CN102138533A - Method for preparing medicago falcata regenerated plant through tissue culture and culture medium - Google Patents
Method for preparing medicago falcata regenerated plant through tissue culture and culture medium Download PDFInfo
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Abstract
The invention discloses a method for preparing medicago falcata regenerated plant through tissue culture and a culture medium. The culture medium for obtaining a medicago falcata callus consists of the following nutrient components in parts by weight: 2830 parts of potassium nitrate by major elements, 1 part of manganese sulfate by trace elements, 6.97 parts of ethylenediaminetetraacetic acid by ferric salts, 10 parts of nicotinic acid by organic components, 100 parts of inositol, 1 to 2 parts of 2,4-dichlorphenoxyacetic acid, and 1 to 2 parts of N6-furfuryladenine. The invention provides a convenient, quick and efficient method for medicago falcata breeding, and the method provided by the invention has certain social benefit and economical benefit, and contributes to the sustainable development of grassland industry in China.
Description
Technical field
The present invention relates to obtain the method and the medium thereof of sickle alfalfa regeneration plant by tissue culture.
Background technology
Sickle alfalfa (Medicago falcata L.) is a perennial wild leguminous forage very important in the pulse family Medicago, mainly is distributed in countries such as China, Mongolia, Russia, Pakistan, India, Turkey.The sickle alfalfa variety source of China is abundant, mainly is distributed in northeast, North China, the Northwest of China.With other kind comparison, sickle alfalfa has outstanding features such as strong stress resistance, cold-resistant, drought-enduring, barren-resistant and life-span be long, suit in arid, cold area plantation, and be the first-selection that northern China is built the high yield and high quality sown pasture.
Times type of sickle alfalfa is seen dliploid (2n=2x=16) and 2 ploidy levels of tetraploid (2n=4x=32) more.In general, tetraploid is compared with dliploid, and it is tall and big to have a plant, grow prolifically, features such as blade increase.Ploidy affects inside plants expression of gene and regulation and control, and then has influence on form and the function of plant.
The group of sickle alfalfa training for many years is very difficult, causes molecular biology and the genetics improvement work stagnation of sickle alfalfa.The key of biological breeding is to set up a tissue culture regenerating system efficiently, with the culture systems of recipient cell behind genetic recipient system that the improvement target is provided and the genetic transformation.
The training of the group of sickle alfalfa begins one's study early, and Daniel Brown in 1985 and Atanas Atanassov have tested cotyledon and the hypocotyl of 67 kinds of clovers, do tissue culture with three-step approach, select the kind that some have high regeneration capacity.Tony H.H.Chen has tested 17 kinds of sickle alfalfas in 1987, also the same with the cotyledon hypocotyl formation callus of having the ability of blade is described.Gilmour has carried out the tissue culture regeneration system in 1987 with the protoplast cultivation of sickle alfalfa.Domestic Ma Haiyan was cultivated with the hypocotyl of Xinjiang Wild sickle alfalfa in 2005, but differentiation rate lower (20-30%).
Summary of the invention
An object of the present invention is to provide a kind of medium that obtains the sickle alfalfa callus.
The medium of acquisition sickle alfalfa callus provided by the present invention, form by the nutrient component of following mass parts:
Macroelement in 2830 parts in potassium nitrate, trace element in 1 part of manganese sulphate, molysite in 6.97 parts of sodium ethylene diamine tetracetates, organic principle in 1 part-2 parts of 10 parts of niacins, 100 parts of inositols, 2,4 dichlorophenoxyacetic acid or 1 part or 2 parts, 1 part-2 parts or 1 part or 2 parts of N6-furfuryladenines;
Described macroelement is made up of following materials in parts by mass:
166 parts of 2830 parts in potassium nitrate, 463 parts in ammonium sulfate, 400 parts of potassium dihydrogen phosphates, 185 parts of epsom salts and calcium chloride dihydrates;
Described trace element is made up of following materials in parts by mass:
0.01 part of 1 part of manganese sulphate, 0.5 part of boric acid, 0.1 part of white vitriol, 0.1 part of potassium iodide, 0.01 part of Sodium Molybdate Dihydrate, 0.02 part of cupric sulfate pentahydrate and CoCL2;
Described molysite is made up of following materials in parts by mass:
6.97 parts of sodium ethylene diamine tetracetates;
Described organic principle is made up of following materials in parts by mass:
10 parts of 10 parts of niacins, 10 parts of thiamine hydrochlorides and puridoxine hydrochlorides.
