CN105210880A - The method of enlightening celebrating wild type crowtoe Fast-propagation - Google Patents
The method of enlightening celebrating wild type crowtoe Fast-propagation Download PDFInfo
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- CN105210880A CN105210880A CN201510717185.2A CN201510717185A CN105210880A CN 105210880 A CN105210880 A CN 105210880A CN 201510717185 A CN201510717185 A CN 201510717185A CN 105210880 A CN105210880 A CN 105210880A
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Abstract
The present invention relates to a kind of enlightening celebrating wild type crowtoe method for quickly breeding, comprise the following steps: the preparation of MS medium, sterilizing: often liter of medium comprises: macroelement 50ml, trace element 5ml, organic element 5ml, molysite 5ml, sucrose 30g, inositol 0.lg, agar powder 8g, pH are 5.8, use high-pressure sterilizing pot, pressurize sterilizing 30min; Sterilization crowtoe seed, aseptic inoculation, as 14d, after seed germination, two panels cotyledon launches, and gets its hypocotyl, segment, is inoculated on the MS medium containing 2,4-D, KT, pH=5.8, and the callus access that subculture is 2 ~ 4 times is containing the MS medium of NAA, 6-BA; When the bud induced grows to 5-8cm, indefinite bud is cut to the little stem section of 1.0cm, each little stem section has an internode at least, little stem section lower end is inserted in the 1/2MS medium containing NAA and carbon dust and takes root.Enlightening of the present invention celebrating wild type crowtoe propagation method, expands that numerous time is short, reproduction coefficient large, improves the reproduction coefficient of enlightening celebrating wild type crowtoe, shortens the breeding cycle.
Description
Technical field
The present invention relates to technical field of plant propagation, be specifically related to a kind of enlightening celebrating wild type crowtoe method for quickly breeding.
Background technology
Crowtoe (LorescomiculatusLinn.) has another name called five leaf grass (bunge bedstraw herb), all has distribution on Yunnan Province of China, Sichuan, Guizhou, Gansu, Hubei and other places.Development in Yunnan livestock breeding needs a large amount of leguminous forages, and from nineteen eighty-three, large-scale leguminous forage is introduced a fine variety, and therefrom filters out 21 kinds, though at various places well-grown, all fails large scale application aborning.Enlightening celebrating wild type crowtoe is widely distributed in the mountain region of the western regions of the Yunnan Province height above sea level 1400 ~ 3400m, woods unoccupied place, wheel have a rest ground, is the important leguminous forage in natural meadow.Enlightening celebrating wild type crowtoe is heat-resisting, drought-enduring, alumite, and its branches and leaves are tender, good palatability, and various livestock eating, cuts and herd dual-purpose.Enlightening celebrating wild type crowtoe is not only good forage, and is excellent soil-and-water conservation effect, nectariferous plant and ornamental plants, has value of exploiting and utilizing in Yunnan.
The cytothesis of crowtoe is best in leguminous forage, all can obtain high-frequency regeneration plant by embryoid approach and indefinite bud approach; Genetic transformation efficiency is relatively high, and transfer-gen plant fast growth.Therefore, crowtoe is the model legume of Study of Exogenous genetic transformation, biological nitrogen fixation mechanism, herbage quality improvement, is also the material carrying out animal vaccine production as bio-reactor.But in introducing and planting practice, find that seedling growth is slow because the history of life of crowtoe is continue to use seminal propagation always; resistance is poor, and seed hard seed rate is high, restricts by factors such as soil, temperature, seasons; germination rate is on the low side, have impact on crowtoe large-scale planting to a certain extent.So, on the basis of conventional breeding, utilizing biotechnology to its in addition genetic improvement, to cultivate the new varieties of high-quality, high yield, to produce and the development of the following grass cultivation of our province has far reaching significance expanding enlightening celebrating wild type crowtoe.Also enlightening celebrating wild type crowtoe is not carried out at present to the large-scale breeding of production, mainly land with seed maturity the mode sprouted in locality and breed, have no the report of its seed asepsis sprouting and tissue cultures.
Summary of the invention
The present invention, in order to solve the problem that enlightening celebrating wild type crowtoe reproduction coefficient is low, growth of seedling is slow, provides a kind of enlightening celebrating wild type crowtoe method for quickly breeding.
