CN105660404A - Method for PEG-resisting alfalfa callus dedifferentiation - Google Patents

Method for PEG-resisting alfalfa callus dedifferentiation Download PDF

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CN105660404A
CN105660404A CN201610082224.0A CN201610082224A CN105660404A CN 105660404 A CN105660404 A CN 105660404A CN 201610082224 A CN201610082224 A CN 201610082224A CN 105660404 A CN105660404 A CN 105660404A
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alfalfa
peg
callus
broken
cultivate
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李波
贾秀峰
赵宇佳
徐婉玉
马赫
陈雪梅
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Qiqihar University
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/008—Methods for regeneration to complete plants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/06—Processes for producing mutations, e.g. treatment with chemicals or with radiation
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
    • A01H4/001—Culture apparatus for tissue culture

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  • Developmental Biology & Embryology (AREA)
  • Engineering & Computer Science (AREA)
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  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Abstract

The invention provides a method for PEG-resisting alfalfa callus dedifferentiation and belongs to the field of biotechnology breeding. The method solves the problems that alfalfa obtained through an existing conventional breeding method is poor in drought resistance and low in transplanting survival rate. The method for PEG-resisting alfalfa callus dedifferentiation comprises the steps that 1, alfalfa calluses subjected to NaN3 mutagenesis and PEG stress treatment are placed into an MS solid culture medium for subculture; 2, a bud differentiation MS culture medium containing NAA and 6-BA is inoculated with the blocky calluses, and the blocky calluses are cultured to obtain alfalfa tissue differentiating and sprouting; 3, the alfalfa tissue sprouting is placed into a rooting culture medium to be cultured to obtain alfalfa tissue differentiating and rooting; 4, alfalfa seedlings are domesticated and transplanted. The method for PEG-resisting alfalfa callus dedifferentiation is simple and easy to implement, the transplanting survival rate is 90% or above, and a drought-resisting breeding material is obtained by means of the method for callus dedifferentiation.

Description

A kind of method that anti-PEG alfalfa callus is broken up again
Technical field
The invention belongs to biotechnology breeding field, it is specifically related to a kind of method that anti-PEG alfalfa callus is broken up again.
Background technology
Alfalfa (MedicagosativaL.) is the good forage extensively distributing in the world and enjoying high reputation, it is called as " king of herbage ", soil can not only be improved, high-quality protein can also be increased, it is the basic substance developing high-quality and efficient livestock industry, also it is one of mankind food plant with health care good for health.
The development of China's alfalfa industry is seriously subject to the restriction of germ plasm resource, lack the seed adapting to different areas alfalfa variety, in order to meet the needs of modernization animal husbandry development, expand the cultivated area of clover, cultivate, breed the alfalfa variety adapting to Different habitats difference use object, become one of main problem of development China alfalfa industry.
Medicago in China breeding adopts the methods such as selection and use, cross-breeding, male sterile line breeding, biotechnology assistant breeding, space breeding usually. Various method all has application, and applying more is selection and use and cross-breeding. The New alfalfa cultivars that China has selected at present all have employed traditional breeding method and method (introducing a fine variety selection, evaluation and screening, cross-breeding). The kind of domestic incubation is all adopt traditional traditional breeding method and method to be bred as, what generally adopt is the breeding methods such as introduction and acclimatization, artificial selection, cross-breeding, selection by mutation, evaluation and screening, and the application of the cell engineering modern biotechnologies such as cell mutation, cytogamy, Hybrids embryo culture in herbage breeding is almost or blank. There is not been reported to utilize the kind that the biotechnologys such as modern plants tissue culture select. At present, there is no the kind that simple Applied Biotechnology is bred as in the middle of the kind of China's registration. More research concentrates on conversion and the genetic expression of resistant gene, to improve the resistance of clover. The alfalfa variety that seed selection adapts to the different ecological conditions such as arid, severe cold, desertification, salinization to land resources of preserving the ecological environment, make full use of, meet market to the demand of grass raising property livestock product and have important realistic meaning.
Summary of the invention
It is an object of the invention to solve the alfalfa drought-resistance that existing conventional breeding method obtains poor, the problem that transplanting survival rate is not high, and the method for alfalfa callus differentiation and bud formation and root again that arid has resistance is provided.
