CN103975859B - A kind of Annual Ryegrass resistance glyphosate plant screening technique - Google Patents
A kind of Annual Ryegrass resistance glyphosate plant screening technique Download PDFInfo
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- CN103975859B CN103975859B CN201410234043.6A CN201410234043A CN103975859B CN 103975859 B CN103975859 B CN 103975859B CN 201410234043 A CN201410234043 A CN 201410234043A CN 103975859 B CN103975859 B CN 103975859B
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Abstract
The invention discloses a kind of shorter Annual Ryegrass resistance glyphosate plant screening technique consuming time.The present invention adopts Annual Ryegrass mature seed as explant, callus is obtained by being inoculated into cultivation 18 ~ 22d in callus inducing medium after Annual Ryegrass mature seed washing and sterilizing, then callus is proceeded in the first callus subculture medium and cultivate 18 ~ 22d, proceeded to cultivation 18 ~ 22d in the second callus subculture medium containing glyphosate again and carried out the screening of resistance glyphosate callus, again the resistance glyphosate callus filtered out is transferred in Calli Differentiation medium and cultivates 18 ~ 22d, when seedling grows to 2 ~ 3cm, 20 ~ 30d is cultivated by the seedling of survival access root media, finally carry out resistance transplantation of seedlings, the plant with glyphosate resistance can be obtained fast by said method, required time is shorter, only need about 100 days.Be adapted at biological technical field to promote the use.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of Annual Ryegrass resistance glyphosate plant screening technique.
Background technology
Annual Ryegrass is annual or gets over year raw plant, and fibrous root is intensive thin and delicate, and stalk is most, uprightly high 50 ~ 70 centimetres.Spike is flat.Annual Ryegrass good palatability, various domestic animal is all liked and searches for food.Annual Ryegrass is suitable for cradling green grass or young crops and raises, modulation high-quality hay, also can grazing utilization, and be also the good bait of breeding fish, each provinces and regions of south China utilize the other plantation in limit, fish pond more, in order to grass carp of feeding.Annual Ryegrass is best in quality, and containing rich in protein, the leafage phase, leaf amount was many because stem stalk is few, and quality is better.Annual Ryegrass is important annual or short-term study of perennial forage grasses, is suitable for as the Winter-Spring crop in field rotation system.
Annual Ryegrass has the good characteristics such as growth is rapid, tillering ability is strong, output is high, barren-resistant, is widely used grass seeds in grassland in South China agricultural production.But, owing to carrying out strong competition between weeds in field and Annual Ryegrass, had a strong impact on the Yield and qualities of Annual Ryegrass.In order to remove weeds in field, adopt the mode of spraying insecticide simultaneously, at present, the agricultural chemicals used is generally glyphosate, but glyphosate can produce poisoning effect for Annual Ryegrass, Annual Ryegrass also kills by serious meeting, therefore, in order to avoid glyphosate does not kill Annual Ryegrass while killing weeds, just need to make Annual Ryegrass have glyphosate resistance, seed selection is needed to have the Annual Ryegrass of glyphosate resistance, to improve the competitiveness of Annual Ryegrass.
At present, the Annual Ryegrass that seed selection has glyphosate resistance adopts following two kinds of modes usually, and first kind of way adopts continuous several times to spray glyphosate, selects resistance plant year after year, finally obtain resistant strain.The method is consuming time longer, as perennial ryegrass is just chosen successfully through 66 generations; The second way utilizes resistance parent and common variety to carry out hybridizing and repeatedly backcrossing, resistant plant is selected under the condition of spraying glyphosate, profit formulates glyphosate resistant crops kind in this way, then must have anti-source, i.e. resistance parent, the acquisition of resistance parent is comparatively difficult, secondly, this mode also needs repeatedly to backcross, consuming time still longer.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of shorter Annual Ryegrass resistance glyphosate plant screening technique consuming time.