The present invention adopts following technical scheme: a kind of enlightening celebrating wild type crowtoe method for quickly breeding, comprises the following steps:
1) preparation of MS medium, sterilizing: often liter of medium comprises mother liquor medicine: macroelement 50ml (potassium nitrate KNO
3, ammonium nitrate NH
4nO
3, potassium dihydrogen phosphate KH
2pO
4, magnesium sulfate MgSO
47H
2o, calcium chloride CaCl
22H
2o), micro-5ml (potassium iodide KI, boric acid H
3bO
3, manganese sulphate MnSO
44H
2o, zinc sulphate ZnSO
47H
2o, sodium molybdate Na
2moO
42H
2o, copper sulphate CuSO
45H
2o, cobalt chloride CoCl
26H
2o), organic element 5ml (inositol, glycine, thiamine hydrochloride VB
1, puridoxine hydrochloride VB
6, nicotinic acid VPP), molysite 5ml (disodium ethylene diamine tetraacetate Na
2.EDTA, ferrous sulfate FeSO
47H
2o).Sucrose 30g, inositol 0.lg, agar powder 8g, pH are 5.8; The preparation of medium: the running water getting about 400ml is poured in little aluminum pot and heated, after water is opened, put into 8g agar powder, heat while stirring, note preventing from being sticking, very hot oven is boiled, again with slow fire heating, until agar all melts namely as well so clear that you can see the bottom, after 25min, close fire, add 30g sucrose, 0.1g inositol and the mother liquor medicine (NH measured
4nO
31650mg/L; KNO
31900mg/L; CaCl
22H
2o440mg/L; MgSO
47H
2o370mg/L; KH
2pO
4170mg/L; KI0.83mg/L; H
3bO
36.2mg/L; MnSO
44H
2o22.3mg/L; ZnSO
47H
2o8.6mg/L; CuSO
45H
2o0.025mg/L; CoCl
26H
2o0.025mg/L; Na
2moO
42H
2o0.25mg/L; FeSO
47H
2o27.8mg/L; Na
2eDTA37.3mg/L; Inositol 100mg/L; Nicotinic acid 0.5mg/L; Puridoxine hydrochloride 0.5mg/L; Thiamine hydrochloride 0.1mg/L; Glycine 2mg/L), stir 5min, pour constant volume in the constant volume beaker of 1000ml into, use the PH of PH measurement amount medium, use NaOH or the HC1 solution furnishing pH value 5.8 of 0.1% when needing to adjust, use high-pressure sterilizing pot, pressurize sterilizing 30min;
2) sterilization crowtoe seed, aseptic inoculation: enlightening is celebrated wild type crowtoe seed and put into gauze, soak (preferably soaking 20min) with 98% concentrated sulfuric acid, taking-up is put into distilled water and cleaned 1min, at least changes water 3 times; Then use 70% ~ 75% alcohol disinfecting 30s, taking-up is put into distilled water and is cleaned 1min, at least changes water 3 times; Finally use 0.1% mercury chloride (mercuric chloride) sterilizing (preferred sterilizing 10min) to use distilled water flushing afterwards 5 ~ 6 times, be placed in culture dish, be then inoculated on the MS medium without hormone, every bottle graft kind 10 seeds, repeat 10 times; In the process of liquid sterilization, jiggle and fill seed-bearing container, liquid is fully contacted with seed; The seed of inoculation is put into culturing room, and cultivation temperature is 25 scholar 2 DEG C, and intensity of illumination is 1500-2000Lux, illumination 10h/d; After inoculation, every day observes 1 time, adds up the germination rate of seed when 14 days.