Described medium is made up of described nutrient component and water;
The equal independent packaging of macroelement, trace element, molysite, organic principle, inositol, 2,4 dichlorophenoxyacetic acid and N6-furfuryladenine described in the described nutrient component.
Described medium is grouped into by following one-tenth:
Potassium nitrate, ammonium sulfate, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, manganese sulphate, boric acid, white vitriol, potassium iodide, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2, sodium ethylene diamine tetracetate, niacin, thiamine hydrochloride, puridoxine hydrochloride, inositol, 2,4 dichlorophenoxyacetic acid and N6-furfuryladenine and water;
Above composition concentration in described medium is respectively:
Potassium nitrate 2.830g/L, ammonium sulfate 0.463g/L, potassium dihydrogen phosphate 0.400g/L, epsom salt 0.185g/L, calcium chloride dihydrate 0.166g/L, manganese sulphate 1mg/L, boric acid 0.5mg/L, white vitriol 0.1mg/L, potassium iodide 0.1mg/L, Sodium Molybdate Dihydrate 0.01mg/L, cupric sulfate pentahydrate 0.02mg/L, CoCL2 0.01mg/L, sodium ethylene diamine tetracetate 6.97mg/L, niacin 10mg/L, thiamine hydrochloride 10mg/L, puridoxine hydrochloride 10mg/L, inositol 100mg/L, 2,4-dichlorphenoxyacetic acid 1mg/L-2mg/L and N6-furfuryladenine 1mg/L-2mg/L, all the other are water.
Another object of the present invention provides a kind of solid culture medium that obtains the sickle alfalfa callus, is made into solid culture medium by the medium of coagulating agent and above-mentioned acquisition sickle alfalfa callus.
The consumption of described coagulating agent makes the medium of above-mentioned acquisition sickle alfalfa callus form solid and gets final product;
The concentration of described coagulating agent in described solid culture medium is 8g/L;
Described coagulating agent is an agar.
Described solid culture medium is following 1) or 2) shown in:
1) concentration of 2,4 dichlorophenoxyacetic acid is that the concentration of 2mg/L and N6-furfuryladenine is 1mg/L in the described solid culture medium;
2) concentration of 2,4 dichlorophenoxyacetic acid is that the concentration of 1mg/L and N6-furfuryladenine is 2mg/L in the described solid culture medium.
The pH value of described medium is 5.8;
Described sickle alfalfa is tetraploid sickle alfalfa or dliploid sickle alfalfa.
Another purpose of the present invention provides a kind of method that obtains the sickle alfalfa callus.
The method of acquisition provided by the present invention sickle alfalfa callus comprises the steps: with above-mentioned 1) shown in medium inducing culture sickle alfalfa explant, obtain the sickle alfalfa callus.
Described method also comprises with above-mentioned 2) shown in the step of medium enrichment culture sickle alfalfa callus;
Described sickle alfalfa explant is the young leaflet tablet that is cut into piece;
Described sickle alfalfa is tetraploid sickle alfalfa or dliploid sickle alfalfa.
Another purpose of the present invention provides a kind of method that obtains the sickle alfalfa regeneration plant.
The method of acquisition provided by the present invention sickle alfalfa regeneration plant may further comprise the steps: the sickle alfalfa callus that said method is obtained is inoculated into and carries out differentiation culture on the differential medium and obtain bud, promptly obtains the sickle alfalfa regeneration plant.
Described differential medium is grouped into by following one-tenth:
Potassium nitrate, ammonium sulfate, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, manganese sulphate, boric acid, white vitriol, potassium iodide, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2, sodium ethylene diamine tetracetate, niacin, thiamine hydrochloride, puridoxine hydrochloride, inositol, N6-furfuryladenine and water;
Above composition concentration in described differential medium is respectively:
Potassium nitrate 2.830g/L, ammonium sulfate 0.463g/L, potassium dihydrogen phosphate 0.400g/L, epsom salt 0.185g/L, calcium chloride dihydrate 0.166g/L, manganese sulphate 1mg/L, boric acid 0.5mg/L, white vitriol 0.1mg/L, potassium iodide 0.1mg/L, Sodium Molybdate Dihydrate 0.01mg/L, cupric sulfate pentahydrate 0.02mg/L, CoCL2 0.01mg/L, sodium ethylene diamine tetracetate 6.97mg/L, niacin 10mg/L, thiamine hydrochloride 10mg/L, puridoxine hydrochloride 10mg/L, inositol 100mg/L and N6-furfuryladenine 0.4mg/L, all the other are water.
