CN103444527B - A kind of growth-promoting cultivation method of Hupeh Fritillary Bulb - Google Patents
A kind of growth-promoting cultivation method of Hupeh Fritillary Bulb Download PDFInfo
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Abstract
The invention discloses a kind of growth-promoting cultivation method of Hupeh Fritillary Bulb, the selection comprising explant with disinfect, the process of material, callus induction and Bud polarization, the propagation of bud, the squamous subculture of callus, culture of rootage, greenhouse hardening and soil management, transplanting and other steps, through above-mentioned process, calli induction rate reaches more than 96%, proliferation times has exceeded 7.3 times, rooting rate reaches more than 98%, final transplanting survival rate reaches more than 98%, and plant strain growth is vigorous, improve soil fertility, Disease management rate.And in cultivating process, the propagation of the propagation of bud and callus is separated process, which enhance the availability of material, improve the survival rate of the two.
Description
Technical field
The present invention relates to the method for tissue culture of a kind of bulb of fritillary, be specifically related to the method for quickly breeding of Hupeh Fritillary Bulb.
Background technology
Hupeh Fritillary Bulb (FritillariahupehensisHsiaoetK.C.Hsia), also known as Hubei Province shellfish, plate shellfish, kiln shellfish, Feng Bei, for Liliaceae (Liliaceae) Fritillaria (FritillariaL.) herbaceos perennial, Hupeh Fritillary Bulb is at Jianshi, hubei, Xuanen one is with cultivation, GAP base kind of bestowing favour now, it is one of medicinal material of greatly developing of Enshi, extensively there is environmental deterioration at present, the problem of soil nutrient deficiency, and the breeding of Hupeh Fritillary Bulb is based on bulb breeding, also scale and seminal propagation can be used, after carrying out vegetative propagation many generations, 1 sexual propagation should be carried out.But because Hupeh Fritillary Bulb is bloomed many, result is few, difficulty receives more seed, seminal propagation is still in experimental stage, because Hupeh Fritillary Bulb has higher medical value, main its bulb edible, large to bulb of fritillary bulb demand at present, direct underground bulb carries out breeding also grows slowly, need 4-5 could grow the size arriving commodity bulb, growth cycle is long, therefore its area under cultivation and output are all very restricted, because the current demand to the bulb of fritillary increases considerably, also when the river rises the boat goes up thereupon for price, have very important significance so breed Hupeh Fritillary Bulb tool fast, to the income improving people, there is very high value.
At present through 20 years of researches, Hupeh Fritillary Bulb has become in the bulb of fritillary the second largest main flow bulb of fritillary commodity being only second to fritillaria thunbergii, utilizes tissue cultures and fast breeding technique to improve reproduction coefficient, expands area under cultivation.Production tool is very helpful.A lot of research has been had at present to bulb of fritillary tissue cultures and fast breeding technique, but relatively less to the research of Hupeh Fritillary Bulb, and do not form the breeding system of complete set.
Summary of the invention
The technical problem that the present invention mainly solves is to provide a kind of growth-promoting cultivation method of Hupeh Fritillary Bulb.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is:
A growth-promoting cultivation method for Hupeh Fritillary Bulb, comprises the following steps:
(1) explant selection with disinfect: first select robust growth, ripe fresh Hupeh Fritillary Bulb bulb ball, peel off scale and the remaining old root of base portion of bulb outermost layer shriveling, first use running water running water 2-3 time, then filter paper suck dry moisture is used with after 50% rifampin, 300 times of immersion bubble 20-30min, then by the strip off in layer of bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, superclean bench is first used 75% alcohol-pickled sterilization 30s, remove alcohol, by rinsed with sterile water 3-4 time, to sterilize 10min with 2% liquor natrii hypochloritis again, aseptic water washing 2-3 time.Add rapidly 3% hydrogen peroxide dipping sterilization 8-10min, often shake beaker in immersion process, then outwell and rise hydrogen peroxide liquid, after aseptic water washing 4-5 time, then soak for subsequent use with sterile water.
(2) process of material: the scale aseptic filter paper of disinfecting is blotted, aseptically scale also had base portion according to upper, middle and lower portion, be cut into the fritter that 3-5mm is square respectively, and upper, middle and lower portion also has the material of base portion to carry out next step cultivation respectively.