3) induction of callus: after seed germination, two panels cotyledon launches as 14d, get its hypocotyl, be cut into the segment that 0.5cm is long, be inoculated in containing 2,4-D (2,4-dichlorphenoxyacetic acid) 2-8mg/L (preferred 8mg/L), KT (kinetin) 1-5mg/L (preferred 3mg/L), on the MS medium of pH=5.8, each treatment combination is in triplicate;
4) bud is differentiation-inducing: the hormone concentration combination medium of induced bud differentiation is: containing the MS medium of NAA (methyl α-naphthyl acetate) 0.5-1.0mg/L (preferred 1mg/L), 6-BA (6-benzyl aminoadenine) 0.5-2.0mg/L (preferred 2mg/L), and after the callus access medium that subculture is 2 ~ 4 times, 18-32d observation bud is long;
5) induction of root: when the bud induced grows to 5-8cm, indefinite bud is cut to the little stem section of 1.0cm, each little stem section has an internode at least, little stem section lower end is inserted in the 1/2MS medium containing 0.05-0.15mg/LNAA (preferred 0.1mg/L) and 0.1-1g/L carbon dust (preferred 1.0g/L) and takes root.
The beneficial effect of the invention and advantage: the present invention, relative to the propagation method of enlightening celebrating wild type crowtoe in prior art, expands that numerous time is short, reproduction coefficient is large.The present invention can improve reproduction coefficient, the shortening breeding cycle of enlightening celebrating wild type crowtoe.
Accompanying drawing explanation
Fig. 1. enlightening celebrating wild type crowtoe seed asepsis sprouting figure of the present invention;
Fig. 2. the induced map of enlightening celebrating wild type crowtoe seed callus of the present invention;
Fig. 3. the differentiation figure of enlightening celebrating wild type crowtoe bud of the present invention;
Fig. 4. enlightening celebrating wild type crowtoe root induction figure of the present invention.
Embodiment
Embodiment 1
1) preparation of medium: (often liter of medium comprises: macroelement 50ml to select MS medium, trace element 5ml, organic element 5ml, molysite 5ml, sugar 30g, inositol 0.lg, agar powder 8g) PH is 5.8, specific as follows: the running water getting about 400ml is poured in little aluminum pot and heated, after water is opened, put into 8g agar powder, heat while stirring, note preventing from being sticking, very hot oven is boiled, heat with slow fire again, until agar all melts namely as well so clear that you can see the bottom, fire is closed after 25min, add 30g sucrose, 0.1g inositol and the mother liquor medicine measured, stir 5min.Pour constant volume in the constant volume beaker of 1000ml into.Use the PH of PH measurement amount medium, when needing to adjust, use NaOH or the HC1 solution furnishing 5.8pH value left and right of 0.1%.Adjustment PH after notes stirring, and attention standing time can not be used too of a specified duration acid or aqueous slkali.
Described mother liquor medicine: macroelement 50ml: potassium nitrate KNO
3, ammonium nitrate NH
4nO
3, potassium dihydrogen phosphate KH
2pO
4, magnesium sulfate MgSO
47H
2o, calcium chloride CaCl
22H
2o); Trace element 5ml: potassium iodide KI, boric acid H
3bO
3, manganese sulphate MnSO
44H
2o, zinc sulphate ZnSO
47H
2o, sodium molybdate Na
2moO
42H
2o, copper sulphate CuSO
45H
2o, cobalt chloride CoCl
26H
2o: organic element 5ml: inositol, glycine, thiamine hydrochloride VB
1, puridoxine hydrochloride VB
6, nicotinic acid VPP; Molysite 5ml: disodium ethylene diamine tetraacetate Na
2.EDTA, ferrous sulfate FeSO
47H
2o.Wherein, NH
4nO
31650mg/L; KNO
31900mg/L; CaCl
22H
2o440mg/L; MgSO
47H
2o370mg/L; KH
2pO
4170mg/L; KI0.83mg/L; H
3bO
36.2mg/L; MnSO
44H
2o22.3mg/L; ZnSO
47H
2o8.6mg/L; CuSO
45H
2o0.025mg/L; CoCl
26H
2o0.025mg/L; Na
2moO
42H
2o0.25mg/L; FeSO
47H
2o27.8mg/L; Na
2eDTA37.3mg/L; Inositol 100mg/L; Nicotinic acid 0.5mg/L; Puridoxine hydrochloride 0.5mg/L; Thiamine hydrochloride 0.1mg/L; Glycine 2mg/L.