The pH value of described differential medium is 5.8;
Described method comprises that also the bud that will obtain is inoculated into the step of cultivating on the root media after described differentiation culture;
Described root media is grouped into by following one-tenth:
Potassium nitrate, ammonium sulfate, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, manganese sulphate, boric acid, white vitriol, potassium iodide, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2, sodium ethylene diamine tetracetate, niacin, thiamine hydrochloride, puridoxine hydrochloride and water;
Above composition concentration in described root media is respectively:
Potassium nitrate 1.415g/L, ammonium sulfate 0.2315g/L, potassium dihydrogen phosphate 0.200g/L, epsom salt 0.0925g/L, calcium chloride dihydrate 0.083g/L, manganese sulphate 1mg/L, boric acid 0.5mg/L, white vitriol 0.1mg/L, potassium iodide 0.1mg/L, Sodium Molybdate Dihydrate 0.01mg/L, cupric sulfate pentahydrate 0.02mg/L, CoCL2 0.01mg/L, sodium ethylene diamine tetracetate 6.97mg/L, niacin 10mg/L, thiamine hydrochloride 10mg/L, puridoxine hydrochloride 10mg/L, all the other are water.
The pH value of described root media is 5.8;
Described sickle alfalfa explant is the young leaflet tablet that is cut into piece;
Described sickle alfalfa is tetraploid sickle alfalfa or dliploid sickle alfalfa.
The present invention carries out the tissue culture experiment from a collection of tetraploid sickle alfalfa that comes from Xinjiang with coming from the Muscovite dliploid sickle alfalfa, therefrom filter out the kind of group training the most efficiently, and set up one and overlap regenerating system efficiently, callus induction rate reaches more than 90%, differentiation rate reaches 60%-70%, planting percent reaches 40%-50%, finishing whole regenerating systems only needed about 10 weeks, can utilize biotechnology such as transgenosis efficiently, apace crop varieties and proterties are carried out genetic improvement subsequently.Because the sickle alfalfa of China mainly is grown in the Inner Mongol and Xinjiang, these places relatively arid are cold, the improvement of sickle alfalfa being carried out cold-resistant drought resisting is very necessary, the present invention provides a kind of convenient, fast and effective method for the sickle alfalfa breeding, have certain social benefit and economic benefit, the sustainable development of China's grass cultivation is had certain contribution.
Description of drawings
Fig. 1 is the growth conditions of callus on inducing culture.
Fig. 2 is callus organizator blast and have blastogenesis to become in proliferated culture medium.
Fig. 3 is callus regeneration bud continued growth and have root to generate on differential medium.
Fig. 4 is that embryo callus grows up to regrowth in root media.
Fig. 5 grows for the sickle alfalfa regrowth moves on in the flowerpot.
Embodiment
Employed experimental technique is conventional method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
Mineral salt are available from Chemical Reagent Co., Ltd., Sinopharm Group and Beijing chemical reagents corporation; 2,4 dichlorophenoxyacetic acid (2,4-D), N6-furfuryladenine (KT) and vitamin is available from SIGMA company; Inositol is available from Chemical Reagent Co., Ltd., Sinopharm Group; Sucrose is available from Beijing northization fine chemicals Co., Ltd, and agar is available from the safe scientific and technical innovation centre of development that shakes, Beijing.
Embodiment 1, obtain tetraploid sickle alfalfa regeneration plant by tissue culture
(public can obtain from China Agricultural University the wild sickle alfalfa of this experiment Xinjiang tetraploid, the non-patent literature of putting down in writing this material is: Wang Hongmei, Wang person of outstanding talent etc., the comparative studies of the wild sickle alfalfa morphological feature of dliploid and tetraploid, the meadow journal, Vol.17 No.4, Jul 2009) be material, pick up from the Ili Prefecture, Xinjiang.