The technical solution adopted for the present invention to solve the technical problems is: this Annual Ryegrass resistance glyphosate plant screening technique, comprises the following steps:
A, employing Annual Ryegrass mature seed are as explant, callus is obtained by being inoculated into cultivation 18 ~ 22d in callus inducing medium after Annual Ryegrass mature seed washing and sterilizing, described callus inducing medium is made up of the 6-BA of 2,4-D and the 0.5 ~ 3mg/L of MS medium, 1 ~ 5mg/L;
B, to be proceeded to by callus in the first callus subculture medium and cultivate 18 ~ 22d, described first callus subculture medium is made up of the 6-BA of 2,4-D and the 0 ~ 0.3mg/L of MS medium, 1 ~ 3mg/L;
C, the callus through step B process is transferred in the second callus subculture medium and cultivates 18 ~ 22d, described second callus subculture medium by MS medium, 1 ~ 3mg/L 2,4-D, the 6-BA of 0 ~ 0.3mg/L, 100 ~ 500mg/L glyphosate form;
D, by after step C process survival callus be transferred in Calli Differentiation medium cultivate 18 ~ 22d, described Calli Differentiation medium is made up of the 6-BA of MS medium, 0.5 ~ 2mg/L;
E, when seedling grows to 2 ~ 3cm, by survival seedling access root media in cultivate 20 ~ 30d, described root media is made up of 1/2MS medium;
F, after seedlings root grows, to be transplanted.
Be further, in step, as described below to the processing mode of Annual Ryegrass mature seed washing and sterilizing: first, Annual Ryegrass mature seed is put into bottle, and in bottle, adds water and washing agent soaks 12 hours, the ratio of described water and washing agent is 1:3, then wash away with clear water the unreal seed swum on liquid level again, then be transferred to superclean bench, first the seed picked out be immersed in 1 ~ 2min in the ethanolic solution of 75%, then forward the HgCl that concentration is 0.1% to
2sterilize in solution 8min, also blots with double-layer sterile filter paper several times after sterilization with aseptic water washing.
Further, the MS medium contained in described callus inducing medium, the first callus subculture medium, the second callus subculture medium, Calli Differentiation medium comprises KNO
3, NH
4nO
3, MgSO47H
2o, KH
2pO
4, CaCl
22H
2o, MnSO
44H
2o, ZnSO
47H
2o, H
3bO
3, KI, NaMoO
42H
2o, CuSO
45H
2o, CoCl
26H
2o, Na
2-EDTA, FeSO
44H
2o, glycine, thiamine hydrochloride, puridoxine hydrochloride, nicotinic acid, inositol, sucrose, agar, the content of each component is as described below: KNO
3for 1900mg/L, NH
4nO
3for 1650mg/L, MgSO47H
2o is 370mg/L, KH
2pO
4for 170mg/L, CaCl
22H
2o is 440mg/L, MnSO
44H
2o is 22.3mg/L, ZnSO
47H
2o is 8.6mg/L, H
3bO
3for 6.2mg/L, KI are 0.83mg/L, NaMoO
42H
2o is 0.25mg/L, CuSO
45H
2o is 0.025mg/L, CoCl
26H
2o is 0.025mg/L, Na
2-EDTA is 37.3mg/L, FeSO
44H
2o is 27.8mg/L, and glycine is 2.0mg/L, and thiamine hydrochloride is 0.1mg/L, and puridoxine hydrochloride is 0.5mg/L, and nicotinic acid is 0.5mg/L, and inositol is 100mg/L, sucrose 30g/L, agar 8g/L, and pH value is 6.0.
Further, in step, contain in described callus inducing medium 2,4-D is 2mg/L, and the 6-BA contained is 1mg/L.
Further, in stepb, contain in described first callus subculture medium 2,4-D is 2mg/L, and the 6-BA contained is 0.2mg/L.
Further, in step C, contain in described second callus subculture medium 2,4-D is 2mg/L, and the 6-BA contained is 0.2mg/L.
Further, in step D, the 6-BA contained in described Calli Differentiation medium is 0.5mg/L.