(3) callus induction and Bud polarization: be seeded in callus inducing medium by the explant that step (2) obtains under sterile conditions, this medium is the GA at 6-BA, the 0.8mg/L that with the addition of 0.6mg/L in MS conventional medium
3, the ZT of the sucrose of 80mg/L, 10g/L agar and 0.4mg/L; Cultivation temperature is 26 DEG C, light application time 13h/d, and intensity of illumination 2200Lx, PH are 6.5, forms callus through 10-15 days, also has part directly to differentiate little thin bud;
(4) propagation of bud: direct differentiation little thin bud out in step (3) is inoculated into 3/4MS+ agar 6.0g/L+NAA0.5mg/L+GA
3on the medium of 0.5mg/L+ZT0.2mg/L+ sucrose 15g/L, cultivation temperature is 25 DEG C, light application time 12h/d, intensity of illumination 2000Lx, PH is 6.5, every bottle graft kind 1 bud, by the clove that grows thickly of induction after 8-12 days, wherein robust growth, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out squamous subculture again.
(5) squamous subculture of callus: the callus formed in step (3) is inoculated into 1/2MS+6-BA2.0mg/L+GA
3on the medium of 0.5mg/L+KT0.5mg/L+ sucrose 20g/L+ agar 5g/L, cultivation temperature is 25 DEG C, light application time 12h/d, and intensity of illumination 2000Lx, PH are 6.5, grow clove through 15-20 days.
(6) culture of rootage: carry out root induction by being inoculated in respectively in following inducing culture one by one through step (4) and step (5) differentiation clove out: 3/4MS medium+agar 8g/L+KT1.2mg/L+ sucrose 20mg/L+ active carbon 2.0mg/L, cultivation temperature is 24 DEG C, periodicity of illumination 12h/d, luminous intensity is 2200lux, pH value is 6.5, treated that clove grew to 2-3cm through 20-25 days high, when root system is more healthy and stronger and when growing 6-10 root, namely start the next stage;
(7) soil matrix management: regulate soil pH value to be 7.0 ~ 8.2, use after mixing with 5 times of volume fine earths after Cosan is sieved, except regulating soil pH value with Cosan, Cosan is then better with peat composed of rotten mosses mixing application effect, or use pine needle to coordinate Cosan to improve soil, wherein pine needle, original soil, muck, Cosan are according to ratio of weight and number 2:3:1:2, or apply in soil saw not, the peat composed of rotten mosses, liver moss and Cosan carry out soil melioration, wherein saw not, the peat composed of rotten mosses, liver moss and Cosan ratio of weight and number be 1:1:1:2;
Increase soil organic matter content: the field planting ditch digging wide 50cm, dark 40cm, organic matter, fertilizer, original soil are improved the soil in the ratio of 1:1:1, organic matter uses the acid peat composed of rotten mosses, if first January more than must be stacked with sawdust, bark, become thoroughly decomposed completely after decomposing and re-use, as employment domestic animals manure become thoroughly decomposed or straw leaf rot to become thoroughly decomposed after the natural fertilizers that formed, note not mixing ash, lime alkaline matter, content reaches more than 3% ~ 5%, to increase the permeability of soil;
Soil covers: can carry out after soil covers seedling planting, by covering uniform fold at bed surface, wide 1 meter, thick 5 ~ 10 centimetres, cover 2 cm thicks more every year later, to keep original thickness, the new sawdust decomposed if application is not rotted, the nitrogenous fertilizer of 50% need be enriched, rot the sawdust decomposed, nitrogen fertilizer amount should correspondingly reduce, cover unregistered land film and can prevent soil water evaporation, control weeds, improve ground temperature, preferably the orchard of drip irrigation facility is being had to apply, cover unregistered land film and cover the organic matter better or front weed killer herbicide of field planting of effect that combines and kill weeds, entirely cultivate field after field planting and sow green manure Trifolium repense, the leguminous plants such as red clover, field can be covered completely after March, soil fertility can be increased again,
(8) greenhouse hardening and transplanting: in order to improve survival rate, before transplanting, first blake bottle is moved to greenhouse, temperature hardening one week when 20-25 DEG C, first in culturing room, cultivation bottle cap is opened during transplanting, taking-up tap water removes the agar of root, avoid damaging root, then 2min is soaked with 2000 times of potassium permanganate, be transplanted in the soil matrix in step (7) after drying, for keeping higher humidity, spray every day 2 times, after growing young leaves, the suitable Nitrogen Top Dressing of visual plant strain growth situation, land for growing field crops is moved to when plant grows to 4-8cm height through 10-20 days, and suitable carries out Shading treatment.