The method that the anti-PEG alfalfa callus of the present invention is broken up again follows these steps to realize:
One, will through NaN3Alfalfa callus (stem section is explant) after mutagenesis and PEG Stress treatment is inoculated in MS solid medium, cultivate at 23～25 DEG C, every sun exposure 12h, 1000Lx intensity of illumination, cultivation 18～22d (my god) complete 1 subculture, repeat succeeding transfer culture repeatedly, obtain the callus after succeeding transfer culture;
Two, the callus after succeeding transfer culture step one obtained is divided into bulk, then it is inoculated on the Bud polarization MS substratum containing NAA and 6-BA, it it is 23～25 DEG C in temperature, every sun exposure 16h, cultivate when 2000～4000Lx illuminance, obtain breaking up the alfalfa tissue sprouted;
Three, the alfalfa tissue that differentiation is sprouted is cut into two sections in budding place, moving after segmentation receives taking 1/2MS or MS as on the root media of minimum medium, it it is 23～25 DEG C in temperature, the illuminance of 1000～2000Lx, cultivate in culturing bottle when illumination 16h every day, obtain differentiating the aseptic seedling of root;
Four, when the aseptic seedling differentiating root grows to 2～3cm and has 4～5 sturdy root systems, in culturing room, cultivation bottleneck is opened, carry out the hardening of 2～5d, then selecting strain height is 4～5cm, and have the seedling replanting of 4～5 sturdy root systems to cultivate in nutrition soil, complete breaking up again of anti-PEG alfalfa callus;
Wherein Bud polarization MS substratum described in step 2 is MS+2.0mg/L6-BA (6-benzyl aminoadenine)+0.2mg/LNAA (naphthylacetic acid)+30g/L sucrose+10g/L agar;
When the root media described in step 3 is minimum medium taking 1/2MS, root media is 1/2MS+0.15mg/LNAA+30g/L sucrose+30g/L agar+0.5g/L gac or 1/2MS+0.05mg/LNAA+1.5mg/LIBA+30g/L sucrose+10g/L agar+0.5g/L gac;
When the root media described in step 3 is minimum medium taking MS, containing 0.05～0.10mg/LNAA, 1.0～1.5mg/LIBA (indolebutyric acid), 0～2mg/L6-BA, 30g/L sucrose, 30g/L agar, the gac of 0.5g/L and 15ml mother liquid of iron salt in root media, wherein said mother liquid of iron salt is by 2～3gFeSO4·7H2O and 3～4gNa2-EDTA (disodium ethylene diamine tetraacetate) is dissolved in and obtaining in 1L deionized water.
The anti-PEG alfalfa callus of the present invention breaks up that method is simple again, the callus used easily obtains embryo callus, easy seedling on the bud of screening and the division culture medium of root, transplanting survival rate height, not by the restriction in season, and the chemical mutagen used has the advantages such as efficient, nontoxic, cheap and use safety, obtain drought resisting breeding material by this method. Research and set up the callus induction of this kind of plant, differentiation culture and plant regeneration, has its realistic meaning to the acquisition setting up the perfect group training system of this kind of alfalfa plant and clover resistance breeding base mateiral.
Accompanying drawing explanation
Fig. 1 is the pictorial diagram that embodiment breaks up the clover seedling obtained again by callus.