Further, in step D, described condition of culture is cultivation temperature 23 ± 2 DEG C, and intensity of illumination is 800 ~ 1000lx, and light application time is 16 hours every days.
Further, the 1/2MS medium of described composition root media comprises KNO
3, NH
4nO
3, MgSO47H
2o, KH
2pO
4, CaCl
22H
2o, MnSO
44H
2o, ZnSO
47H
2o, H
3bO
3, KI, NaMoO
42H
2o, CuSO
45H
2o, CoCl
26H
2o, Na
2-EDTA, FeSO
44H
2o, glycine, thiamine hydrochloride, puridoxine hydrochloride, nicotinic acid, inositol, sucrose, agar, the content of each component is as described below: KNO
3for 950mg/L, NH
4nO
3for 825mg/L, MgSO47H
2o is 185mg/L, KH
2pO
4for 85mg/L, CaCl
22H
2o is 220mg/L, MnSO
44H
2o is 11.15mg/L, ZnSO
47H
2o is 4.3mg/L, H
3bO
3for 3.1mg/L, KI are 0.415mg/L, NaMoO
42H
2o is 0.125mg/L, CuSO
45H
2o is 0.0125mg/L, CoCl
26H
2o is 0.0125mg/L, Na
2-EDTA is 37.3mg/L, FeSO
44H
2o is 27.8mg/L, and glycine is 2.0mg/L, and thiamine hydrochloride is 0.1mg/L, and puridoxine hydrochloride is 0.5mg/L, and nicotinic acid is 0.5mg/L, and inositol is 100mg/L, sucrose 30g/L, agar 8g/L, and pH value is 6.0.
Further, in step C, the glyphosate contained in described second callus subculture medium is 500mg/L.
Beneficial effect of the present invention is: the present invention adopts Annual Ryegrass mature seed as explant, callus is obtained by being inoculated into cultivation 18 ~ 22d in callus inducing medium after Annual Ryegrass mature seed washing and sterilizing, then callus is proceeded in the first callus subculture medium and cultivate 18 ~ 22d, proceeded to cultivation 18 ~ 22d in the second callus subculture medium containing glyphosate again and carried out the screening of resistance glyphosate callus, again the resistance glyphosate callus filtered out is transferred in Calli Differentiation medium and cultivates 18 ~ 22d, when seedling grows to 2 ~ 3cm, 20 ~ 30d is cultivated by the seedling of survival access root media, finally carry out resistance transplantation of seedlings, the plant with glyphosate resistance can be obtained fast by said method, required time is shorter, only need about 100 days.
Embodiment
The present invention adopts Annual Ryegrass mature seed as explant, callus is obtained by being inoculated into cultivation 18 ~ 22d in callus inducing medium after Annual Ryegrass mature seed washing and sterilizing, then callus is proceeded in the first callus subculture medium and cultivate 18 ~ 22d, proceeded to cultivation 18 ~ 22d in the second callus subculture medium containing glyphosate again and carried out the screening of resistance glyphosate callus, again the resistance glyphosate callus filtered out is transferred in Calli Differentiation medium and cultivates 18 ~ 22d, when seedling grows to 2 ~ 3cm, 20 ~ 30d is cultivated by the seedling of survival access root media, finally carry out resistance transplantation of seedlings, the plant with glyphosate resistance can be obtained fast by said method, required time is shorter, only need about 100 days.Concrete, Annual Ryegrass resistance glyphosate plant screening technique of the present invention, specifically comprises the following steps:
A, employing Annual Ryegrass mature seed are as explant, callus is obtained by being inoculated into cultivation 18 ~ 22d in callus inducing medium after Annual Ryegrass mature seed washing and sterilizing, described callus inducing medium is made up of the 6-BA of 2,4-D and the 0.5 ~ 3mg/L of MS medium, 1 ~ 5mg/L;
B, to be proceeded to by callus in the first callus subculture medium and cultivate 18 ~ 22d, described first callus subculture medium is made up of the 6-BA of 2,4-D and the 0 ~ 0.3mg/L of MS medium, 1 ~ 3mg/L;
C, the callus through step B process is transferred in the second callus subculture medium and cultivates 18 ~ 22d, described second callus subculture medium by MS medium, 1 ~ 3mg/L 2,4-D, the 6-BA of 0 ~ 0.3mg/L, 100 ~ 500mg/L glyphosate form;
D, by after step C process survival callus be transferred in Calli Differentiation medium cultivate 18 ~ 22d, described Calli Differentiation medium is made up of the 6-BA of MS medium, 0.5 ~ 2mg/L;
E, when seedling grows to 2 ~ 3cm, by survival seedling access root media in cultivate 20 ~ 30d, described root media is made up of 1/2MS medium;
F, after seedlings root grows, to be transplanted.
