CN103444527A - Culture method for promoting growth of Fritillaria hupehensis Hsiao et K. C. Hsia - Google Patents
Culture method for promoting growth of Fritillaria hupehensis Hsiao et K. C. Hsia Download PDFInfo
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Abstract
The invention discloses a culture method for promoting the growth of Fritillaria hupehensis Hsiao et K. C. Hsia. The culture method comprises the steps of selecting and disinfecting an explant, treating a material, performing induction and bud initiation on calluses, proliferating buds, performing secondary culture on the calluses, performing rooting culture, performing greenhouse seedling exercising and soil management, transplanting and the like. Through the treatment, the formation rate of the calluses reaches more than 96%, the proliferation exceeds 7.3 times, the rooting rate reaches more than 98%, the final transplanting survival rate reaches more than 98%, the plants grow vigorously, and the soil fertility and the disease control rate are improved. Moreover, the proliferation of the buds and the proliferation of the calluses during culture are separated, so that the utilization ratio of the material is improved, and the survival rates of the buds and the calluses are improved.
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 (Fritillaria hupehensis Hsiao et K. C. Hsia), claim again Hubei Province shellfish, the plate shellfish, the kiln shellfish, Feng Bei, for Liliaceae (Liliaceae) Fritillaria (Fritillaria L .) herbaceos perennial, Hupeh Fritillary Bulb is at Jianshi, hubei, Xuanen one is with cultivation, the GAP base kind of bestowing favour now, it is one of medicinal material of greatly developing of Enshi, extensively exist at present environmental deterioration, the problem of soil nutrient deficiency, and the breeding of Hupeh Fritillary Bulb be take the bulb breeding as main, also available scale and seminal propagation, after carrying out vegetative propagation many generations, should carry out 1 sexual propagation.But because Hupeh Fritillary Bulb is bloomed many, result is few, difficulty is received more seed, seminal propagation is still in experimental stage, because Hupeh Fritillary Bulb has higher medical value, main edible its bulb, large to bulb of fritillary bulb demand at present, directly with underground bulb, bred and also grown slowly, need 4-5 could grow the size to the commodity bulb, growth cycle is long, therefore its area under cultivation and output all are 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, so breeding fast the Hupeh Fritillary Bulb tool has very important significance, the income that improves people is had to very high value.
Through 20 years of researches, Hupeh Fritillary Bulb has become in the bulb of fritillary the second largest main flow bulb of fritillary commodity that are only second to fritillaria thunbergii at present, utilizes tissue cultivation and fast breeding technique can improve reproduction coefficient, enlarges area under cultivation.To producing tool, be very helpful.At present the cultivation of bulb of fritillary tissue and fast breeding technique have been had to a lot of research, but relatively less to the research of Hupeh Fritillary Bulb, and do not form a set of complete breeding system.
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 kind of growth-promoting cultivation method of Hupeh Fritillary Bulb comprises the following steps:
(1) selection of explant with disinfect: at 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 with running water flowing water, rinse 2-3 time, then with after 300 times of immersion bubble 20-30 min of 50% rifampin, using the filter paper suck dry moisture, then the strip off in layer by the bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, first use alcohol-pickled sterilization 30 s of 75 % on superclean bench, remove alcohol, by rinsed with sterile water 3-4 time, again with the 2% liquor natrii hypochloritis 10min that sterilizes, aseptic water washing 2-3 time.Add rapidly 3 % hydrogen peroxide dipping sterilization 8-10 min, often shake beaker in immersion process, then outwell and rise hydrogen peroxide liquid, with after aseptic water washing 4-5 time, then soak standby with sterile water.
(2) processing of material: the scale of disinfecting is blotted with aseptic filter paper, under aseptic condition, scale is also had to base portion according to upper, middle and lower section, be cut into respectively the square fritter of 3-5mm, and upper, middle and lower section also has the material of base portion to carry out respectively next step cultivation.