Embodiment
Embodiment one: the method that the anti-PEG alfalfa callus of present embodiment is broken up again follows these steps to realize:
One, will through NaN3Alfalfa callus (stem section is explant) after mutagenesis and PEG Stress treatment is inoculated in MS solid medium, cultivate at 23～25 DEG C, every sun exposure 12h, 1000Lx intensity of illumination, cultivation 18～22d (my god) complete 1 subculture, repeat succeeding transfer culture repeatedly, obtain the callus after succeeding transfer culture;
Two, the callus after succeeding transfer culture step one obtained is divided into bulk, then it is inoculated on the Bud polarization MS substratum containing NAA and 6-BA, it it is 23～25 DEG C in temperature, every sun exposure 16h, cultivate when 2000～4000Lx illuminance, obtain breaking up the alfalfa tissue sprouted;
Three, the alfalfa tissue that differentiation is sprouted is cut into two sections in budding place, moving after segmentation receives taking 1/2MS or MS as on the root media of minimum medium, it it is 23～25 DEG C in temperature, the illuminance of 1000～2000Lx, cultivate in culturing bottle when illumination 16h every day, obtain differentiating the aseptic seedling of root;
Four, when the aseptic seedling differentiating root grows to 2～3cm and has 4～5 sturdy root systems, in culturing room, cultivation bottleneck is opened, carry out the hardening of 2～5d, then selecting strain height is 4～5cm, and have the seedling replanting of 4～5 sturdy root systems to cultivate in nutrition soil, complete breaking up again of anti-PEG alfalfa callus;
Wherein Bud polarization MS substratum described in step 2 is MS+2.0mg/L6-BA (6-benzyl aminoadenine)+0.2mg/LNAA (naphthylacetic acid)+30g/L sucrose+10g/L agar;
When the root media described in step 3 is minimum medium taking 1/2MS, root media is 1/2MS+0.15mg/LNAA+30g/L sucrose+30g/L agar+0.5g/L gac or 1/2MS+0.05mg/LNAA+1.5mg/LIBA+30g/L sucrose+10g/L agar+0.5g/L gac;
When the root media described in step 3 is minimum medium taking MS, containing 0.05～0.10mg/LNAA, 1.0～1.5mg/LIBA (indolebutyric acid), 0～2mg/L6-BA, 30g/L sucrose, 30g/L agar, the gac of 0.5g/L and 15ml mother liquid of iron salt in root media, wherein said mother liquid of iron salt is by 2～3gFeSO4·7H2O and 3～4gNa2-EDTA (disodium ethylene diamine tetraacetate) is dissolved in and obtaining in 1L deionized water.
Present embodiment step one is the succeeding transfer culture process of callus, and step 2 carries out the differentiation of callus bud, and step 3 carries out the differentiation of callus root, finally carries out domestication and the transplanting of seedling.
Present embodiment utilizes plant tissue culture technique that Herba Medicaginis stem section carries out the induction of callus, sodiumazide chemomorphosis and PEG-6000 simulation of infiltration drought stress, obtains the alfalfa callus of anti-PEG. The differentiation of callus refers to that callus is when ambient conditions meets, and Cell redifferentiation forms the meristematic tissue of bud and root, and develops into the process of whole plant by it. The differentiation of alfalfa callus is the step setting up clover regeneration system key. Callus seriously affects its ability broken up again after repeatedly succeeding transfer culture, mutagenic treatment, environment stress process, under suitable isolated culture condition, by the induction of exogenous hormone, alfalfa callus can be differentiated to form regrowth, designed by composite test, filter out suitable bud and the combination of Furcation defects exogenous hormone.
Embodiment two: present embodiment and embodiment one the difference is that described in step one through NaN3Alfalfa callus after mutagenesis and PEG Stress treatment follows these steps to realize: alfalfa callus is divided into bulk, and then proceeding to containing concentration is 200mg/LNaN3Liquid Induced medium in carry out shaking culture, the callus after mutagenesis is obtained after static 1h, the callus after mutagenesis is proceeded to the L-Hyp containing 8mmol/L (L-oxyproline) again and mass percentage be 30%PEG the MS substratum containing PEG in cultivate, the callus of survival proceeds in MS solid medium again and cultivates, and cultivates successively and carry out alternately screening on the MS substratum containing PEG and MS solid medium.Other step and parameter are identical with embodiment one.
Embodiment three: present embodiment and embodiment one or two the difference is that the MS solid medium described in step one be MS+0.5mg/L6-BA (6-benzylaminopurine)+2.0mg/L2,4-D (2,4-dichlorphenoxyacetic acid)+30g sucrose+0.8% agar, pH=5.8. Other step and parameter are identical with embodiment one or two.
The percentage composition of present embodiment agar refers to mass percentage.
Embodiment four: one of present embodiment and embodiment one to three are divided into 0.5cm the difference is that the callus after step 2 succeeding transfer culture3Bulk. Other step and one of parameter and embodiment one to three are identical.
Embodiment five: one of present embodiment and embodiment one to four are cultivated the difference is that the alfalfa tissue that differentiation step 2 obtained is sprouted proceeds in strong bud substratum again, strong bud substratum is: MS+2.0mg/L6-BA+30g/L sucrose+10g/L agar. Other step and one of parameter and embodiment one to four are identical.