In above-mentioned Annual Ryegrass resistance glyphosate plant screening technique process, in step, as described below to the processing mode of Annual Ryegrass mature seed washing and sterilizing: first, Annual Ryegrass mature seed is put into bottle, and in bottle, add water and washing agent soaks 12 hours, the ratio of described water and washing agent is 1:3, then wash away with clear water the unreal seed swum on liquid level again, then superclean bench is transferred to, first the seed picked out is immersed in 1 ~ 2min in the ethanolic solution of 75%, then forwards the HgCl that concentration is 0.1% to
2sterilize in solution 8min, also blots with double-layer sterile filter paper several times after sterilization with aseptic water washing.
The MS medium contained in described callus inducing medium, the first callus subculture medium, the second callus subculture medium, Calli Differentiation medium comprises KNO
3, NH
4nO
3, MgSO47H
2o, KH
2pO
4, CaCl
22H
2o, MnSO
44H
2o, ZnSO
47H
2o, H
3bO
3, KI, NaMoO
42H
2o, CuSO
45H
2o, CoCl
26H
2o, Na
2-EDTA, FeSO
44H
2o, glycine, thiamine hydrochloride, puridoxine hydrochloride, nicotinic acid, inositol, sucrose, agar, the content of each component is as described below: KNO
3for 1900mg/L, NH
4nO
3for 1650mg/L, MgSO47H
2o is 370mg/L, KH
2pO
4for 170mg/L, CaCl
22H
2o is 440mg/L, MnSO
44H
2o is 22.3mg/L, ZnSO
47H
2o is 8.6mg/L, H
3bO
3for 6.2mg/L, KI are 0.83mg/L, NaMoO
42H
2o is 0.25mg/L, CuSO
45H
2o is 0.025mg/L, CoCl
26H
2o is 0.025mg/L, Na
2-EDTA is 37.3mg/L, FeSO
44H
2o is 27.8mg/L, and glycine is 2.0mg/L, and thiamine hydrochloride is 0.1mg/L, and puridoxine hydrochloride is 0.5mg/L, and nicotinic acid is 0.5mg/L, and inositol is 100mg/L, sucrose 30g/L, agar 8g/L, and pH value is 6.0.This formula is specially for Annual Ryegrass configuration, more suitable Annual Ryegrass tissue cultures.
To be inoculated in different callus inducing mediums after multiple Annual Ryegrass mature seed washing and sterilizing, the MS medium that its different callus tissue culture base contains is identical, its contain 2, the mass concentration of 4-D and 6-BA is as shown in table 1 below, then dark culturing 20d in the culturing room of 25 ± 2 DEG C, observe the quality of callus and add up callus number calculating callus induction rate, its result is as shown in table 1:
Table 1
Note: the different lowercase of same column represents the result of LSD method Multiple range test: difference 2 when capitalization represents that 6-BA concentration is identical, significant difference between 4-D concentration, significant difference (P<0.05) when lowercase represents that 2,4-D concentration is identical between different 6-BA concentration.