The invention has the beneficial effects as follows:
(1) complete tissue culture propagating system is established, for the fast breeding of Hupeh Fritillary Bulb is laid a solid foundation;
(2) before explant is disinfected, first steep 20-30min with by 50% rifampin, 300 times of immersions, sterilization is more thorough like this, and calli induction rate is improved;
(3) the upper, middle and lower portion of Hupeh Fritillary Bulb also has the differentiation capability of base portion different, is also had in upper, middle and lower portion the material of base portion to carry out next step respectively and cultivates, be conducive to the time relatively unification of the formation callus of material, be conducive to management;
(4) propagation of the propagation of bud and callus is separated process, the availability of material can be improved like this, the survival rate of both raisings.
(5) improve soil fertility, reduce the generation of damage by disease and insect.
Embodiment
Below preferred embodiment of the present invention is described in detail, can be easier to make advantages and features of the invention be readily appreciated by one skilled in the art, thus more explicit defining is made to protection scope of the present invention.
Embodiment 1:
A growth-promoting cultivation method for Hupeh Fritillary Bulb, comprises the following steps:
(1) explant selection with disinfect: first select robust growth, ripe fresh Hupeh Fritillary Bulb bulb ball, peel off scale and the remaining old root of base portion of bulb outermost layer shriveling, first use running water running water 2 times, then filter paper suck dry moisture is used with after 50% rifampin, 300 times of immersion bubble 20min, then by the strip off in layer of bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, superclean bench is first used 75% alcohol-pickled sterilization 30s, remove alcohol, by rinsed with sterile water 3 times, to sterilize 10min with 2% liquor natrii hypochloritis again, aseptic water washing 2 times.Add rapidly 3% hydrogen peroxide dipping sterilization 8min, often shake beaker in immersion process, then outwell and rise hydrogen peroxide liquid, after aseptic water washing 4 times, then soak for subsequent use with sterile water;
(2) process of material: the scale aseptic filter paper of disinfecting is blotted, aseptically scale also had base portion according to upper, middle and lower portion, be cut into the fritter that 3-5mm is square respectively, and upper, middle and lower portion also has the material of base portion to carry out next step cultivation respectively;
(3) callus induction and Bud polarization: be seeded in callus inducing medium by the explant that step (2) obtains under sterile conditions, this medium is the GA at 6-BA, the 0.8mg/L that with the addition of 0.6mg/L in MS conventional medium
3, the ZT of the sucrose of 80mg/L, 10g/L agar and 0.4mg/L; Cultivation temperature is 26 DEG C, light application time 13h/d, intensity of illumination 2200Lx, PH is 6.5, the material on top formed callus through 15 days, and the material at middle part formed callus through 13 days, and the material of bottom formed callus through 11 days, the material of base portion formed callus through 12 days, and the material of its middle and lower part and base portion also has part directly to differentiate little thin bud;
(4) propagation of bud: direct differentiation little thin bud out in step (3) is inoculated into 3/4MS+ agar 6.0g/L+NAA0.5mg/L+GA
3on the medium of 0.5mg/L+ZT0.2mg/L+ sucrose 15g/L, cultivation temperature is 25 DEG C, light application time 12h/d, intensity of illumination 2000Lx, PH is 6.5, every bottle graft kind 1 bud, by the clove that grows thickly of induction after 8-12 days, wherein robust growth, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out squamous subculture again;
(5) squamous subculture of callus: by the callus formed in step (3) be inoculated into 1/2MS+6-BA2.0mg/L+GA
3on the medium of 0.5mg/L+KT0.5mg/L+ sucrose 20g/L+ agar 5g/L, cultivation temperature is 25 DEG C, light application time 12h/d, and intensity of illumination 2000Lx, PH are 6.5, grow clove through 15-20 days;
(6) culture of rootage: carry out root induction by being inoculated in respectively in following inducing culture one by one through step (4) and step (5) differentiation clove out: 3/4MS medium+agar 8g/L+KT1.2mg/L+ sucrose 20mg/L+ active carbon 2.0mg/L, cultivation temperature is 24 DEG C, periodicity of illumination 12h/d, luminous intensity is 2200lux, pH value is 6.5, treated that clove grew to 2-3cm through 20-25 days high, when root system is more healthy and stronger and when growing 6-10 root, namely start the next stage;
(7) greenhouse hardening and transplanting: in order to improve survival rate, before transplanting, first blake bottle is moved to greenhouse, temperature hardening one week when 20-25 DEG C, first in culturing room, cultivation bottle cap is opened during transplanting, taking-up tap water removes the agar of root, avoid damaging root, then 2min is soaked with 2000 times of potassium permanganate, be transplanted to after drying by humus soil, vermiculite, perlitic mixed proportion is on the seedbed of 1: 2: 1, for keeping higher humidity, spray every day 2 times, after growing young leaves, the suitable Nitrogen Top Dressing of visual plant strain growth situation, land for growing field crops is moved to when plant grows to 4-8cm height through 10-20 days, and suitable carries out Shading treatment.
