CN103975859A - Method for screening glyphosate resisting lolium multiflorum plants - Google Patents
Method for screening glyphosate resisting lolium multiflorum plants Download PDFInfo
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- CN103975859A CN103975859A CN201410234043.6A CN201410234043A CN103975859A CN 103975859 A CN103975859 A CN 103975859A CN 201410234043 A CN201410234043 A CN 201410234043A CN 103975859 A CN103975859 A CN 103975859A
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Abstract
The invention discloses a method for screening glyphosate resisting lolium multiflorum plants with shorter consumed time. The method comprises the steps of adopting mature lolium multiflorum seeds as explants, cleaning and sterilizing the mature lolium multiflorum seeds, then, inoculating to a callus induction culture medium, culturing for 18-22 days so as to obtain a callus, then, transferring the callus into a first callus subculture medium, culturing for 18-22 days, then, transferring to a second callus subculture medium containing glyphosate, culturing for 18-22 days, then, carrying out glyphosate resisting callus screening, then, transferring screened glyphosate resisting calluses into a callus differentiation culture medium, culturing for 18-22 days, transferring survival seedlings into a rooting culture medium when the seedlings grow to 2-3cm, culturing for 20-30 days, and finally, transplanting resistant seedlings. The plants with glyphosate resistance can be rapidly obtained by the method, and the required time is relatively short and is just about one hundred days; the method is suitable for being popularized and applied to the field of biotechnologies.
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 a year raw plant more, 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 animals are all liked and search for food.Annual Ryegrass is suitable for cradling green grass or young crops to be raised, and modulation high-quality hay, also can herd utilization, is also the good bait of breeding fish, and each provinces and regions of south China utilize the other plantation in limit, fish pond more, in order to the grass carp of feeding.Annual Ryegrass is best in quality, contains rich in protein, and 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 output and the quality of Annual Ryegrass.In order to remove weeds in field, adopt the mode of spraying insecticide simultaneously, at present, the agricultural chemicals using is generally glyphosate, but glyphosate can produce poisoning effect for Annual Ryegrass, serious meeting is also killed Annual Ryegrass, therefore, for fear of glyphosate, when killing weeds, do not kill Annual Ryegrass, just need to make Annual Ryegrass there is glyphosate resistance, need seed selection to there is 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 conventionally, and first kind of way is to adopt continuous several times to spray glyphosate, selects resistance plant year after year, finally obtains resistant strain.The method is consuming time longer, as just chosen successfully through 66 generations in perennial ryegrass; The second way is to utilize resistance parent and common variety hybridize and repeatedly backcross, under the condition of spraying glyphosate, select resistant plant, profit is formulated glyphosate resistant crops kind in this way, must have anti-source, be resistance parent, resistance parent's acquisition is difficulty comparatively, 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, by being inoculated into after Annual Ryegrass mature seed washing and sterilizing, in callus inducing medium, cultivating 18～22d and obtain callus, described callus inducing medium is by 2 of MS medium, 1～5mg/L, and the 6-BA of 4-D and 0.5～3mg/L forms;
B, callus is proceeded in the first callus subculture medium and cultivates 18～22d, described the first callus subculture medium is by 2 of MS medium, 1～3mg/L, and the 6-BA of 4-D and 0～0.3mg/L forms;
C, the callus that process step B is processed are transferred in the second callus subculture medium and cultivate 18～22d, described the second callus subculture medium is by 2 of MS medium, 1～3mg/L, and the 6-BA of 4-D, 0～0.3mg/L, the glyphosate of 100～500mg/L form;
D, the callus of survival after step C processes is transferred in Calli Differentiation medium and cultivates 18～22d, described Calli Differentiation medium is comprised of the 6-BA of MS medium, 0.5～2mg/L;
E, when seedling grows to 2～3cm, in seedling access root media that will survival, cultivate 20～30d, described root media is comprised of 1/2MS medium;
F, after seedlings root grows, by its transplanting.