Embodiment six: one of present embodiment and embodiment one to five the difference is that the mother liquid of iron salt described in step 3 be by 2.78gFeSO4·7H2O and 3.73gNa2-EDTA (disodium ethylene diamine tetraacetate) is dissolved in and obtaining in 1L deionized water. Other step and one of parameter and embodiment one to five are identical.
Embodiment seven: present embodiment and embodiment six the difference is that the root media of step 3 be MS+1.5mg/LIBA+0.05mg/LNAA+2.00mg/L6-BA+15ml mother liquid of iron salt+30g/L sucrose+10g/L agar+0.5g/L gac. Other step and parameter are identical with embodiment six.
Present embodiment is by increasing the content of molysite in root media, it is possible to increase the culture cycle of alfalfa tissue.
Embodiment eight: one of present embodiment and embodiment one to seven the difference is that step 3 be 23～25 DEG C in temperature, the high light illumination of 2000～4000Lx, alfalfa callus is made to turn green, it is beneficial to acquisition embryo callus, in culturing bottle, when illumination 16h every day, carries out cultivation 22～28d. Other step and one of parameter and embodiment one to seven are identical.
Embodiment nine: one of present embodiment and embodiment one to eight are cultivated when ambient moisture is 70%～80% the difference is that in step 4 seedling replanting to nutrition soil. Other step and one of parameter and embodiment one to eight are identical.
Embodiment one: the method that the anti-PEG alfalfa callus of the present embodiment is broken up again follows these steps to realize:
One, will through NaN3Alfalfa callus (stem section is explant) after mutagenesis and PEG Stress treatment is inoculated in MS solid medium, cultivate at 25 DEG C, every day, diffused light irradiated 12h, 1000Lx intensity of illumination cultivation 20d (my god) complete 1 subculture, repeat succeeding transfer culture 2 times, obtain the callus after succeeding transfer culture;
Two, the callus after succeeding transfer culture step one obtained is divided into 0.5cm3Block, then it is inoculated on Bud polarization MS substratum (see table 1) containing different ratios NAA and 6-BA, it it is 25 DEG C in temperature, every sun exposure 16h, cultivate when 3000Lx illuminance, obtaining breaking up the alfalfa tissue sprouted, and then move and receive in strong bud substratum and cultivate, strong bud substratum is: the agar of the sucrose+10g/L of the 6-BA+30g/L of MS+2.0mg/L;
Three, the alfalfa tissue that differentiation is sprouted is cut into two sections in budding place, two sections are moved respectively and receive on different root media (see table 2), it it is 25 DEG C in temperature, the illuminance of 1000Lx, cultivate in culturing bottle when illumination 16h every day, obtaining differentiating the aseptic seedling of root, originally root growth is less, it is necessary to switching 2-3 rear of taking second place just can grow abundant root;
Four, when the aseptic seedling differentiating root grows to 2～3cm and has 4～5 sturdy root systems, in culturing room, cultivation bottleneck is opened, carry out the hardening of 2～5d, then selecting strain height is 4～5cm, and have the test-tube seedling transplanting of 4～5 sturdy root systems to cultivate in nutrition soil, complete breaking up again of anti-PEG alfalfa callus.
First the present embodiment chooses the full Medicago sativa CV.Aohan seed of seed, through 0.1% mercuric chloride solution sterilization 5min, with after aseptic water washing 3～4 times, alfalfa seed being placed in the fixing substratum of MS, namely aseptic seedlings is obtained after the incubator of 23 ± 2 DEG C is cultivated one week, then aseptic seedlings is taken out from substratum, it is placed on aseptic filter paper, it is cut into the stem section of 1cm, it is inoculated in MS solid medium (MS+0.5mg/L6-BA+2.0mg/L2, 4-D+30g sucrose+0.8% agar, pH=5.8) in, cultivate at 25 DEG C, diffused light irradiates 12h every day, choose and grown 20d white, crisp callus. through NaN in step one3Alfalfa callus after mutagenesis and PEG Stress treatment is that alfalfa callus is divided into 0.5cm3Uniform blocks, proceed to NaN3In the liquid nutrient medium of 200mg/L, 10 pieces every bottle, 70r/min shaking culture 1h, after static 1h, the callus of survival is carried out succeeding transfer culture, callus after mutagenesis proceeds to the MS substratum containing L-Hyp8mmol/L and 30%PEG (PEG-6000) (the same inducing culture of other composition) and carries out alternately screening, culture condition is the same, the callus enlarged culturing of survival.
