CN103975860A - Method for constructing lolium multiflorum tissue culture regeneration system - Google Patents
Method for constructing lolium multiflorum tissue culture regeneration system Download PDFInfo
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Abstract
The invention discloses a method for constructing a lolium multiflorum tissue culture regeneration system. The method can be used for providing a technical reference for the selection and breeding of excellent lolium multiflorum varieties. 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 callus subculture medium, culturing for 18-22 days, then, transferring to a callus differentiation culture medium, culturing for 18-22 days, transferring seedlings into a rooting culture medium when the seedlings grow to 2-3cm, and culturing for 20-30 days. The efficient lolium multiflorum tissue culture regeneration system is constructed by the method, so that a basis for constructing an efficient genetic transformation system of lolium multiflorum tissue is laid, and a technical reference for the selection and breeding of excellent lolium multiflorum tissue varieties is provided; 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 method for building up of Annual Ryegrass Tissue Culture Regeneration System.
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 improve Annual Ryegrass field dust removal rate, reduce herbage production cost, need the good Annual Ryegrass of Cultivars, to improve the competitiveness of Annual Ryegrass, but, there is not yet so far about setting up Annual Ryegrass Tissue Culture Regeneration System and transgenic technology correlative study, can not provide Technical Reference for seed selection Annual Ryegrass improved seeds.
Summary of the invention
Technical problem to be solved by this invention is to provide and a kind ofly can provides for seed selection Annual Ryegrass improved seeds the method for building up of the Annual Ryegrass Tissue Culture Regeneration System of Technical Reference.
The technical solution adopted for the present invention to solve the technical problems is: the method for building up of this Annual Ryegrass Tissue Culture Regeneration System, 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 callus subculture medium and cultivates 18～22d, described 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 Calli Differentiation medium and cultivate 18～22d, and described Calli Differentiation medium is comprised of the 6-BA of MS medium, 0.5～2mg/L;
D, when seedling grows to 2～3cm, seedling is accessed in root media and cultivates 20～30d, described root media is comprised of 1/2MS medium;
E, 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 collection several times and blot with double-layer sterile filter paper.
Further, the MS medium containing in described callus inducing medium, 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 induction of callus, contain 2,4-D is 2mg/L, the 6-BA containing is 1mg/L.
Further, in step B, in described callus subculture medium, contain 2,4-D is 2mg/L, the 6-BA containing is 0.2mg/L.
Further, in step C, the 6-BA containing in described Calli Differentiation medium is 1mg/L.
Further, in step C, described condition of culture is 25 ± 2 ℃ of cultivation temperature, intensity of illumination 1000lx.
Further, the 1/2MS medium that described root media contains 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 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.
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, by MS medium, 2 of 1～5mg/L, the callus inducing medium that the 6-BA of 4-D and 0.5～3mg/L forms can improve inductivity greatly, then callus is proceeded in callus subculture medium and cultivates 18～22d, by MS medium, 2 of 1～3mg/L, the callus subculture medium that the 6-BA of 4-D and 0～0.3mg/L forms can make callus breed the fastest and quality is good, be transferred to again in Calli Differentiation medium and cultivated 18～22d, by MS medium, the Calli Differentiation medium that the 6-BA of 0.5～2mg/L forms can improve differentiation rate greatly, when seedling grows to 2～3cm, seedling is accessed and in root media, cultivates 20～30d, the root media being formed by 1/2MS medium greatly the rooting rate of seedling and root system comparatively healthy and strong, by said method, set up efficient Annual Ryegrass Tissue Culture Regeneration System, for setting up the organizationally efficient genetic conversion system of Annual Ryegrass, lay the foundation, for seed selection Annual Ryegrass, organize improved seeds Technical Reference is provided.
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, by MS medium, 2 of 1～5mg/L, the callus inducing medium that the 6-BA of 4-D and 0.5～3mg/L forms can improve inductivity greatly, then callus is proceeded in callus subculture medium and cultivates 18～22d, by MS medium, 2 of 1～3mg/L, the callus subculture medium that the 6-BA of 4-D and 0～0.3mg/L forms can make callus breed the fastest and quality is good, be transferred to again in Calli Differentiation medium and cultivated 18～22d, by MS medium, the Calli Differentiation medium that the 6-BA of 0.5～2mg/L forms can improve differentiation rate greatly, when seedling grows to 2～3cm, seedling is accessed and in root media, cultivates 20～30d, the root media being formed by 1/2MS medium greatly the rooting rate of seedling and root system comparatively healthy and strong, by said method, set up efficient Annual Ryegrass Tissue Culture Regeneration System, for setting up the organizationally efficient genetic conversion system of Annual Ryegrass, lay the foundation, for seed selection Annual Ryegrass, organize improved seeds Technical Reference is provided.Concrete, the method for building up of Annual Ryegrass Tissue Culture Regeneration System 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, the 6-BA of 4-D and 0.5～3mg/L forms, described 2,4-D is 2,4-dichlorphenoxyacetic acid 2, the abbreviation of 4-dichlorophenoxy-acetic acid, described 6-BA is the abbreviation of 6-benzylaminopurine 6-Benzylaminopurine;
B, callus is proceeded in callus subculture medium and cultivates 18～22d, described 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 Calli Differentiation medium and cultivate 18～22d, and described Calli Differentiation medium is comprised of the 6-BA of MS medium, 0.5～2mg/L;
D, when seedling grows to 2～3cm, seedling is accessed in root media and cultivates 20～30d, described root media is comprised of 1/2MS medium;
E, after seedlings root grows, by its transplanting.