Experimental procedure is as follows:
1, the cultivation of seed treatment and aseptic seedling
Choose 80 in the Xinjiang Wild sickle alfalfa seed of full seed, handled about 8 minutes with the concentrated sulfuric acid, treat to have on kind of the skin stain to occur, the sucking-off concentrated sulfuric acid is with aseptic washing 5 times.Then in super-clean bench with soaking in 5% aqueous sodium hypochlorite solution 15 minutes, use aseptic washing 5 times again; Then it is inoculated on 1/2 MS medium (operation in the super-clean bench).Place group training chamber, condition of culture is: cultivation temperature is room temperature (25 ℃); Photoperiod is illumination 16 hours/dark 8 hours, and intensity of illumination is 5000Lux.
2, the selection of explant
The young leaflet tablet of choosing the sickle alfalfa aseptic seedling about 3 weeks is as explant.
3, callus induces and keeps
In super-clean bench, will be cut into about 3mm with the blade of the bacterium of going out as the young leaflet tablet of the sickle alfalfa aseptic seedling of explant
2Fritter, be inoculated on the callus of induce medium and cultivate.About two all callus generate (Fig. 1), callus is transferred on the callus proliferated culture medium cultivates.Callus of induce medium and callus proliferated culture medium all are transferred to 5.8 with pH before sterilization, sterilising conditions be 121 ℃ following 20 minutes.Condition of culture is identical with step 1.
Three repetitions are established in experiment, results averaged, and each repeats to establish 50 explants.
Callus induction rate (%)=reality forms callus number/inoculation explant number * 100%.
The result: callus induction rate is 90.7%.
The callus of induce medium is grouped into by following one-tenth:
Potassium nitrate, ammonium sulfate, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, manganese sulphate, boric acid, white vitriol, potassium iodide, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2, sodium ethylene diamine tetracetate, niacin, thiamine hydrochloride, puridoxine hydrochloride, inositol, 2,4 dichlorophenoxyacetic acid and N6-furfuryladenine and water;
Above composition concentration in described callus of induce medium is respectively:
Potassium nitrate 2.830g/L, ammonium sulfate 0.463g/L, potassium dihydrogen phosphate 0.400g/L, epsom salt 0.185g/L, calcium chloride dihydrate 0.166g/L, manganese sulphate 1mg/L, boric acid 0.5mg/L, white vitriol 0.1mg/L, potassium iodide 0.1mg/L, Sodium Molybdate Dihydrate: 0.01mg/L, cupric sulfate pentahydrate 0.02mg/L, CoCL2 0.01mg/L, sodium ethylene diamine tetracetate 6.97mg/L, niacin 10mg/L, thiamine hydrochloride 10mg/L, puridoxine hydrochloride 10mg/L, inositol 100mg/L, 2,4-dichlorphenoxyacetic acid 2mg/L and N6-furfuryladenine 1mg/L, all the other are water.
The pH value of callus of induce medium is 5.8.
The callus proliferated culture medium is grouped into by following one-tenth:
Potassium nitrate, ammonium sulfate, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, manganese sulphate, boric acid, white vitriol, potassium iodide, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2, sodium ethylene diamine tetracetate, niacin, thiamine hydrochloride, puridoxine hydrochloride, inositol, 2,4 dichlorophenoxyacetic acid and N6-furfuryladenine and water;
Above composition concentration in described callus proliferated culture medium is respectively:
Potassium nitrate 2.830g/L, ammonium sulfate 0.463g/L, potassium dihydrogen phosphate 0.400g/L, epsom salt 0.185g/L, calcium chloride dihydrate 0.166g/L, manganese sulphate 1mg/L, boric acid 0.5mg/L, white vitriol 0.1mg/L, potassium iodide 0.1mg/L, Sodium Molybdate Dihydrate: 0.01mg/L, cupric sulfate pentahydrate 0.02mg/L, CoCL2 0.01mg/L, sodium ethylene diamine tetracetate 6.97mg/L, niacin 10mg/L, thiamine hydrochloride 10mg/L, puridoxine hydrochloride 10mg/L, inositol 100mg/L, 2,4-dichlorphenoxyacetic acid 1mg/L and N6-furfuryladenine 2mg/L, all the other are water.
The pH value of callus proliferated culture medium is 5.8.