Through above-mentioned process, calli induction rate reaches 94%, and proliferation times reaches 6.3 times, and rooting rate reaches 96%, and final transplanting survival rate reaches 94%, and plant strain growth is vigorous.
embodiment 2
A growth-promoting cultivation method for Hupeh Fritillary Bulb, comprises the following steps:
(1) explant selection with disinfect: first select robust growth, ripe fresh Hupeh Fritillary Bulb bulb ball, peel off scale and the remaining old root of base portion of bulb outermost layer shriveling, first use running water running water 3 times, then filter paper suck dry moisture is used with after 50% rifampin, 300 times of immersion bubble 30min, then by the strip off in layer of bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, superclean bench is first used 75% alcohol-pickled sterilization 30s, remove alcohol, by rinsed with sterile water 4 times, to sterilize 10min with 2% liquor natrii hypochloritis again, aseptic water washing 3 times.Add rapidly 3% hydrogen peroxide dipping sterilization 10min, often shake beaker in immersion process, then outwell and rise hydrogen peroxide liquid, after aseptic water washing 5 times, then soak for subsequent use with sterile water;
(2) process of material: the scale aseptic filter paper of disinfecting is blotted, aseptically scale also had base portion according to upper, middle and lower portion, be cut into the fritter that 3-5mm is square respectively, and upper, middle and lower portion also has the material of base portion to carry out next step cultivation respectively.
(3) callus induction and Bud polarization: be seeded in callus inducing medium by the explant that step (2) obtains under sterile conditions, this medium is the GA at 6-BA, the 0.8mg/L that with the addition of 0.6mg/L in MS conventional medium
3, the ZT of the sucrose of 80mg/L, 10g/L agar and 0.4mg/L; Cultivation temperature is 26 DEG C, light application time 13h/d, intensity of illumination 2200Lx, PH is 6.5, the material on top formed callus through 14 days, and the material at middle part formed callus through 12 days, and the material of bottom formed callus through 10 days, the material of base portion formed callus through 12 days, and the material of its middle and lower part and base portion also has part directly to differentiate little thin bud;
(4) propagation of bud: direct differentiation little thin bud out in step (3) is inoculated into 3/4MS+ agar 6.0g/L+NAA0.5mg/L+GA
3on the medium of 0.5mg/L+ZT0.2mg/L+ sucrose 15g/L, cultivation temperature is 25 DEG C, light application time 12h/d, intensity of illumination 2000Lx, PH is 6.5, every bottle graft kind 1 bud, by the clove that grows thickly of induction after 8-12 days, wherein robust growth, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out squamous subculture again.
