propagation corn leaf aphids, it is characterised in that This method comprises the following steps：A, the culture matrix of live body plant is prepared：By loam and Nutrition Soil by volume (1~3):1 is well mixed, obtains Formula soil be culture matrix；B, plant is cultivated：Susceptible aphid corn variety seeds are sowed in culture matrix, then in daytime in growth cabinet 25~31 DEG C of temperature, 18~20 DEG C of night temperature, humidity 55~70%, intensity of illumination 9600Lx, light application time are trained under the conditions of 7~12 hours Support the growth of plant；C, captive breeding aphid：Treat that plant was grown to the heart stage of two leaf one and be inoculated with corn leaf aphids worm sources, afterwards in growth cabinet In 7~12 hours 25~31 DEG C of daytime temperature, 18~20 DEG C of night temperature, humidity 55~70%, intensity of illumination 9600Lx, illumination conditions, Continue on the premise of cultivating plant growth, carry out the captive breeding of corn leaf aphids.
- 3. the method according to claim 2 using live body plant hand-feeding propagation corn leaf aphids, it is characterised in that： The volume ratio of the formula soil medium loam and Nutrition Soil is 3:1.
- 4. the method according to claim 2 using live body plant hand-feeding propagation corn leaf aphids, it is characterised in that： The susceptible aphid corn variety includes kind entitled S221, PH6WC, JS06330 corn material.
- 5. according to the method using live body plant hand-feeding propagation corn leaf aphids described in claim 2, its feature exists In：Step b, the condition of culture described in c in growth cabinet is 28 DEG C of daytime temperature, 20 DEG C of night temperature, humidity 70%, illumination 10 hours.
- 6. the method according to claim 2 using live body plant hand-feeding propagation corn leaf aphids, it is characterised in that： The inoculum concentration of the corn leaf aphids worm sources is that every plant of plant is inoculated with least 3 corn leaf aphids.
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| CN201711065453.2A CN107593626B (en) | 2017-11-02 | 2017-11-02 | Method for breeding corn aphids by using living corn plants |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108174824A (en) * | 2018-01-31 | 2018-06-19 | 南阳师范学院 | A kind of low-density cultural method for keeping the lonely female aptery state of aphid of acyrthosiphum pisim |
| CN109601483A (en) * | 2018-12-11 | 2019-04-12 | 安阳工学院 | A kind of method for breeding making corn leaf aphids life table |
| CN114271240A (en) * | 2022-01-14 | 2022-04-05 | 北京市农林科学院 | Breeding method of adelphocoris suturalis |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108174824A (en) * | 2018-01-31 | 2018-06-19 | 南阳师范学院 | A kind of low-density cultural method for keeping the lonely female aptery state of aphid of acyrthosiphum pisim |
| CN108174824B (en) * | 2018-01-31 | 2020-11-13 | 南阳师范学院 | Low-density breeding method for keeping wingless state of pea aphid parthenogenetic female aphid |
| CN109601483A (en) * | 2018-12-11 | 2019-04-12 | 安阳工学院 | A kind of method for breeding making corn leaf aphids life table |
| CN114271240A (en) * | 2022-01-14 | 2022-04-05 | 北京市农林科学院 | Breeding method of adelphocoris suturalis |
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