4, somatic embryo induces and breaks up
Through a successive transfer culture, through the growth in 3 weeks, embryo callus subculture (Fig. 2) appears in some callus again; The callus that embryo callus subculture occurs is divided into 3mm
2Fritter, be transferred on the differential medium and induce, condition of culture is identical with above-mentioned steps 1; After about 20 days, the somatic embryo differentiation is sprouted and root (Fig. 3).
Three repetitions are established in experiment, results averaged, and each repeats to establish 30 embryo callus subcultures.
The callus of callus differentiation rate (%)=differentiation and bud formation/inoculation callus number * 100%.
The result: the callus differentiation rate is 68.9%.
Differential medium is grouped into by following one-tenth:
Potassium nitrate, ammonium sulfate, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, manganese sulphate, boric acid, white vitriol, potassium iodide, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2, sodium ethylene diamine tetracetate, niacin, thiamine hydrochloride, puridoxine hydrochloride, inositol, N6-furfuryladenine and water;
Above composition concentration in described differential medium is respectively:
Potassium nitrate 2.830g/L, ammonium sulfate 0.463g/L, potassium dihydrogen phosphate 0.400g/L, epsom salt 0.185g/L, calcium chloride dihydrate 0.166g/L, manganese sulphate 1mg/L, boric acid 0.5mg/L, white vitriol 0.1mg/L, potassium iodide 0.1mg/L, Sodium Molybdate Dihydrate: 0.01mg/L, cupric sulfate pentahydrate 0.02mg/L, CoCL2 0.01mg/L, sodium ethylene diamine tetracetate 6.97mg/L, niacin 10mg/L, thiamine hydrochloride 10mg/L, puridoxine hydrochloride 10mg/L, inositol 100mg/L and N6-furfuryladenine 0.4mg/L, all the other are water;
The pH value of described differential medium is 5.8.
5, regeneration bud takes root and transplants
The sickle alfalfa regeneration bud generally all has the formation of root in differentiation.It is forwarded to during to about 1cm and cultivate root on the root media (1/2 SH medium) and grow when root is long, can carry out the transplanting of regrowth (Fig. 4) at this moment to about 5cm.The method that regrowth is transplanted is: distilled water is added to did not just have medium, sealing the film half opening, to place group training chamber condition (be that cultivation temperature is room temperature (25 ℃); Photoperiod is illumination 16 hours/dark 8 hours; The intensity of illumination on culture surface is 5000Lux) 3 days, standard-sized sheet seals film placement 3 days then, under tap, medium is rinsed well carefully again, regrowth is transplanted under similarity condition, cultivates (Fig. 5) in the flowerpot then, finish the process of whole sickle alfalfa regeneration.
Three repetitions are established in experiment, results averaged, and each repeats to establish 50 regeneration buds.
Planting percent (%)=grow up to regeneration bud number * 100% of the bud/inoculation of regrowth.
The result: planting percent is 46.7%.
Described root media is grouped into by following one-tenth:
Potassium nitrate, ammonium sulfate, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, manganese sulphate, boric acid, white vitriol, potassium iodide, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2, sodium ethylene diamine tetracetate, niacin, thiamine hydrochloride, puridoxine hydrochloride;
Above composition concentration in described root media is respectively:
Potassium nitrate 1.415g/L, ammonium sulfate 0.2315g/L, potassium dihydrogen phosphate 0.200g/L, epsom salt 0.0925g/L, calcium chloride dihydrate 0.083g/L, manganese sulphate 1mg/L, boric acid 0.5mg/L, white vitriol 0.1mg/L, potassium iodide 0.1mg/L, Sodium Molybdate Dihydrate: 0.01mg/L, cupric sulfate pentahydrate 0.02mg/L, CoCL2 0.01mg/L, sodium ethylene diamine tetracetate 6.97mg/L, niacin 10mg/L, thiamine hydrochloride 10mg/L, puridoxine hydrochloride 10mg/L, all the other are water.
The pH value of described root media is 5.8.
Embodiment 2, obtain dliploid Russia sickle alfalfa regeneration plant by tissue culture
Dliploid Russia sickle alfalfa PI502449 derives from U.S. USDA-ARS Plant GermplasmQuarantine Center.