From table 1, different 2, comparatively significantly (P<0.05), the Annual Ryegrass callus induction rate difference of different 6-BA concentration induction also comparatively significantly (P<0.05) for the Annual Ryegrass callus induction rate difference of 4-D concentration induction.When contain in described induction of callus 2, the frequency of embryonic callus induction when 6-BA that 4-D is 2mg/L, contain is 1mg/L is the highest, reach 60.42%, and callus growth speed is fast, quality is best, in faint yellow, fine and close, comparatively large (diameter >1cm) particle.
Proceed in different callus subculture mediums by cultivating the callus obtained through callus inducing medium, the MS medium that its different callus subculture medium contains is identical, its contain 2, the mass concentration of 4-D and 6-BA is as shown in table 2 below, then dark culturing 20d in culturing room, observe the quality of callus and add up callus final weight, then calculate value-added coefficient, its result is as shown in table 2:
Table 2
Squamous subculture Main Function is that callus is increased, and is more conducive to turning out more regeneration plant.As shown in Table 2, contain in described callus subculture medium 2, the 4-D averages of value-added coefficient when be 2mg/L, 6-BA being 0.2mg/L are maximum, and callus is flaxen dense granule.Therefore, in stepb, contain in described first callus subculture medium 2,4-D is 2mg/L, and the 6-BA contained is 0.2mg/L.Same, in step C, contain in described second callus subculture medium 2,4-D is 2mg/L, and the 6-BA contained is 0.2mg/L.
On the second callus subculture medium callus being inoculated into variable concentrations glyphosate after 5d, some callus start to occur brownization, there is yellow liquid in some callus bottoms, particle becomes loose, moistening gradually, in water soaking mode, along with the passing of incubation time, some callus gradually brownization are even dead, some callus gradually become milky or white, add up survival rate and the melting brown rate of callus after 20d, as shown in table 3:
Table 3
Note: often the different lowercase alphabet in row end is shown in the significant difference in 0.05 level.
As shown in Table 3, the screening of variable concentrations glyphosate has remarkable impact to the growth of Annual Ryegrass callus, and along with the increase of glyphosate concentration, Annual Ryegrass callus survival rate constantly reduces, and melting brown rate constantly increases.When glyphosate concentration is lower than 500mg/L, Annual Ryegrass callus survival rate is higher than 50%; When concentration is 1000mg/L, survival rate only 6.67%; Concentration be 5000mg/L and above time, survival rate is 0.Therefore, the glyphosate Cmax that Annual Ryegrass callus can bear is 1000mg/L.
By in the Calli Differentiation medium of survival after the second callus subculture medium containing variable concentrations glyphosate is cultivated, cultivation temperature 25 ± 2 DEG C, intensity of illumination 1000lx, the callus of 5d rear section survival starts the bud point occurring peak green, after 10d, Calli Differentiation reaches maximum, differentiation resistance seedling is out light yellow green, and color is shallow compared with the Seedling Color without screening; Part seedling is withered and yellow shape, and through verification experimental verification, when the 6-BA contained in described Calli Differentiation medium is 0.5mg/L, differentiation rate reaches the highest, adds up the differentiation rate of callus after 20d, as shown in table 4:
Table 4
Note: often the different lowercase alphabet in row end is shown in the significant difference in 0.05 level.
As shown in Table 4, after glyphosate screening, Annual Ryegrass phenylacetic acid significantly reduces; Glyphosate concentration is higher, and differentiation rate is lower; Differentiation seedling qualitatively with the seedling breaking up out under normal circumstances difference to some extent.When glyphosate concentration reaches 500mg/L, differentiation rate is 15.38%.When glyphosate concentration reaches 1000mg/L, differentiation rate is 0, and callus is continued growth, but does not occur differentiation.