(5) squamous subculture of callus: the callus formed in step (3) is inoculated into 1/2MS+6-BA2.0mg/L+GA
3on the medium of 0.5mg/L+KT0.5mg/L+ sucrose 20g/L+ agar 5g/L, cultivation temperature is 25 DEG C, light application time 12h/d, and intensity of illumination 2000Lx, PH are 6.5, grow clove through 15-20 days;
(6) culture of rootage: carry out root induction by being inoculated in respectively in following inducing culture one by one through step (4) and step (5) differentiation clove out: 3/4MS medium+agar 8g/L+KT1.2mg/L+ sucrose 20mg/L+ active carbon 2.0mg/L, cultivation temperature is 24 DEG C, periodicity of illumination 12h/d, luminous intensity is 2200lux, pH value is 6.5, treated that clove grew to 2-3cm through 20-25 days high, when root system is more healthy and stronger and when growing 6-10 root, namely start the next stage;
(7) greenhouse hardening and transplanting: in order to improve survival rate, before transplanting, first blake bottle is moved to greenhouse, temperature hardening one week when 20-25 DEG C, first in culturing room, cultivation bottle cap is opened during transplanting, taking-up tap water removes the agar of root, avoid damaging root, then 2min is soaked with 2000 times of potassium permanganate, be transplanted to after drying by humus soil, vermiculite, perlitic mixed proportion is on the seedbed of 1: 2: 1, for keeping higher humidity, spray every day 2 times, after growing young leaves, the suitable Nitrogen Top Dressing of visual plant strain growth situation, land for growing field crops is moved to when plant grows to 4-8cm height through 10-20 days, and suitable carries out Shading treatment.
Embodiment 3:
A growth-promoting cultivation method for Hupeh Fritillary Bulb, is characterized in that, comprise the following steps:
(1) explant selection with disinfect: first select robust growth, ripe fresh Hupeh Fritillary Bulb bulb ball, peel off scale and the remaining old root of base portion of bulb outermost layer shriveling, first use running water running water 2-3 time, then filter paper suck dry moisture is used with after 50% rifampin, 300 times of immersion bubble 20-30min, then by the strip off in layer of bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, superclean bench is first used 75% alcohol-pickled sterilization 30s, remove alcohol, by rinsed with sterile water 3-4 time, to sterilize 10min with 2% liquor natrii hypochloritis again, aseptic water washing 2-3 time.Add rapidly 3% hydrogen peroxide dipping sterilization 8-10min, often shake beaker in immersion process, then outwell and rise hydrogen peroxide liquid, after aseptic water washing 4-5 time, then soak for subsequent use with sterile water;
(2) process of material: the scale aseptic filter paper of disinfecting is blotted, aseptically scale also had base portion according to upper, middle and lower portion, be cut into the fritter that 3-5mm is square respectively, and upper, middle and lower portion also has the material of base portion to carry out next step cultivation respectively;
(3) callus induction and Bud polarization: be seeded in callus inducing medium by the explant that step (2) obtains under sterile conditions, this medium is the GA at 6-BA, the 0.8mg/L that with the addition of 0.6mg/L in MS conventional medium
3, the ZT of the sucrose of 80mg/L, 10g/L agar and 0.4mg/L; Cultivation temperature is 26 DEG C, light application time 13h/d, and intensity of illumination 2200Lx, PH are 6.5, forms callus through 10-15 days, also has part directly to differentiate little thin bud;
(4) propagation of bud: direct differentiation little thin bud out in step (3) is inoculated into 3/4MS+ agar 6.0g/L+NAA0.5mg/L+GA
3on the medium of 0.5mg/L+ZT0.2mg/L+ sucrose 15g/L, cultivation temperature is 25 DEG C, light application time 12h/d, intensity of illumination 2000Lx, PH is 6.5, every bottle graft kind 1 bud, by the clove that grows thickly of induction after 8-12 days, wherein robust growth, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out squamous subculture again;
(5) squamous subculture of callus: by the callus formed in step (3) be inoculated into 1/2MS+6-BA2.0mg/L+GA
3on the medium of 0.5mg/L+KT0.5mg/L+ sucrose 20g/L+ agar 5g/L, cultivation temperature is 25 DEG C, light application time 12h/d, and intensity of illumination 2000Lx, PH are 6.5, grow clove through 15-20 days;
(6) culture of rootage: carry out root induction by being inoculated in respectively in following inducing culture one by one through step (4) and step (5) differentiation clove out: 3/4MS medium+agar 8g/L+KT1.2mg/L+ sucrose 20mg/L+ active carbon 2.0mg/L, cultivation temperature is 24 DEG C, periodicity of illumination 12h/d, luminous intensity is 2200lux, pH value is 6.5, treated that clove grew to 2-3cm through 20-25 days high, when root system is more healthy and stronger and when growing 6-10 root, namely start the next stage;