(3) callus induction and bud differentiation: during the explant under aseptic condition, step (2) obtained is seeded in callus inducing medium, this medium is to add the 6-BA of 0.6mg/L, the GA of 0.8mg/L in the MS conventional medium
3, the ZT of the sucrose of 80mg/L, 10 g/L agar and 0.4 mg/L; Cultivation temperature is 26 ℃, light application time 13h/d, and intensity of illumination 2 200 Lx, PH is 6.5, through 10-15 days formation callus, also has part directly to differentiate little thin bud;
(4) propagation of bud: the little thin bud directly broken up out in step (3) is inoculated into to 3/4MS+ agar 6.0g/L+ NAA 0. 5mg/L+GA
30.5mg/L+ on the medium of ZT 0.2mg/L+sucrose 15g/L, cultivation temperature is 25 ℃, light application time 12h/d, intensity of illumination 2 000 Lx, PH is 6.5,1 bud of every bottle graft kind, the clove that grows thickly that will induce after 8-12 days, robust growth wherein, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out the subculture cultivation again.
(5) subculture of callus is cultivated: the callus formed in step (3) is inoculated into to 1/2MS+6-BA2.0 mg/L+GA
30.5mg/L+ on the medium of KT 0.5mg/L+sucrose 20g/L+agar 5 g/L, cultivation temperature is 25 ℃, light application time 12h/d, and intensity of illumination 2 000 Lx, PH is 6.5, through within 15-20 days, growing clove.
(6) culture of rootage: will be inoculated in respectively one by one in following inducing culture and carry out root induction 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 ℃, periodicity of illumination 12h/d, luminous intensity is 2200lux, pH value is 6.5, high through within 20-25 days, treating that clove grows to 2-3cm, when root system is more healthy and stronger and grow 6-10 root, start the next stage;
(7) soil matrix management: regulating the soil pH value is 7.0～8.2, after mixing with 5 times of volume fine earths after Cosan is sieved, use, except regulating the soil pH value with Cosan, Cosan is better with peat composed of rotten mosses mixing application effect, perhaps use pine needle to coordinate Cosan to be improved soil, wherein pine needle, original soil, muck, Cosan are according to ratio of weight and number 2:3:1:2, perhaps in soil, apply saw not, the peat composed of rotten mosses, liver moss and Cosan carry out soil melioration, wherein saw is not, the ratio of weight and number of the peat composed of rotten mosses, liver moss and Cosan is 1:1:1:2;
Increase soil organic matter content: the field planting ditch that digs wide 50 cm, dark 40 cm, organic matter, fertilizer, original soil are improved the soil in the ratio of 1:1:1, the acid peat composed of rotten mosses of organic matter, if more than must first stacking January with sawdust, bark, become thoroughly decomposed fully after decomposing and re-use, as the employment domestic animals manure becomes thoroughly decomposed or straw leaf rot the to become thoroughly decomposed natural fertilizers of rear formation, note not mixing ash, lime alkaline matter, content reaches more than 3%～5%, to increase the permeability of soil;
Soil covers: soil can carry out after covering seedling planting, by the covering uniform fold at bed surface, wide 1 meter, thick 5～10 centimetres, cover again 2 cm thicks every year later, to keep original thickness, the new sawdust decomposed if application is not rotted, need enrich 50% nitrogenous fertilizer, the sawdust decomposed rots, nitrogen fertilizer amount should correspondingly reduce, cover the unregistered land film and can prevent soil water evaporation, control weeds, improve ground temperature, preferably the orchard application of drip irrigation facility is being arranged, covering the unregistered land film is combined with the organic phase of covering before the better or field planting of effect and kills weeds with weed killer herbicide, entirely cultivate field after field planting and sow green manure Bai Sanye, the leguminous plants such as red clover, can cover field fully after March, can increase soil fertility again,
(8) greenhouse hardening and transplanting: in order to improve survival rate, at first blake bottle was moved to greenhouse before transplanting, temperature hardening one week in the time of 20-25 ℃, first in culturing room, open the cultivation bottle cap during transplanting, the agar that removes root is rinsed in taking-up with running water, avoid damaging root, then soak 2min with 2000 times of potassium permanganate, after drying, be transplanted in the soil matrix in step (7), for keeping higher humidity, spray every day 2 times, after growing young leaves, the suitable Nitrogen Top Dressing of visual plant strain growth situation, when growing to 4-8cm, plant moved to land for growing field crops through 10-20 days when high, and the suitable processing of being shaded.