1, the cultivation of seed treatment and aseptic seedling
Choose 80 in the Russian sickle alfalfa PI502449 seed of full seed, handled about 8 minutes with the concentrated sulfuric acid, treat to have on kind of the skin stain to occur, the sucking-off concentrated sulfuric acid is with aseptic washing 5 times.Then in super-clean bench with soaking in 5% aqueous sodium hypochlorite solution 15 minutes, use aseptic washing 5 times again; Then it is inoculated on 1/2 MS medium (operation in the super-clean bench).Place group training chamber, condition of culture is: cultivation temperature is room temperature (25 ℃); Photoperiod is illumination 16 hours/dark 8 hours, and intensity of illumination is 5000Lux.
2, the selection of explant
The young leaflet tablet of choosing the sickle alfalfa aseptic seedling about 3 weeks is as explant.
3, callus induces and keeps
In super-clean bench, will be cut into the fritter of about 3mm2 with the blade of the bacterium of going out as the young leaflet tablet of the sickle alfalfa aseptic seedling of explant, be inoculated on the callus of induce medium and cultivate.About two all callus generate, and callus is transferred on the callus proliferated culture medium cultivates.Callus of induce medium and callus proliferated culture medium all are transferred to 5.8 with pH before sterilization, sterilising conditions be 121 ℃ following 20 minutes.Condition of culture is identical with step 1.
Three repetitions are established in experiment, results averaged, and each repeats to establish 50 explants.
Callus induction rate (%)=reality forms callus number/inoculation explant number * 100%.
The result: callus induction rate is 89.3%.
Identical among the composition of callus of induce medium and the concentration of each composition and the embodiment 1; Identical among the pH value of callus of induce medium and the embodiment 1.
Identical among the composition of callus proliferated culture medium and the concentration of each composition and the embodiment 1; Identical among the pH value of callus proliferated culture medium and the embodiment 1.
4, somatic embryo induces and breaks up
Through a successive transfer culture, through the growth in 3 weeks, embryo callus subculture appears in some callus again; The callus that embryo callus subculture occurs is divided into 3mm
2Fritter, be transferred on the differential medium and induce, condition of culture is identical with above-mentioned steps 2; After about 20 days, the somatic embryo differentiation is sprouted and root.
Three repetitions are established in experiment, results averaged, and each repeats to establish 50 embryo callus subcultures.
The callus of callus differentiation rate (%)=differentiation and bud formation/inoculation callus number * 100%.
The result: the callus differentiation rate is 37.8%.
Identical among the composition of differential medium and the concentration of each composition and the embodiment 1; Identical among the pH value of differential medium and the embodiment 1.
5, regeneration bud takes root and transplants
The sickle alfalfa regeneration bud generally all has the formation of root in differentiation.It is forwarded to during to about 1cm and cultivate root on the root media (1/2 SH medium) and grow when root is long, can carry out the transplanting of regrowth at this moment to about 5cm.The method that regrowth is transplanted is: with identical among the embodiment 1.
Three repetitions are established in experiment, results averaged, and each repeats to establish 30 regeneration buds.
Planting percent (%)=grow up to regeneration bud number * 100% of the bud/inoculation of regrowth.
The result: planting percent is 21.1%.
Identical among the composition of root media and the concentration of each composition and the embodiment 1; Identical among the pH value of root media and the embodiment 1.
Claims (10)
1. medium that obtains the sickle alfalfa callus, form by the nutrient component of following mass parts:
Macroelement in 2830 parts in potassium nitrate, trace element in 1 part of manganese sulphate, molysite in 6.97 parts of sodium ethylene diamine tetracetates, organic principle in 1 part-2 parts of 10 parts of niacins, 100 parts of inositols, 2,4 dichlorophenoxyacetic acid or 1 part or 2 parts, 1 part-2 parts or 1 part or 2 parts of N6-furfuryladenines;
Described macroelement is made up of following materials in parts by mass:
166 parts of 2830 parts in potassium nitrate, 463 parts in ammonium sulfate, 400 parts of potassium dihydrogen phosphates, 185 parts of epsom salts and calcium chloride dihydrates;
Described trace element is made up of following materials in parts by mass:
0.01 part of 1 part of manganese sulphate, 0.5 part of boric acid, 0.1 part of white vitriol, 0.1 part of potassium iodide, 0.01 part of Sodium Molybdate Dihydrate, 0.02 part of cupric sulfate pentahydrate and CoCL2;
Described molysite is made up of following materials in parts by mass:
6.97 parts of sodium ethylene diamine tetracetates;
Described organic principle is made up of following materials in parts by mass:
10 parts of 10 parts of niacins, 10 parts of thiamine hydrochlorides and puridoxine hydrochlorides.