(7) soil matrix management: regulate soil pH value to be 7.0 ~ 8.2, use after mixing with 5 times of volume fine earths after Cosan is sieved, except regulating soil pH value with Cosan, Cosan is then better with peat composed of rotten mosses mixing application effect, or use pine needle to coordinate Cosan to improve soil, wherein pine needle, original soil, muck, Cosan are according to ratio of weight and number 2:3:1:2, or apply in soil saw not, the peat composed of rotten mosses, liver moss and Cosan carry out soil melioration, wherein saw not, the peat composed of rotten mosses, liver moss and Cosan ratio of weight and number be 1:1:1:2;
Increase soil organic matter content: the field planting ditch digging wide 50cm, dark 40cm, organic matter, fertilizer, original soil are improved the soil in the ratio of 1:1:1, organic matter uses the acid peat composed of rotten mosses, if first January more than must be stacked with sawdust, bark, become thoroughly decomposed completely after decomposing and re-use, as employment domestic animals manure become thoroughly decomposed or straw leaf rot to become thoroughly decomposed after the natural fertilizers that formed, note not mixing ash, lime alkaline matter, content reaches more than 3% ~ 5%, to increase the permeability of soil;
Soil covers: can carry out after soil covers seedling planting, by covering uniform fold at bed surface, wide 1 meter, thick 5 ~ 10 centimetres, cover 2 cm thicks more every year later, to keep original thickness, the new sawdust decomposed if application is not rotted, the nitrogenous fertilizer of 50% need be enriched, rot the sawdust decomposed, nitrogen fertilizer amount should correspondingly reduce, cover unregistered land film and can prevent soil water evaporation, control weeds, improve ground temperature, preferably the orchard of drip irrigation facility is being had to apply, cover unregistered land film and cover the organic matter better or front weed killer herbicide of field planting of effect that combines and kill weeds, entirely cultivate field after field planting and sow green manure Trifolium repense, the leguminous plants such as red clover, field can be covered completely after March, soil fertility can be increased again,
(8) greenhouse hardening and transplanting: in order to improve survival rate, before transplanting, first blake bottle is moved to greenhouse, temperature hardening one week when 20-25 DEG C, first in culturing room, cultivation bottle cap is opened during transplanting, taking-up tap water removes the agar of root, avoid damaging root, then 2min is soaked with 2000 times of potassium permanganate, be transplanted in the soil matrix in step (7) after drying, for keeping higher humidity, spray every day 2 times, after growing young leaves, the suitable Nitrogen Top Dressing of visual plant strain growth situation, land for growing field crops is moved to when plant grows to 4-8cm height through 10-20 days, and suitable carries out Shading treatment.
Through above-mentioned process, calli induction rate reaches more than 96%, and proliferation times has exceeded 7.3 times, rooting rate reaches more than 98%, final transplanting survival rate reaches more than 98%, and plant strain growth is vigorous, improves soil fertility 10.2%, Disease management rate 21%.
The foregoing is only embodiments of the invention; not thereby the scope of the claims of the present invention is limited; every utilize description of the present invention to do equivalent structure or equivalent flow process conversion; or be directly or indirectly used in other relevant technical fields, be all in like manner included in scope of patent protection of the present invention.
Claims (1)
1. a growth-promoting cultivation method for Hupeh Fritillary Bulb, is characterized in that, comprise the following steps:
(1) explant selection with disinfect: first select robust growth, ripe fresh Hupeh Fritillary Bulb bulb ball, peel off scale and the remaining old root of base portion of bulb outermost layer shriveling, first use running water running water 2-3 time, then filter paper suck dry moisture is used with after 50% rifampin, 300 times of immersion bubble 20-30min, then by the strip off in layer of bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, superclean bench is first used 75% alcohol-pickled sterilization 30s, remove alcohol, by rinsed with sterile water 3-4 time, to sterilize 10min with 2% liquor natrii hypochloritis again, aseptic water washing 2-3 time, add rapidly 3% hydrogen peroxide dipping sterilization 8-10min, often shake beaker in immersion process, then outwell hydrogen peroxide liquid, after aseptic water washing 4-5 time, then soak for subsequent use with sterile water,
(2) process of material: the scale aseptic filter paper of disinfecting is blotted, aseptically scale also had base portion according to upper, middle and lower portion, be cut into the fritter that 3-5mm is square respectively, and upper, middle and lower portion also has the material of base portion to carry out next step cultivation respectively;