Different 6-BA and the NAA proportioning Bud polarization of table 1
Determine that the best substratum of sprouting is: MS+2.0mg/L6-BA+0.2mg/LNAA+30g/L sucrose+10g/L agar by table 1. If when the quantity of required bud is very big, it is necessary to enlarged culturing, can being cut by bud at bud bifurcated place, move to receive and continue on the suitableeest strong bud substratum to cultivate, period notes replaced medium, otherwise bud may because nutritive ingredient exhausts Necrosis.
After the present embodiment step 2 callus cultivates for some time on substratum is sprouted in induction, it is possible to find that the callus of original yellow-green colour turns green gradually, the substratum that continuation cultivation for some time can find that there is grows budlet. Move in termination process with a small amount of callus at bud, have the callus do not sprouted can continue shifting and receive substratum of sprouting and continue induction and sprout. Step 3 adds gac in the medium in rooting process, thus manufactures dark environment for taking root.
The present embodiment step 3 bud is transplanted to after cultivating for some time in root media, observes the root finding that there is white and bears, and originally root growth is less, it is necessary to switching 2-3 rear of taking second place just can grow abundant root, and situation about taking root under substratum different ratio is in table 2. Root media: MS+1.5mg/LIBA+0.05mg/LNAA+2.00mg/L6-BA+15mL mother liquid of iron salt+30g/L sucrose+10g/L agar+0.5g/L gac is the suitableeest root media, and wherein mother liquid of iron salt is by 2.78gFeSO4·7H2O and 3.73gNa2-EDTA (disodium ethylene diamine tetraacetate) is dissolved in and obtaining in 1L deionized water. The seedling turned out not only root length, many, strong, and culture cycle is long. The present embodiment seedling replanting surviving rate can reach more than 90%.
The substratum of table 2 different ratio hormone is taken root situation
The active significant difference before and after mutagenesis of the proline content of the alfalfa-leaves of following table display mutagenesis and non-mutagenesis, soluble sugar content, POD, after mutagenesis, proline content, soluble sugar content, POD activity obviously increase, and show the enhancing of the clover seedling drought-resistant ability of differentiation.
Table 3 alfalfa-leaves proline content, soluble sugar content and POD are active

Claims (9)

1. the method that an anti-PEG alfalfa callus is broken up again, it is characterised in that be realize through the following steps:
One, will through NaN3Alfalfa callus after mutagenesis and PEG Stress treatment is inoculated in MS solid medium, cultivates, every sun exposure 12h at 23～25 DEG C, 1000Lx intensity of illumination, cultivate 18～22d and complete 1 subculture, repeat succeeding transfer culture repeatedly, obtain the callus after succeeding transfer culture;
Two, the callus after succeeding transfer culture step one obtained is divided into bulk, then it is inoculated on the Bud polarization MS substratum containing NAA and 6-BA, it it is 23～25 DEG C in temperature, every sun exposure 16h, cultivate when 2000～4000Lx illuminance, obtain breaking up the alfalfa tissue sprouted;
Three, the alfalfa tissue that differentiation is sprouted is cut into two sections in budding place, moving after segmentation receives taking 1/2MS or MS as on the root media of minimum medium, it it is 23～25 DEG C in temperature, the illuminance of 1000～2000Lx, cultivate in culturing bottle when illumination 16h every day, obtain differentiating the aseptic seedling of root;
Four, when the aseptic seedling differentiating root grows to 2～3cm and has 4～5 sturdy root systems, in culturing room, cultivation bottleneck is opened, carry out the hardening of 2～5d, then selecting strain height is 4～5cm, and have the seedling replanting of 4～5 sturdy root systems to cultivate in nutrition soil, complete breaking up again of anti-PEG alfalfa callus;
Wherein Bud polarization MS substratum described in step 2 is MS+2.0mg/L6-BA+0.2mg/LNAA+30g/L sucrose+10g/L agar;
When the root media described in step 3 is minimum medium taking 1/2MS, root media is 1/2MS+0.15mg/LNAA+30g/L sucrose+30g/L agar+0.5g/L gac or 1/2MS+0.05mg/LNAA+1.5mg/LIBA+30g/L sucrose+10g/L agar+0.5g/L gac;
When the root media described in step 3 is minimum medium taking MS, containing 0.05～0.10mg/LNAA, 1.0～1.5mg/LIBA, 0～2mg/L6-BA, 30g/L sucrose, 30g/L agar, the gac of 0.5g/L and 15ml mother liquid of iron salt in root media, wherein said mother liquid of iron salt is by 2～3gFeSO4·7H2O and 3～4gNa2-EDTA is dissolved in and obtaining in 1L deionized water.