The packing of medium: will packing while hot after medium boils, 1000ml medium packing 30 bottles, the amount of each bottle is as far as possible identical, can not too much can not be very little.Note during packing medium not being poured on bottle mouth wall, avoid a day after stain, if desired packing while stirring.Encapsulation bottle cap is tightened, and writes kinds of culture medium with waterproof recording pen, prepares sterilizing.
The sterilizing of medium: use high-pressure sterilizing pot, open pot cover, mention drum, adds water to waterline (can not dry combustion method autoclave, can cause danger).Put install the Cans of medium and inoculating appliance etc. in people's drum, cover pot cover, diagonal angle screwing screw, switch on power heating, when rising to 0.05MPa, closes power switch, open vent valve exit back " 0 " close vent valve afterwards, then to turn on the power switch.When being raised to 0.l0MPa atmospherically, pressurize sterilizing 30min, then powered-down switch, open air valve, after air pressure goes back to " 0 ", open pot cover, take out medium, be placed in basket (balance of trying one's best during placement for subsequent use, ramp-like after avoiding culture medium solidifying), basket is placed shady and cool clean place, and the medium that sterilizing is good standing time can not more than 1 month.
2) sterilization crowtoe seed, aseptic inoculation: enlightening is celebrated wild type crowtoe seed and put into gauze, soak 20min with 98% concentrated sulfuric acid, taking-up is put into distilled water and cleaned 1min, at least changes water 3 times; Then use 70% ~ 75% alcohol disinfecting 30s, taking-up is put into distilled water and is cleaned 1min, at least changes water 3 times; Use distilled water flushing 5 ~ 6 times after finally using 0.1% mercury chloride (mercuric chloride) sterilizing 10min, be placed in culture dish, be then inoculated on the MS medium without hormone, every bottle graft kind 10 seeds, repeat 10 times; In the process of liquid sterilization, jiggle and fill seed-bearing container, liquid is fully contacted with seed; The seed of inoculation is put into culturing room, and cultivation temperature is 25 scholar 2 DEG C, and intensity of illumination is 1500-2000Lux, illumination 10h/d; After inoculation, every day observes 1 time, adds up the germination rate of seed when 14 days.
3) induction of callus: after seed germination, two panels cotyledon launches as 14d, get its hypocotyl, be cut into the segment that 0.5cm is long, be inoculated in containing 2,4-D (2,4-dichlorphenoxyacetic acid) 8mg/L, KT (kinetin) 3mg/L, pH=5.8 MS medium on, each treatment combination is in triplicate;
4) bud is differentiation-inducing: the hormone concentration combination medium of induced bud differentiation is: containing the MS medium of NAA (methyl α-naphthyl acetate) 1mg/L, 6-BA (6-benzyl aminoadenine) 2mg/L, and after the callus access medium that subculture is 2 ~ 4 times, 18-32d observation bud is long;
5) induction of root: when the bud induced grows to 5-8cm, is cut to the little stem section of 1.0cm by indefinite bud, each little stem section has an internode at least, little stem section lower end is inserted in the 1/2MS medium containing 0.1mg/LNAA and 1g/L carbon dust and takes root.
Embodiment 2
Step 1), step 2) with embodiment 1,
3) induction of callus: after seed germination, two panels cotyledon launches as 14d, get its hypocotyl, be cut into the segment that 0.5cm is long, be inoculated in containing 2,4-D (2,4-dichlorphenoxyacetic acid) 2mg/L, KT (kinetin) 1mg/L, pH=5.8 MS medium on, each treatment combination is in triplicate;
4) bud is differentiation-inducing: the hormone concentration combination medium of induced bud differentiation is: containing the MS medium of NAA (methyl α-naphthyl acetate) 0.5mg/L, 6-BA (6-benzyl aminoadenine) 0.5mg/L, and after the callus access medium that subculture is 2 ~ 4 times, 18-32d observation bud is long;
5) induction of root: when the bud induced grows to 5-8cm, is cut to the little stem section of 1.0cm by indefinite bud, each little stem section has an internode at least, little stem section lower end is inserted in the 1/2MS medium containing 0.05mg/LNAA and 0.1g/L carbon dust and takes root.