Be further, in steps A, processing mode to Annual Ryegrass mature seed washing and sterilizing is as described below: first, Annual Ryegrass mature seed is put into bottle, and in bottle, add water and washing agent and soak 12 hours, the ratio of described water and washing agent is 1:3, then with clear water, wash away the unreal seed swimming on liquid level again, then be transferred to superclean bench, first the seed of picking out be immersed in to 1～2min in 75% ethanolic solution, then to forward concentration to be 0.1% HgCl
2the 8min that sterilizes in solution, after sterilization with aseptic water washing several times and blot with double-layer sterile filter paper.
Further, the MS medium containing 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 is 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 steps A, in described callus inducing medium, contain 2,4-D is 2mg/L, the 6-BA containing is 1mg/L.
Further, in step B, in described the first callus subculture medium, contain 2,4-D is 2mg/L, the 6-BA containing is 0.2mg/L.
Further, in step C, in described the second callus subculture medium, contain 2,4-D is 2mg/L, the 6-BA containing is 0.2mg/L.
Further, in step D, the 6-BA containing in described Calli Differentiation medium is 0.5mg/L.
Further, in step D, described condition of culture is 23 ± 2 ℃ of cultivation temperature, 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 is 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 containing in described the second callus subculture medium is 500mg/L.
Beneficial effect of the present invention is: the present invention adopts Annual Ryegrass mature seed as explant, by being inoculated into after Annual Ryegrass mature seed washing and sterilizing, in callus inducing medium, cultivating 18～22d and obtain callus, then callus is proceeded in the first callus subculture medium and cultivates 18～22d, proceeded to again and in the second callus subculture medium that contains glyphosate, cultivate 18～22d and carry out the screening of resistance glyphosate callus, again the resistance glyphosate callus filtering out is transferred in Calli Differentiation medium and cultivates 18～22d, when seedling grows to 2～3cm, to in the seedling access root media of survival, cultivate 20～30d, finally carry out resistance transplantation of seedlings, by said method, can obtain fast the plant with glyphosate resistance, required time is shorter, only need about 100 days.
Embodiment
The present invention adopts Annual Ryegrass mature seed as explant, by being inoculated into after Annual Ryegrass mature seed washing and sterilizing, in callus inducing medium, cultivating 18～22d and obtain callus, then callus is proceeded in the first callus subculture medium and cultivates 18～22d, proceeded to again and in the second callus subculture medium that contains glyphosate, cultivate 18～22d and carry out the screening of resistance glyphosate callus, again the resistance glyphosate callus filtering out is transferred in Calli Differentiation medium and cultivates 18～22d, when seedling grows to 2～3cm, to in the seedling access root media of survival, cultivate 20～30d, finally carry out resistance transplantation of seedlings, by said method, can obtain fast the plant with glyphosate resistance, 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, by being inoculated into after Annual Ryegrass mature seed washing and sterilizing, in callus inducing medium, cultivating 18～22d and obtain callus, described callus inducing medium is by 2 of MS medium, 1～5mg/L, and the 6-BA of 4-D and 0.5～3mg/L forms;
B, callus is proceeded in the first callus subculture medium and cultivates 18～22d, described the first callus subculture medium is by 2 of MS medium, 1～3mg/L, and the 6-BA of 4-D and 0～0.3mg/L forms;
C, the callus that process step B is processed are transferred in the second callus subculture medium and cultivate 18～22d, described the second callus subculture medium is by 2 of MS medium, 1～3mg/L, and the 6-BA of 4-D, 0～0.3mg/L, the glyphosate of 100～500mg/L form;
D, the callus of survival after step C processes is transferred in Calli Differentiation medium and cultivates 18～22d, described Calli Differentiation medium is comprised of the 6-BA of MS medium, 0.5～2mg/L;
E, when seedling grows to 2～3cm, in seedling access root media that will survival, cultivate 20～30d, described root media is comprised of 1/2MS medium;
F, after seedlings root grows, by its transplanting.