(3) callus induction and Bud polarization: under sterile conditions the explant that step (2) obtains is seeded in callus inducing medium, this medium is the ZT of the sucrose of the GA3 of 6-BA, the 0.8mg/L that with the addition of 0.6mg/L in MS conventional medium, 80mg/L, 10g/L agar and 0.4mg/L; Cultivation temperature is 26 DEG C, light application time 13h/d, and intensity of illumination 2200Lx, PH are 6.5, forms callus through 10-15 days, also has part directly to differentiate little thin bud;
(4) propagation of bud: direct differentiation little thin bud out in step (3) is inoculated on the medium of 3/4MS+ agar 6.0g/L+NAA0.5mg/L+GA30.5mg/L+ZT0.2mg/L+ sucrose 15g/L, cultivation temperature is 25 DEG C, light application time 12h/d, intensity of illumination 2000Lx, PH is 6.5, every bottle graft kind 1 bud, by the clove that grows thickly of induction after 8-12 days, wherein robust growth, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out squamous subculture again;
(5) squamous subculture of callus: the callus formed in step (3) is inoculated on the medium of 1/2MS+6-BA2.0mg/L+GA30.5mg/L+KT0.5mg/L+ sucrose 20g/L+ agar 5g/L, cultivation temperature is 25 DEG C, light application time 12h/d, intensity of illumination 2000Lx, PH is 6.5, grows clove through 15-20 days;
(6) culture of rootage: carry out root induction by being inoculated in respectively in following inducing culture one by one through step (4) and step (5) differentiation clove out: 3/4MS medium+agar 8g/L+KT1.2mg/L+ sucrose 20mg/L+ active carbon 2.0mg/L, cultivation temperature is 24 DEG C, periodicity of illumination 12h/d, luminous intensity is 2200lux, pH value is 6.5, treated that clove grew to 2-3cm through 20-25 days high, when root system is more healthy and stronger and when growing 6-10 root, namely start the next stage;
(7) soil matrix management: regulate soil pH value to be 7.0 ~ 8.2, use after mixing with 5 times of volume fine earths after Cosan is sieved, soil pH value is regulated with Cosan, or Cosan mixes with the peat composed of rotten mosses uses adjustment soil pH value, or use pine needle to coordinate Cosan to improve soil, wherein pine needle, original soil, muck, Cosan are according to ratio of weight and number 2:3:1:2, or apply in soil saw not, the peat composed of rotten mosses, liver moss and Cosan carry out soil melioration, wherein saw not, the peat composed of rotten mosses, liver moss and Cosan ratio of weight and number be 1:1:1:2;
Increase soil organic matter content: the field planting ditch digging wide 50cm, dark 40cm, organic matter, fertilizer, original soil are improved the soil in the ratio of 1:1:1, organic matter uses the acid peat composed of rotten mosses, if first January more than must be stacked with sawdust, bark, become thoroughly decomposed completely after decomposing and re-use, as employment domestic animals manure become thoroughly decomposed or straw leaf rot to become thoroughly decomposed after the natural fertilizers that formed, note not mixing ash, lime alkaline matter, content reaches more than 3% ~ 5%, to increase the permeability of soil;
Soil covers: can carry out after soil covers seedling planting, by covering uniform fold at bed surface, wide 1 meter, thick 5 ~ 10 centimetres, cover 2 cm thicks more every year later, to keep original thickness, the new sawdust decomposed if application is not rotted, the nitrogenous fertilizer of 50% need be enriched, rot the sawdust decomposed, nitrogen fertilizer amount should correspondingly reduce, cover unregistered land film and can prevent soil water evaporation, control weeds, improve ground temperature, covering unregistered land film combines with covering organic matter or weeds killed by the front weed killer herbicide of field planting, entirely cultivate field after field planting and sow green manure Trifolium repense, the leguminous plants such as red clover, field can be covered completely after March, soil fertility can be increased again,
(8) greenhouse hardening and transplanting: in order to improve survival rate, before transplanting, first blake bottle is moved to greenhouse, temperature hardening one week when 20-25 DEG C, first in culturing room, cultivation bottle cap is opened during transplanting, taking-up tap water removes the agar of root, avoid damaging root, then 2min is soaked with 2000 times of potassium permanganate, be transplanted in the soil matrix in step (7) after drying, for keeping higher humidity, spray every day 2 times, after growing young leaves, the suitable Nitrogen Top Dressing of visual plant strain growth situation, land for growing field crops is moved to when plant grows to 4-8cm height through 10-20 days, and suitable carries out Shading treatment.
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