2. medium according to claim 1 is characterized in that:
Described medium is grouped into by following one-tenth:
Potassium nitrate, ammonium sulfate, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, manganese sulphate, boric acid, white vitriol, potassium iodide, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2, sodium ethylene diamine tetracetate, niacin, thiamine hydrochloride, puridoxine hydrochloride, inositol, 2,4 dichlorophenoxyacetic acid and N6-furfuryladenine and water;
Above composition concentration in described medium is respectively:
Potassium nitrate 2.830g/L, ammonium sulfate 0.463g/L, potassium dihydrogen phosphate 0.400g/L, epsom salt 0.185g/L, calcium chloride dihydrate 0.166g/L, manganese sulphate 1mg/L, boric acid 0.5mg/L, white vitriol 0.1mg/L, potassium iodide 0.1mg/L, Sodium Molybdate Dihydrate 0.01mg/L, cupric sulfate pentahydrate 0.02mg/L, CoCL2 0.01mg/L, sodium ethylene diamine tetracetate 6.97mg/L, niacin 10mg/L, thiamine hydrochloride 10mg/L, puridoxine hydrochloride 10mg/L, inositol 100mg/L, 2,4 dichlorophenoxyacetic acid 1mg/L-2mg/L and N6-furfuryladenine 1mg/L-2mg/L.
3. solid culture medium that obtains the sickle alfalfa callus is made into solid culture medium by coagulating agent and claim 1 or 2 described medium.
4. solid culture medium according to claim 3 is characterized in that:
The concentration of described coagulating agent in described solid culture medium is 8g/L;
Described coagulating agent is an agar.
5. according to claim 3 or 4 described solid culture mediums, it is characterized in that: described solid culture medium is following 1) or 2) shown in:
1) concentration of 2,4 dichlorophenoxyacetic acid is that the concentration of 2mg/L and N6-furfuryladenine is 1mg/L in the described solid culture medium;
2) concentration of 2,4 dichlorophenoxyacetic acid is that the concentration of 1mg/L and N6-furfuryladenine is 2mg/L in the described solid culture medium.
6. according to arbitrary described medium among the claim 1-5, it is characterized in that:
The pH value of described medium is 5.8;
Described sickle alfalfa is tetraploid sickle alfalfa or dliploid sickle alfalfa.
7. method that obtains the sickle alfalfa callus comprises the steps: with in the claim 5 1) shown in medium inducing culture sickle alfalfa explant, obtain the sickle alfalfa callus.
8. method according to claim 7 is characterized in that:
Described method also comprises with in the claim 5 2) shown in the step of medium enrichment culture sickle alfalfa callus;
Described sickle alfalfa explant is the young leaflet tablet that is cut into piece;
Described sickle alfalfa is tetraploid sickle alfalfa or dliploid sickle alfalfa.
9. method that obtains the sickle alfalfa regeneration plant may further comprise the steps: the sickle alfalfa callus that the method described in claim 7 or 8 is obtained is inoculated into and carries out differentiation culture on the differential medium and obtain bud, promptly obtains the sickle alfalfa regeneration plant.