When seedling grows to 2 ~ 3cm, by the seedling of survival access root media, described root media is only made up of 1/2MS medium, comprises KNO
3, NH
4nO
3, MgSO47H
2o, KH
2pO
4, CaCl
22H
2o, MnSO
44H
2o, ZnSO
47H
2o, H
3bO
3, KI, NaMoO
42H
2o, CuSO
45H
2o, CoCl
26H
2o, Na
2-EDTA, FeSO
44H
2o, glycine, thiamine hydrochloride, puridoxine hydrochloride, nicotinic acid, inositol, sucrose, agar, the content of each component is as described below: KNO
3for 950mg/L, NH
4nO
3for 825mg/L, MgSO47H
2o is 185mg/L, KH
2pO
4for 85mg/L, CaCl
22H
2o is 220mg/L, MnSO
44H
2o is 11.15mg/L, ZnSO
47H
2o is 4.3mg/L, H
3bO
3for 3.1mg/L, KI are 0.415mg/L, NaMoO
42H
2o is 0.125mg/L, CuSO
45H
2o is 0.0125mg/L, CoCl
26H
2o is 0.0125mg/L, Na
2-EDTA is 37.3mg/L, FeSO
44H
2o is 27.8mg/L, and glycine is 2.0mg/L, and thiamine hydrochloride is 0.1mg/L, and puridoxine hydrochloride is 0.5mg/L, and nicotinic acid is 0.5mg/L, and inositol is 100mg/L, sucrose 30g/L, agar 8g/L, and pH value is 6.0.
The seedling of survival is accessed after in above-mentioned root media, cultivation temperature be 23 ± 2 DEG C, intensity of illumination is 800 ~ 1000lx, light application time cultivates 20 ~ 30d under being condition 16 hours every days, then rooting rate is observed, show through experimental study, in the root media be only made up of 1/2MS medium, root system is generally creamy white, and rooting rate is the highest.Cultivate 20 ~ 30d by the seedling of survival access root media, treat that root system is healthy and strong, be full of after at the bottom of conical flask, then carry out transplanting and carry out Resistance Identification.During Resistance Identification, the regeneration plant that the callus getting 500mg/L glyphosate process survival breaks up, when after it is transplanted, regeneration plant height reaches about 15cm, it is sprayed to the glyphosate of variable concentrations, after 3d there is the symptom of chemical damage of different brackets in regeneration plant, after 8d, the symptom of chemical damage of regeneration plant substantially no longer changes, and its result statistics is as shown in table 5:
Table 5
Observe after 3d and find, the concentration of glyphosate improves, and the symptom of chemical damage of resistant calli regeneration plant is serious gradually, and poisoning grade also improves constantly, but does not occur the situation of the whole strain death of plant.When glyphosate concentration is equal to or less than 100mg/L, there is not obvious symptom of chemical damage in test plant; When glyphosate concentration is equal to or greater than 500mg/L, plant starts to occur symptom of chemical damage in test, but symptom of chemical damage is very not serious.The symptom of chemical damage of the resistant calli regeneration plant performance after 8d substantially no longer changes, and symptom of chemical damage increases the weight of with the raising of glyphosate concentration, and poisoning grade also improves constantly.When glyphosate concentration is equal to or less than 100mg/L, symptom of chemical damage is comparatively slight, and poisoning grade is also only 1 grade or 0 grade; When glyphosate concentration is 500mg/L, plant starts whole strain chlorisis and turns yellow, and dead leaf area reaches more than 40% ~ 70%; When glyphosate concentration is 1000mg/L, the whole strain of plant is more seriously withered, and dead leaf area reaches 70% ~ 90%.When glyphosate concentration be 5000mg/L and above time, the whole strain of plant is seriously withered and yellow, and dead leaf area reaches more than 95% even dead.Therefore, 1000mg/L is resistant calli regeneration plant maximal tolerable concentration.
From table 3, table 4, table 5, when the concentration of glyphosate is between 100 ~ 500mg/L, glyphosate has certain coercive to Annual Ryegrass callus, sudden change can be produced by evoked callus, and the callus of surviving under these two kinds of concentration also has differentiation capability, can produce regeneration plant.Therefore, to Annual Ryegrass callus carry out glyphosate coerce screening suitable concentration be 100 ~ 500mg/L.Further, in step C, the glyphosate contained in described second callus subculture medium is preferably 500mg/L.