The invention has the beneficial effects as follows:
(1) set up complete tissue culture propagating system, for the fast breeding of Hupeh Fritillary Bulb is laid a solid foundation;
(2) at first by 300 times of immersions of 50% rifampin, steeped 20-30 min before explant is disinfected, sterilization is more thorough like this, and the formation rate of callus is improved;
(3) the upper, middle and lower section of Hupeh Fritillary Bulb also has the differentiation capability difference of base portion, also has the material of base portion to carry out respectively next step upper, middle and lower section and cultivates, and the time of formation callus that is conducive to material is more unified, is conducive to management;
(4) propagation of the propagation of bud and callus is separated to processing, can improve like this availability of material, improve the survival rate of the two.
(5) improve soil fertility, lowered the generation of damage by disease and insect.
Embodiment
Below preferred embodiment of the present invention is described in detail, thereby so that advantages and features of the invention can be easier to be it will be appreciated by those skilled in the art that, protection scope of the present invention is made to more explicit defining.
Embodiment 1:
A kind of growth-promoting cultivation method of Hupeh Fritillary Bulb comprises the following steps:
(1) selection of explant with disinfect: at 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 with running water flowing water, rinse 2 times, use the filter paper suck dry moisture after then by 300 times of immersions of 50% rifampin, steeping 20 min, then the strip off in layer by the bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, first use alcohol-pickled sterilization 30 s of 75 % on superclean bench, remove alcohol, by rinsed with sterile water 3 times, again with the 2% liquor natrii hypochloritis 10min that sterilizes, aseptic water washing 2 times.Add rapidly 3 % hydrogen peroxide dipping sterilization 8 min, often shake beaker in immersion process, then outwell and rise hydrogen peroxide liquid, with after aseptic water washing 4 times, then soak standby with sterile water;
(2) processing of material: the scale of disinfecting is blotted with aseptic filter paper, under aseptic condition, scale is also had to base portion according to upper, middle and lower section, be cut into respectively the square fritter of 3-5mm, and upper, middle and lower section also has the material of base portion to carry out respectively next step cultivation;
(3) callus induction and bud differentiation: during the explant under aseptic condition, step (2) obtained is seeded in callus inducing medium, this medium is to add the 6-BA of 0.6mg/L, the GA of 0.8mg/L in the MS conventional medium
3, the ZT of the sucrose of 80mg/L, 10 g/L agar and 0.4 mg/L; Cultivation temperature is 26 ℃, light application time 13h/d, intensity of illumination 2 200 Lx, 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 was through 11 days formation callus, 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: the little thin bud directly broken up out in step (3) is inoculated into to 3/4MS+ agar 6.0g/L+ NAA 0. 5mg/L+GA
30.5mg/L+ on the medium of ZT 0.2mg/L+sucrose 15g/L, cultivation temperature is 25 ℃, light application time 12h/d, intensity of illumination 2 000 Lx, PH is 6.5,1 bud of every bottle graft kind, the clove that grows thickly that will induce after 8-12 days, robust growth wherein, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out the subculture cultivation again;
(5) subculture of callus is cultivated: by the callus that forms in step (3) be inoculated into 1/2MS+6-BA2.0 mg/L+GA
30.5mg/L+ on the medium of KT 0.5mg/L+sucrose 20g/L+agar 5 g/L, cultivation temperature is 25 ℃, light application time 12h/d, and intensity of illumination 2 000 Lx, PH is 6.5, through within 15-20 days, growing clove;
(6) culture of rootage: will be inoculated in respectively one by one in following inducing culture and carry out root induction 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 ℃, periodicity of illumination 12h/d, luminous intensity is 2200lux, pH value is 6.5, high through within 20-25 days, treating that clove grows to 2-3cm, when root system is more healthy and stronger and grow 6-10 root, start the next stage;
(7) greenhouse hardening and transplanting: in order to improve survival rate, at first blake bottle was moved to greenhouse before transplanting, temperature hardening one week in the time of 20-25 ℃, first in culturing room, open the cultivation bottle cap during transplanting, the agar that removes root is rinsed in taking-up with running water, avoid damaging root, then soak 2min with 2000 times of potassium permanganate, after drying, be transplanted to by humus soil, vermiculite, on the seedbed that perlitic mixed proportion is 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, when growing to 4-8cm, plant moved to land for growing field crops through 10-20 days when high, and the suitable processing of being shaded.