2. the method that a kind of anti-PEG alfalfa callus according to claim 1 is broken up again, it is characterised in that described in step one through NaN3Alfalfa callus after mutagenesis and PEG Stress treatment follows these steps to realize: alfalfa callus is divided into bulk, and then proceeding to containing concentration is 200mg/LNaN3Liquid Induced medium in carry out shaking culture, the callus after mutagenesis is obtained after static 1h, the callus after mutagenesis is proceeded to the L-Hyp containing 8mmol/L again and mass percentage be 30%PEG the MS substratum containing PEG in cultivate, the callus of survival proceeds in MS solid medium again and cultivates, and cultivates successively and carry out alternately screening on the MS substratum containing PEG and MS solid medium.
3. the method that a kind of anti-PEG alfalfa callus according to claim 1 is broken up again, it is characterised in that the MS solid medium described in step one is MS+0.5mg/L6-BA+2.0mg/L2,4-D+30g sucrose+0.8% agar, pH=5.8.
4. the method that a kind of anti-PEG alfalfa callus according to claim 1 is broken up again, it is characterised in that the callus after step 2 succeeding transfer culture is divided into 0.5cm3Bulk.
5. the method that a kind of anti-PEG alfalfa callus according to claim 1 is broken up again, it is characterized in that the alfalfa tissue that differentiation step 2 obtained is sprouted proceeds in strong bud substratum again to cultivate, strong bud substratum is: MS+2.0mg/L6-BA+30g/L sucrose+10g/L agar.
6. the method that a kind of anti-PEG alfalfa callus according to claim 1 is broken up again, it is characterised in that the mother liquid of iron salt described in step 3 is by 2.78gFeSO4·7H2O and 3.73gNa2-EDTA is dissolved in and obtaining in 1L deionized water.
7. the method that a kind of anti-PEG alfalfa callus according to claim 6 is broken up again, it is characterised in that the root media of step 3 is MS+1.5mg/LIBA+0.05mg/LNAA+2.00mg/L6-BA+15ml mother liquid of iron salt+30g/L sucrose+10g/L agar+0.5g/L gac.
8. the method that a kind of anti-PEG alfalfa callus according to claim 1 is broken up again, it is characterised in that step 3 is 23～25 DEG C in temperature, the high light illumination of 2000～4000Lx, carries out cultivation 22～28d in culturing bottle when illumination 16h every day.
9. the method that a kind of anti-PEG alfalfa callus according to claim 1 is broken up again, it is characterised in that cultivate when ambient moisture is 70%～80% in step 4 seedling replanting to nutrition soil.
CN201610082224.0A 2016-02-05 2016-02-05 Method for PEG-resisting alfalfa callus dedifferentiation Pending CN105660404A (en)

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CN110786236A (en) * 2019-05-09 2020-02-14 山东农业大学 Method for improving drought resistance of chrysanthemum by PEG (polyethylene glycol) in-vitro culture
CN110663551A (en) * 2019-11-05 2020-01-10 天津农学院 Method for cultivating stress-resistant novel cauliflower variety
CN113349057A (en) * 2021-07-12 2021-09-07 锡林郭勒职业学院 Method for improving salt-tolerant growth capacity of forage grass seedlings in saline-alkali soil
CN113349057B (en) * 2021-07-12 2022-11-29 锡林郭勒职业学院 Method for improving salt-tolerant growth capacity of forage grass seedlings in saline-alkali soil

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