Embodiment 3
Step 1), step 2) with embodiment 1,
3) induction of callus: after seed germination, two panels cotyledon launches as 14d, get its hypocotyl, be cut into the segment that 0.5cm is long, be inoculated in containing 2,4-D (2,4-dichlorphenoxyacetic acid) 4mg/L, KT (kinetin) 5mg/L, pH=5.8 MS medium on, each treatment combination is in triplicate;
4) bud is differentiation-inducing: the hormone concentration combination medium of induced bud differentiation is: containing the MS medium of NAA (methyl α-naphthyl acetate) 1.5mg/L, 6-BA (6-benzyl aminoadenine) 0.5mg/L, and after the callus access medium that subculture is 2 ~ 4 times, 18-32d observation bud is long;
5) induction of root: when the bud induced grows to 5-8cm, is cut to the little stem section of 1.0cm by indefinite bud, each little stem section has an internode at least, little stem section lower end is inserted in the 1/2MS medium containing 0.15mg/LNAA and 1g/L carbon dust and takes root.
Comparative example
Step 1) with embodiment 1,
2). seed disinfection and Seed treatment:
The process of table 1 seed asepsis sprouting and the design of disinfecting time composite test
Enlightening is celebrated wild type crowtoe seed and put into gauze, soak with 98% concentrated sulfuric acid, taking-up is put into distilled water and is cleaned 1min, at least changes water 3 times; Then use 70% ~ 75% alcohol disinfecting 30s, taking-up is put into distilled water and is cleaned 1min, at least changes water 3 times; Use distilled water flushing 5 ~ 6 times after finally using 0.1% mercury chloride (mercuric chloride) sterilizing, be placed in culture dish, be then inoculated on the MS medium without hormone, every bottle graft kind 10 seeds, repeat 10 times.In the process of liquid sterilization, jiggle and fill seed-bearing container, liquid is fully contacted with seed.The seed of inoculation is put into culturing room, and cultivation temperature is 25 scholar 2 DEG C, and intensity of illumination is 1500-2000Lux, illumination 10h/d.After inoculation, every day observes 1 time, adds up the germination rate of seed when 14 days.
Seed germination is medium based on MS, and by true seed according to 98% concentrated sulfuric acid different disposal time and different disinfecting time combination (see table 1) of 0.1% mercuric chloride, each treatment combination repeats 3 times.Relatively the impact of different time combination on crowtoe leaf seed germination, obtains the combined method of best seed treatment and disinfecting time.Result shows, the process disinfecting time combined method of combination 11 (98% concentrated sulfuric acid seed soaking 20min+0.1% mercuric chloride sterilizing 10min) is conducive to Yunnan Wild crowtoe true seed axenic germination most.
3) induction of callus:
The hormone concentration experimental scheme (mg/L) that table 2 evoked callus generates
As 14d, after seed germination, two panels cotyledon launches, get its hypocotyl, be cut into the segment that 0.5cm is long, be inoculated in containing 2,4-D (2,4-dichlorphenoxyacetic acid), on the MS medium (pH=5.8) of KT (kinetin), 6-BA (6-benzyl aminoadenine) and NAA (methyl α-naphthyl acetate) (see table 2), each treatment combination in triplicate, compares the impact that hormon concentration combination is induced crowtoe hypocotyledonery axis callus.Result shows, the hormone concentration combination of NAA and 6-BA is unfavorable for inducing the hypocotyl of Yunnan Wild crowtoe true seed seedling to generate callus.Combination 7,8,9,10,11,12 all reaches the more raw rate of 100%, and pole is significantly higher than other combinations.But from growth potential, find that callus of induce is after 23 days in observed and recorded, the 11 callus yellowish derived comparatively are combined in combination 7,8,9,10,12, and brownization had situation is serious, and the callus growing way that combination 11 induces is best.Therefore obtaining best evoked callus culture medium prescription is: MS+2,4-D8mg/L+KT3mg/L.
4) bud is differentiation-inducing
This stage adopts based on MS medium, by the basic element of cell division of variable concentrations and the various combination of auximone, has been 5 contrast experiments.Result shows that 2,4-D and KT is unfavorable for the induction of bud; After the callus access medium that subculture is 2 ~ 4 times there is larger strong green bud point on surface and periphery in 18d, on strong green bud point, clump bud is formed after 32d, be beneficial to the hormone concentration producing Multiple Buds and be combined as 18 and No. 21, the hormone concentration combination medium of best induced bud differentiation is: MS+NAA1.0mg/L+6-BA2.0mg/L.