In above-mentioned Annual Ryegrass resistance glyphosate plant screening technique process, in steps A, processing mode to Annual Ryegrass mature seed washing and sterilizing is as described below: first, Annual Ryegrass mature seed is put into bottle, and in bottle, add water and washing agent and soak 12 hours, the ratio of described water and washing agent is 1:3, then with clear water, wash away the unreal seed swimming on liquid level again, then be transferred to superclean bench, first the seed of picking out is immersed in to 1～2min in 75% ethanolic solution, then to forward concentration to be 0.1% HgCl
2the 8min that sterilizes in solution, after sterilization with aseptic water washing several times and blot with double-layer sterile filter paper.
The MS medium containing 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 is 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 for Annual Ryegrass, to configure specially, and more suitable Annual Ryegrass tissue is cultivated.
To after a plurality of Annual Ryegrass mature seed washing and sterilizings, be inoculated in different callus inducing mediums, the MS medium that its different callus medium 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 ℃, observe the quality of callus and add up callus number and calculate callus induction rate, its result is as shown in table 1:
Table 1
Note: the different lowercases of same column represent LSD method multiple ratio result: 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, the Annual Ryegrass callus induction rate difference of 4-D concentration induction is (P<0.05) significantly, and the Annual Ryegrass callus induction rate difference of different 6-BA concentration inductions is significantly (P<0.05) also.In described induction of callus, contain 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, be faint yellow, fine and close, large (diameter >1cm) particle.
By cultivating through callus inducing medium the callus obtaining, proceed in different callus subculture mediums, 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 calculating value-added coefficient, its result is as shown in table 2:
Table 2
It is to make callus amplification that subculture is cultivated Main Function, is more conducive to turn out more regeneration plant.As shown in Table 2, in described callus subculture medium, contain 2,4-D be 2mg/L, 6-BA while being 0.2mg/L value-added coefficient average maximum, and callus is flaxen dense granule.Therefore, in step B, in described the first callus subculture medium, contain 2,4-D is 2mg/L, the 6-BA containing is 0.2mg/L.Same, in step C, in described the second callus subculture medium, contain 2,4-D is 2mg/L, the 6-BA containing is 0.2mg/L.
Callus is inoculated on the second callus subculture medium of variable concentrations glyphosate after 5d, some callus start to occur brownization, there is yellow liquid in some callus bottom, it is loose, moistening that particle becomes gradually, is 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 brown rate of callus after 20d, as shown in table 3:
Table 3
Note: the different lowercases in every row end are illustrated 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 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 is 5000mg/L and when above, and 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 that contains variable concentrations glyphosate is cultivated, 25 ± 2 ℃ of cultivation temperature, intensity of illumination 1000lx, the callus of 5d rear section survival starts to occur the bud point of peak green, after 10d, Calli Differentiation reaches maximum, differentiation resistance seedling is out light yellow green, and color is of light color without the seedling that crosses screening; Part seedling is withered and yellow shape, and through verification experimental verification, the 6-BA containing in described Calli Differentiation medium is 0.5mg/L, and it is the highest that differentiation rate reaches, and adds up the differentiation rate of callus after 20d, as shown in table 4:
Table 4
Note: the different lowercases in every row end are illustrated 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; The seedling of differentiation qualitatively with the difference to some extent of seedling out of differentiation under normal circumstances.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 access root media of survival, described root media is only comprised 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 is 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.
After the seedling of survival is accessed in above-mentioned root media, in cultivation temperature, be that 23 ± 2 ℃, intensity of illumination are that 800～1000lx, light application time are under condition, to cultivate 20～30d 16 hours every days, then observe rooting rate, through experimental study, show, in the root media only being formed by 1/2MS medium, root system is generally creamy white, and rooting rate is the highest.To in the seedling access root media of survival, cultivate 20～30d, treat that root system is healthy and strong, after being full of at the bottom of conical flask, then transplanting and carry out Resistance Identification.During Resistance Identification, get 500mg/L glyphosate and process the regeneration plant that the callus of survival breaks up, when after it is transplanted, regeneration plant height reaches 15cm left and right, 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
After 3d, observe 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, test plant starts to occur symptom of chemical damage, 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 slighter, and poisoning grade is only also 1 grade or 0 grade; When glyphosate concentration is 500mg/L, plant starts the flavescence of whole strain chlorisis, and dead leaf area reaches more than 40%～70%; When glyphosate concentration is 1000mg/L, the whole strain of plant is more withered, and dead leaf area reaches 70%～90%.When glyphosate concentration is 5000mg/L and when above, the whole strain of plant is withered and yellow serious, and dead leaf area reaches more than 95% even death.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 time, glyphosate has certain coercive to Annual Ryegrass callus, can produce sudden change by evoked callus, and the callus of surviving also has differentiation capability, can produce regeneration plant under these two kinds of concentration.Therefore, to Annual Ryegrass callus carry out glyphosate coerce screening suitable concentration be 100～500mg/L.Further, in step C, the glyphosate containing in described the second callus subculture medium is preferably 500mg/L.