10. method according to claim 9 is characterized in that:
Described differential medium is grouped into by following one-tenth:
Potassium nitrate, ammonium sulfate, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, manganese sulphate, boric acid, white vitriol, potassium iodide, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2, sodium ethylene diamine tetracetate, niacin, thiamine hydrochloride, puridoxine hydrochloride, inositol, N6-furfuryladenine and water;
Above composition concentration in described differential medium is respectively:
Potassium nitrate 2.830g/L, ammonium sulfate 0.463g/L, potassium dihydrogen phosphate 0.400g/L, epsom salt 0.185g/L, calcium chloride dihydrate 0.166g/L, manganese sulphate 1mg/L, boric acid 0.5mg/L, white vitriol 0.1mg/L, potassium iodide 0.1mg/L, Sodium Molybdate Dihydrate 0.01mg/L, cupric sulfate pentahydrate 0.02mg/L, CoCL2 0.01mg/L, sodium ethylene diamine tetracetate 6.97mg/L, niacin 10mg/L, thiamine hydrochloride 10mg/L, puridoxine hydrochloride 10mg/L, inositol 100mg/L and N6-furfuryladenine 0.4mg/L;
The pH value of described differential medium is 5.8;
Described method comprises that also the bud that will obtain is inoculated into the step of cultivating on the root media after described differentiation culture;
Described root media is grouped into by following one-tenth:
Potassium nitrate, ammonium sulfate, potassium dihydrogen phosphate, epsom salt, calcium chloride dihydrate, manganese sulphate, boric acid, white vitriol, potassium iodide, Sodium Molybdate Dihydrate, cupric sulfate pentahydrate, CoCL2, sodium ethylene diamine tetracetate, niacin, thiamine hydrochloride, puridoxine hydrochloride and water;
Above composition concentration in described root media is respectively:
Potassium nitrate 1.415g/L, ammonium sulfate 0.2315g/L, potassium dihydrogen phosphate 0.200g/L, epsom salt 0.0925g/L, calcium chloride dihydrate 0.083g/L, manganese sulphate 1mg/L, boric acid 0.5mg/L, white vitriol 0.1mg/L, potassium iodide 0.1mg/L, Sodium Molybdate Dihydrate 0.01mg/L, cupric sulfate pentahydrate 0.02mg/L, CoCL2 0.01mg/L, sodium ethylene diamine tetracetate 6.97mg/L, niacin 10mg/L, thiamine hydrochloride 10mg/L, puridoxine hydrochloride 10mg/L;
The pH value of described root media is 5.8;
Described sickle alfalfa explant is the young leaflet tablet that is cut into piece;
Described sickle alfalfa is tetraploid sickle alfalfa or dliploid sickle alfalfa.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103843663A (en) * | 2014-03-14 | 2014-06-11 | 中国农业科学院北京畜牧兽医研究所 | Method for promoting rooting of alfalfa tissue culture seedlings |
| CN116548307A (en) * | 2023-05-12 | 2023-08-08 | 青岛农业大学 | Leaf-induction-based alfalfa regeneration method and culture medium thereof |
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|---|---|---|---|---|
| CN1618279A (en) * | 2004-07-01 | 2005-05-25 | 南开大学 | Method for quick asexual reproduction of alfalfa by utilizing embryoid |
| CN1759666A (en) * | 2005-10-28 | 2006-04-19 | 吉林省农业科学院 | A kind of method that improves incidence rate of somatic embryo of clover |
| CN101703002A (en) * | 2009-11-03 | 2010-05-12 | 江苏农林职业技术学院 | Culture medium for overcoming variety and genetype restriction in regeneration culture of alfalfa high frequency somatic embryos |
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Patent Citations (3)
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|---|---|---|---|---|
| CN1618279A (en) * | 2004-07-01 | 2005-05-25 | 南开大学 | Method for quick asexual reproduction of alfalfa by utilizing embryoid |
| CN1759666A (en) * | 2005-10-28 | 2006-04-19 | 吉林省农业科学院 | A kind of method that improves incidence rate of somatic embryo of clover |
| CN101703002A (en) * | 2009-11-03 | 2010-05-12 | 江苏农林职业技术学院 | Culture medium for overcoming variety and genetype restriction in regeneration culture of alfalfa high frequency somatic embryos |
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| 《分子植物育种》 20061231 金淑梅等 苜蓿愈伤组织高频再生遗传和转化体系的建立 571-578 1-10 第4卷, 第4期 * |
| 《草地学报》 19941231 白静仁等 野生黄花苜蓿叶肉原生质体培养和植株再生 59-63 1-10 第2卷, 第1期 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103843663A (en) * | 2014-03-14 | 2014-06-11 | 中国农业科学院北京畜牧兽医研究所 | Method for promoting rooting of alfalfa tissue culture seedlings |
| CN103843663B (en) * | 2014-03-14 | 2015-07-15 | 中国农业科学院北京畜牧兽医研究所 | Method for promoting rooting of alfalfa tissue culture seedlings |
| CN116548307A (en) * | 2023-05-12 | 2023-08-08 | 青岛农业大学 | Leaf-induction-based alfalfa regeneration method and culture medium thereof |
| CN116548307B (en) * | 2023-05-12 | 2024-01-26 | 青岛农业大学 | Leaf-induction-based alfalfa regeneration method and culture medium thereof |
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