Table 3 hormone is to the hormone concentration experimental scheme (mg/L) of Multiple Buds
5) induction of root
When the bud induced grows to 5-8cm, indefinite bud is cut to about 1.0cm little stem section (each little stem section has an internode at least), little stem section lower end is inserted in the 1/2MS medium containing NAA and carbon dust.
Table 4 variable concentrations NAA and carbon dust are on the impact of crowtoe rooting rate
Little stem section is in root media, and the concentration hormone combinations of 1/2MS+NAA0.10mg/L+ carbon dust 1.0g/L is put up the best performance, and quantity of taking root is many and speed is fast.Start to sprout lateral bud after little stem section 4d, take root after 8d, after 30, plant height can reach 8cm, and main root reaches 6mm.
Claims (7)
1. an enlightening celebrating wild type crowtoe method for quickly breeding, is characterized in that: comprise the following steps:
1) preparation of MS medium, sterilizing: often liter of medium comprises: macroelement 50ml, micro-5ml, organic element 5ml, molysite 5ml, sucrose 30g, inositol 0.lg, and agar powder 8g, pH are 5.8, use high-pressure sterilizing pot, pressurize sterilizing 30min;
2) sterilization crowtoe seed, aseptic inoculation: enlightening is celebrated wild type crowtoe seed and put into gauze, soak with 98% concentrated sulfuric acid, taking-up is put into distilled water and cleaned 1min, at least changes water 3 times; Then use 70% 75% alcohol disinfecting 30s, take out and put into distilled water and clean 1min, at least change water 3 times; Finally use distilled water flushing 5 ~ 6 times with after 0.1% mercury chloride sterilizing, be placed in culture dish, be then inoculated on the MS medium without hormone, every bottle graft kind 10 seeds, repeat 10 times; In the process of liquid sterilization, jiggle and fill seed-bearing container, liquid is fully contacted with seed; The seed of inoculation is put into culturing room, and cultivation temperature is 25 scholar 2 DEG C, and intensity of illumination is 1500-2000Lux, illumination 10h/d; After inoculation, every day observes 1 time, adds up the germination rate of seed when 14 days;
3) induction of callus: after seed germination, two panels cotyledon launches as 14d, get its hypocotyl, be cut into the segment that 0.5cm is long, be inoculated in containing 2,4-D(2,4-dichlorphenoxyacetic acid) 2-8mg/L, KT(kinetin) 1-5mg/L, pH=5.8 MS medium on, each treatment combination is in triplicate;
4) bud is differentiation-inducing: the hormone concentration combination medium of induced bud differentiation is: containing NAA(methyl α-naphthyl acetate) 0.5-1.0mg/L, 6-BA(6-benzyl aminoadenine) the MS medium of 0.5-2.0mg/L, after the callus access medium that subculture is 2 ~ 4 times, to observe bud long for 18-32d;
5) induction of root: when the bud induced grows to 5-8cm, is cut to the little stem section of 1.0cm by indefinite bud, each little stem section has an internode at least, little stem section lower end is inserted in the 1/2MS medium containing 0.05-0.15mg/LNAA and 0.1-1g/L carbon dust and takes root.
2. a kind of enlightening celebrating wild type crowtoe method for quickly breeding as claimed in claim 1, is characterized in that: in step 1) in often liter of medium: nutrient composition content mg/L,
Macroelement: 1900 potassium nitrate KNO
3, 1650 ammonium nitrate NH
4nO
3, 170 potassium dihydrogen phosphate KH
2pO
4, 370 magnesium sulfate MgSO
47H
2o, 440 calcium chloride CaCl
22H
2o;
Trace element: 0.83 potassium iodide KI, 6.2 boric acid H
3bO
3, 22.3 manganese sulphate MnSO
44H
2o, 8.6 zinc sulphate ZnSO
47H
2o, 0.25 sodium molybdate Na
2moO
42H
2o, 0.025 copper sulphate CuSO
45H
2o, 0.025 cobalt chloride CoCl
26H
2o;
Organic element: 100 inositols, 2 glycine, 0.1 thiamine hydrochloride VB
1, 0.5 puridoxine hydrochloride VB
6, 0.5 nicotinic acid VPP;
Molysite: 37.3 disodium ethylene diamine tetraacetate Na
2.EDTA, 27.8 ferrous sulfate FeSO
47H
2o.