Claims (6)
1. an Annual Ryegrass resistance glyphosate plant screening technique, is characterized in that comprising the following steps:
A, employing Annual Ryegrass mature seed are as explant, callus is obtained by being inoculated into cultivation 18 ~ 22d in callus inducing medium after Annual Ryegrass mature seed washing and sterilizing, described callus inducing medium is made up of the 6-BA of 2,4-D and the 1mg/L of MS medium, 2mg/L;
B, to be proceeded to by callus in the first callus subculture medium and cultivate 18 ~ 22d, described first callus subculture medium is made up of the 6-BA of 2,4-D and the 0.2mg/L of MS medium, 2mg/L;
C, the callus through step B process is transferred in the second callus subculture medium and cultivates 18 ~ 22d, described second callus subculture medium by MS medium, 2mg/L 2,4-D, the 6-BA of 0.2mg/L, 100 ~ 500mg/L glyphosate form;
D, by after step C process survival callus be transferred in Calli Differentiation medium cultivate 18 ~ 22d, described condition of culture is cultivation temperature 23 ± 2 DEG C, intensity of illumination is 800 ~ 1000lx, light application time is 16 hours every days, and described Calli Differentiation medium is made up of the 6-BA of MS medium, 0.5 ~ 2mg/L;
E, when seedling grows to 2 ~ 3cm, by survival seedling access root media in cultivate 20 ~ 30d, described root media is made up of 1/2MS medium, obtains described 1/2MS medium after the macroelement of MS medium and trace element being reduced by half;
F, after seedlings root grows, to be transplanted.
2. Annual Ryegrass resistance glyphosate plant screening technique as claimed in claim 1, it is characterized in that: in step, as described below to the processing mode of Annual Ryegrass mature seed washing and sterilizing: first, Annual Ryegrass mature seed is put into bottle, and in bottle, add water and washing agent soaks 12 hours, the ratio of described water and washing agent is 1:3, then wash away with clear water the unreal seed swum on liquid level again, then superclean bench is transferred to, first the seed picked out is immersed in 1 ~ 2min in the ethanolic solution of 75%, forward the HgCl that concentration is 0.1% again to
2sterilize in solution 8min, also blots with double-layer sterile filter paper several times after sterilization with aseptic water washing.
3. Annual Ryegrass resistance glyphosate plant screening technique as claimed in claim 1, is characterized in that: the MS medium contained in described callus inducing medium, the first callus subculture medium, the second callus subculture medium, Calli Differentiation medium is by KNO
3, NH
4nO
3, MgSO47H
2o, KH
2pO
4, CaCl
22H
2o, MnSO
44H
2o, ZnSO
47H
2o, H
3bO
3, KI, NaMoO
42H
2o, CuSO
45H
2o, CoCl
26H
2o, Na
2-EDTA, FeSO
44H
2o, glycine, thiamine hydrochloride, puridoxine hydrochloride, nicotinic acid, inositol, sucrose, agar form, and the content of each component is as described below: KNO
3for 1900mg/L, NH
4nO
3for 1650mg/L, MgSO47H
2o is 370mg/L, KH
2pO
4for 170mg/L, CaCl
22H
2o is 440mg/L, MnSO
44H
2o is 22.3mg/L, ZnSO
47H
2o is 8.6mg/L, H
3bO
3for 6.2mg/L, KI are 0.83mg/L, NaMoO
42H
2o is 0.25mg/L, CuSO
45H
2o is 0.025mg/L, CoCl
26H
2o is 0.025mg/L, Na
2-EDTA is 37.3mg/L, FeSO
44H
2o is 27.8mg/L, and glycine is 2.0mg/L, and thiamine hydrochloride is 0.1mg/L, and puridoxine hydrochloride is 0.5mg/L, and nicotinic acid is 0.5mg/L, and inositol is 100mg/L, sucrose 30g/L, agar 8g/L, and pH value is 6.0.