Claims (10)
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, by being inoculated into after Annual Ryegrass mature seed washing and sterilizing, in callus inducing medium, cultivating 18～22d and obtain callus, described callus inducing medium is by 2 of MS medium, 1～5mg/L, and the 6-BA of 4-D and 0.5～3mg/L forms;
B, callus is proceeded in the first callus subculture medium and cultivates 18～22d, described the first callus subculture medium is by 2 of MS medium, 1～3mg/L, and the 6-BA of 4-D and 0～0.3mg/L forms;
C, the callus that process step B is processed are transferred in the second callus subculture medium and cultivate 18～22d, described the second callus subculture medium is by 2 of MS medium, 1～3mg/L, and the 6-BA of 4-D, 0～0.3mg/L, the glyphosate of 100～500mg/L form;
D, the callus of survival after step C processes is transferred in Calli Differentiation medium and cultivates 18～22d, described Calli Differentiation medium is comprised of the 6-BA of MS medium, 0.5～2mg/L;
E, when seedling grows to 2～3cm, in seedling access root media that will survival, cultivate 20～30d, described root media is comprised of 1/2MS medium;
F, after seedlings root grows, by its transplanting.
2. Annual Ryegrass resistance glyphosate plant screening technique as claimed in claim 1, it is characterized in that: in steps A, processing mode to Annual Ryegrass mature seed washing and sterilizing is as described below: first, Annual Ryegrass mature seed is put into bottle, and in bottle, add water and washing agent and soak 12 hours, the ratio of described water and washing agent is 1:3, then with clear water, wash away the unreal seed swimming on liquid level again, then be transferred to superclean bench, first the seed of picking out is immersed in to 1～2min in 75% ethanolic solution, forward again concentration to and be 0.1% HgCl
2the 8min that sterilizes in solution, after sterilization with aseptic water washing several times and blot with double-layer sterile filter paper.
3. Annual Ryegrass resistance glyphosate plant screening technique as claimed in claim 1, is characterized in that: the MS medium containing 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 is 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, is characterized in that: in steps A, in described callus inducing medium, contain 2,4-D is 2mg/L, the 6-BA containing is 1mg/L.
5. Annual Ryegrass resistance glyphosate plant screening technique as claimed in claim 4, is characterized in that: in step B, in described the first callus subculture medium, contain 2,4-D is 2mg/L, the 6-BA containing is 0.2mg/L.
6. Annual Ryegrass resistance glyphosate plant screening technique as claimed in claim 5, is characterized in that: in step C, in described the second callus subculture medium, contain 2,4-D is 2mg/L, the 6-BA containing is 0.2mg/L.
7. Annual Ryegrass resistance glyphosate plant screening technique as claimed in claim 6, is characterized in that: in step D, the 6-BA containing in described Calli Differentiation medium is 0.5mg/L.
8. Annual Ryegrass resistance glyphosate plant screening technique as claimed in claim 7, is characterized in that: in step D, described condition of culture is 23 ± 2 ℃ of cultivation temperature, and intensity of illumination is 800～1000lx, and light application time is 16 hours every days.
9. Annual Ryegrass resistance glyphosate plant screening technique as claimed in claim 8, is characterized in that: 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 is 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.
10. Annual Ryegrass resistance glyphosate plant screening technique as claimed in claim 9, is characterized in that: in step C, the glyphosate containing in described the second callus subculture medium is 500mg/L.
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| CN103975859B (en) | 2016-02-10 |
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