3. a kind of enlightening celebrating wild type crowtoe method for quickly breeding as claimed in claim 1 or 2, it is characterized in that: the preparation of MS medium in step 1): specifically comprise the following steps, the running water getting about 400ml is poured in little aluminum pot and is heated, after water is opened, put into 8g agar powder, heat while stirring, note preventing from being sticking, very hot oven is boiled, heat with slow fire again, until agar all melts namely as well so clear that you can see the bottom, fire is closed after 25min, add 30g sucrose, 0.1g inositol and the macroelement 50ml measured, trace element 5ml, organic element 5ml, molysite 5ml, stir 5min, pour constant volume in the constant volume beaker of 1L into, pH meter is used to measure the pH of medium, NaOH or the HC1 solution furnishing pH value 5.8 of 0.1% is used when needing to adjust.
4. a kind of enlightening celebrating wild type crowtoe method for quickly breeding as claimed in claim 3, is characterized in that: step 2) utilize 98% concentrated sulfuric acid to soak crowtoe seed 20min, taking-up is put into distilled water and is cleaned 1min, at least changes water 3 times; Then use 70% 75% alcohol disinfecting 30s, take out and put into distilled water and clean 1min, at least change water 3 times; Finally use distilled water flushing 5 ~ 6 times with after 0.1% mercury chloride sterilizing 10min.
5. a kind of enlightening celebrating wild type crowtoe method for quickly breeding as described in claim 1,2 or 4, it is characterized in that: step 3) is two panels cotyledon expansion after seed germination as 14d, get its hypocotyl, be cut into the segment that 0.5cm is long, be inoculated in containing 2,4-D(2,4-dichlorphenoxyacetic acid) 8mg/L, KT(kinetin) 3mg/L, on the MS medium of pH=5.8, each treatment combination in triplicate.
6. a kind of enlightening celebrating wild type crowtoe method for quickly breeding as claimed in claim 5, it is characterized in that: the hormone concentration combination medium of step 4) induced bud differentiation is: containing NAA(methyl α-naphthyl acetate) 1.0mg/L, 6-BA(6-benzyl aminoadenine) the MS medium of 2.0mg/L, after the callus access medium that subculture is 2 ~ 4 times, to observe bud long for 18-32d.
7. a kind of enlightening celebrating wild type crowtoe method for quickly breeding as claimed in claim 6, is characterized in that: take root in the 1/2MS concentration hormone combinations root media of step 5) little stem Duan Han NAA0.10mg/L, carbon dust 1.0g/L.
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| CN116034877A (en) * | 2023-04-03 | 2023-05-02 | 云南农业大学 | Fast propagation method and application of Baimai root of Zhongdian |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020173037A1 (en) * | 2001-03-29 | 2002-11-21 | Pati Pratap Kumar | Efficient method of protoplast culture |
| CN103155858A (en) * | 2011-12-08 | 2013-06-19 | 深圳市农科集团有限公司 | Rapid lotus corniculatus l. breeding method |
-
2015
- 2015-10-28 CN CN201510717185.2A patent/CN105210880A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20020173037A1 (en) * | 2001-03-29 | 2002-11-21 | Pati Pratap Kumar | Efficient method of protoplast culture |
| CN103155858A (en) * | 2011-12-08 | 2013-06-19 | 深圳市农科集团有限公司 | Rapid lotus corniculatus l. breeding method |
Non-Patent Citations (2)
| Title |
|---|
| 何奕昆等: "百脉根的体细胞胚胎发生与高频率植株再生", 《四川师范大学学报（自然科学报）》 * |
| 韩阳等: "百脉根植株高频再生体系的建立", 《辽宁大学学报自然科学版》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116034877A (en) * | 2023-04-03 | 2023-05-02 | 云南农业大学 | Fast propagation method and application of Baimai root of Zhongdian |
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