4. Annual Ryegrass resistance glyphosate plant screening technique as claimed in claim 3, it is characterized in that: in step D, the 6-BA contained in described Calli Differentiation medium is 0.5mg/L.
5. Annual Ryegrass resistance glyphosate plant screening technique as claimed in claim 4, is characterized in that: the 1/2MS medium of described composition root media is by KNO
3, NH
4nO
3, MgSO47H
2o, KH
2pO
4, CaCl
22H
2o, MnSO
44H
2o, ZnSO
47H
2o, H
3bO
3, KI, NaMoO
42H
2o, CuSO
45H
2o, CoCl
26H
2o, Na
2-EDTA, FeSO
44H
2o, glycine, thiamine hydrochloride, puridoxine hydrochloride, nicotinic acid, inositol, sucrose, agar form, and the content of each component is as described below: KNO
3for 950mg/L, NH
4nO
3for 825mg/L, MgSO47H
2o is 185mg/L, KH
2pO
4for 85mg/L, CaCl
22H
2o is 220mg/L, MnSO
44H
2o is 11.15mg/L, ZnSO
47H
2o is 4.3mg/L, H
3bO
3for 3.1mg/L, KI are 0.415mg/L, NaMoO
42H
2o is 0.125mg/L, CuSO
45H
2o is 0.0125mg/L, CoCl
26H
2o is 0.0125mg/L, Na
2-EDTA is 37.3mg/L, FeSO
44H
2o is 27.8mg/L, and glycine is 2.0mg/L, and thiamine hydrochloride is 0.1mg/L, and puridoxine hydrochloride is 0.5mg/L, and nicotinic acid is 0.5mg/L, and inositol is 100mg/L, sucrose 30g/L, agar 8g/L, and pH value is 6.0.
6. Annual Ryegrass resistance glyphosate plant screening technique as claimed in claim 5, it is characterized in that: in step C, the glyphosate contained in described second callus subculture medium is 500mg/L.
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| CN1732758A (en) * | 2005-07-21 | 2006-02-15 | 北京林业大学 | Method for establishing high frequency regenerating system of perennial ryegrass |
| CN102450214A (en) * | 2010-10-21 | 2012-05-16 | 中国科学院成都生物研究所 | Screening and preserving method for Lolium L. embryogenic callus |
| CN103314860A (en) * | 2013-07-08 | 2013-09-25 | 中国科学院武汉植物园 | Method for improving perennial ryegrass callus regeneration rate |
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| US8604280B2 (en) * | 2009-10-19 | 2013-12-10 | J.R. Simplot Company | Glyphosate tolerant perennial ryegrass named ‘replay’ |
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| CN1732758A (en) * | 2005-07-21 | 2006-02-15 | 北京林业大学 | Method for establishing high frequency regenerating system of perennial ryegrass |
| CN102450214A (en) * | 2010-10-21 | 2012-05-16 | 中国科学院成都生物研究所 | Screening and preserving method for Lolium L. embryogenic callus |
| CN103314860A (en) * | 2013-07-08 | 2013-09-25 | 中国科学院武汉植物园 | Method for improving perennial ryegrass callus regeneration rate |
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| Title |
|---|
| High frequency plant regeneration from mature seed-derived callus of Italian ryegrass (Lolium multiflorum) cultivars;Ki-Won Lee et.al.,;《African Journal of Biotechnology》;20091215;第8卷(第24期);第6829页左栏第4-8段、右栏第1-2、4段，第6830页左栏第2段 * |
| In vitro selection of glyphosate-tolerant variants from long-term callus cultures of Zoysia matrella [L.] Merr;Shu Chen et.al.,;《Plant Cell Tiss Organ Cult》;20120605;第111卷;摘要 * |
| 原种黑麦草组培再生体系建立初探;郑若男等;《安徽农业科学》;20131231;第41卷(第15期);第6608-6610页 * |
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