In the method for building up process of above-mentioned Annual Ryegrass Tissue Culture Regeneration System, in steps A, processing mode to Annual Ryegrass mature seed washing and sterilizing can adopt various ways, in order to make cleaning performance, it is best that bactericidal effect reaches, the present invention adopts mode 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, also blots with double-layer sterile filter paper for 3 times with aseptic water washing after sterilization.
The MS medium containing in described callus inducing medium, 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.
The callus of processing through callus subculture medium is transferred in different Calli Differentiation medium and cultivates 20d, the MS medium that its different Calli Differentiation medium contains is identical, the mass concentration of the 6-BA that it contains is as shown in table 3 below, then under 25 ± 2 ℃ of cultivation temperature, intensity of illumination 1000lx condition, cultivate 20d, add up green seedling differentiation rate, average bud is counted, its result is 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.
Callus is inoculated in after differential medium 3d, and some flaxen callus start to grow the tender green bud point of children; Some callus continued growths increase but do not occur green bud point, through 7d left and right, cultivate, and green bud point grows up to the seedling of many tiny upright or roomy bendings; There is differentiation in the callus that continued growth increases, as shown in Table 3, the 6-BA containing in described Calli Differentiation medium is 1mg/L, and it is the highest that differentiation rate reaches, and its average number reaches 66.18%, and average bud is counted also more, reaches 57.83.
When seedling grows to 2～3cm, seedling is accessed in root media, described root media is only comprised of 1/2MS medium, and described 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, glycine is 2.0mg/L, thiamine hydrochloride is 0.1mg/L, puridoxine hydrochloride is 0.5mg/L, nicotinic acid is 0.5mg/L, inositol is 100mg/L, sucrose 30g/L, agar 8g/L, pH value is 6.0, after seedling is accessed in above-mentioned root media, in cultivation temperature, it is 23 ± 2 ℃, intensity of illumination is 800～1000lx, light application time is 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, rooting rate is up to being 93.33%.
Claims (9)
1. a method for building up for Annual Ryegrass Tissue Culture Regeneration System, 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 callus subculture medium and cultivates 18～22d, described 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 Calli Differentiation medium and cultivate 18～22d, and described Calli Differentiation medium is comprised of the 6-BA of MS medium, 0.5～2mg/L;
D, when seedling grows to 2～3cm, seedling is accessed in root media and cultivates 20～30d, described root media is comprised of 1/2MS medium;
E, after seedlings root grows, by its transplanting.
2. the method for building up of Annual Ryegrass Tissue Culture Regeneration System 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. the method for building up of Annual Ryegrass Tissue Culture Regeneration System as claimed in claim 1, is characterized in that: the MS medium containing in described callus inducing medium, 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. the method for building up of Annual Ryegrass Tissue Culture Regeneration System 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. the method for building up of Annual Ryegrass Tissue Culture Regeneration System as claimed in claim 4, is characterized in that: in step B, in described callus subculture medium, contain 2,4-D is 2mg/L, the 6-BA containing is 0.2mg/L.
6. the method for building up of Annual Ryegrass Tissue Culture Regeneration System as claimed in claim 5, is characterized in that: in step C, the 6-BA containing in described Calli Differentiation medium is 1mg/L.
7. the method for building up of Annual Ryegrass Tissue Culture Regeneration System as claimed in claim 6, is characterized in that: in step C, described condition of culture is 25 ± 2 ℃ of cultivation temperature, intensity of illumination 1000lx.
8. the method for building up of Annual Ryegrass Tissue Culture Regeneration System as claimed in claim 7, 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.
9. the method for building up of Annual Ryegrass Tissue Culture Regeneration System as claimed in claim 8, 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.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105684894A (en) * | 2014-11-27 | 2016-06-22 | 北京林业大学 | An inducing method for a perennial lolium perenne callus |
| CN109234306A (en) * | 2018-09-25 | 2019-01-18 | 四川农业大学 | A kind of method for building up of diploid crowtoe transformation system |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105684894A (en) * | 2014-11-27 | 2016-06-22 | 北京林业大学 | An inducing method for a perennial lolium perenne callus |
| CN105684894B (en) * | 2014-11-27 | 2017-11-14 | 北京林业大学 | A kind of abductive approach of English ryegrass callus |
| CN109234306A (en) * | 2018-09-25 | 2019-01-18 | 四川农业大学 | A kind of method for building up of diploid crowtoe transformation system |
| CN113207691A (en) * | 2021-05-26 | 2021-08-06 | 四川农业大学 | Method for establishing seashore paspalum tissue culture regeneration system |
| CN117561978A (en) * | 2023-12-28 | 2024-02-20 | 中国农业大学 | Culture medium for improving oat tissue culture efficiency, preparation and application thereof |
| CN117561978B (en) * | 2023-12-28 | 2024-04-26 | 中国农业大学 | Culture medium for improving oat tissue culture efficiency, preparation and application thereof |
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