Through above-mentioned processing, the formation rate of callus reaches 94%, and the propagation multiple has reached 6.3 times, and rooting rate has reached 96%, and final transplanting survival rate has reached 94%, and plant strain growth is vigorous.
embodiment 2
A kind of growth-promoting cultivation method of Hupeh Fritillary Bulb comprises the following steps:
(1) selection of explant with disinfect: at 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 with running water flowing water, rinse 3 times, use the filter paper suck dry moisture after then by 300 times of immersions of 50% rifampin, steeping 30 min, then the strip off in layer by the bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, first use alcohol-pickled sterilization 30 s of 75 % on superclean bench, remove alcohol, by rinsed with sterile water 4 times, again with the 2% liquor natrii hypochloritis 10min that sterilizes, aseptic water washing 3 times.Add rapidly 3 % hydrogen peroxide dipping sterilization 10 min, often shake beaker in immersion process, then outwell and rise hydrogen peroxide liquid, with after aseptic water washing 5 times, then soak standby with sterile water;
(2) processing of material: the scale of disinfecting is blotted with aseptic filter paper, under aseptic condition, scale is also had to base portion according to upper, middle and lower section, be cut into respectively the square fritter of 3-5mm, and upper, middle and lower section also has the material of base portion to carry out respectively next step cultivation.
(3) callus induction and bud differentiation: during the explant under aseptic condition, step (2) obtained is seeded in callus inducing medium, this medium is to add the 6-BA of 0.6mg/L, the GA of 0.8mg/L in the MS conventional medium
3, the ZT of the sucrose of 80mg/L, 10 g/L agar and 0.4 mg/L; Cultivation temperature is 26 ℃, light application time 13h/d, intensity of illumination 2 200 Lx, 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 was through 10 days formation callus, 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: the little thin bud directly broken up out in step (3) is inoculated into to 3/4MS+ agar 6.0g/L+ NAA 0. 5mg/L+GA
30.5mg/L+ on the medium of ZT 0.2mg/L+sucrose 15g/L, cultivation temperature is 25 ℃, light application time 12h/d, intensity of illumination 2 000 Lx, PH is 6.5,1 bud of every bottle graft kind, the clove that grows thickly that will induce after 8-12 days, robust growth wherein, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out the subculture cultivation again.
(5) subculture of callus is cultivated: the callus formed in step (3) is inoculated into to 1/2MS+6-BA2.0 mg/L+GA
30.5mg/L+ on the medium of KT 0.5mg/L+sucrose 20g/L+agar 5 g/L, cultivation temperature is 25 ℃, light application time 12h/d, and intensity of illumination 2 000 Lx, PH is 6.5, through within 15-20 days, growing clove;
(6) culture of rootage: will be inoculated in respectively one by one in following inducing culture and carry out root induction 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 ℃, periodicity of illumination 12h/d, luminous intensity is 2200lux, pH value is 6.5, high through within 20-25 days, treating that clove grows to 2-3cm, when root system is more healthy and stronger and grow 6-10 root, start the next stage;
(7) greenhouse hardening and transplanting: in order to improve survival rate, at first blake bottle was moved to greenhouse before transplanting, temperature hardening one week in the time of 20-25 ℃, first in culturing room, open the cultivation bottle cap during transplanting, the agar that removes root is rinsed in taking-up with running water, avoid damaging root, then soak 2min with 2000 times of potassium permanganate, after drying, be transplanted to by humus soil, vermiculite, on the seedbed that perlitic mixed proportion is 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, when growing to 4-8cm, plant moved to land for growing field crops through 10-20 days when high, and the suitable processing of being shaded.
Embodiment 3:
A kind of growth-promoting cultivation method of Hupeh Fritillary Bulb, is characterized in that, comprises the following steps:
(1) selection of explant with disinfect: at 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 with running water flowing water, rinse 2-3 time, then with after 300 times of immersion bubble 20-30 min of 50% rifampin, using the filter paper suck dry moisture, then the strip off in layer by the bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, first use alcohol-pickled sterilization 30 s of 75 % on superclean bench, remove alcohol, by rinsed with sterile water 3-4 time, again with the 2% liquor natrii hypochloritis 10min that sterilizes, aseptic water washing 2-3 time.Add rapidly 3 % hydrogen peroxide dipping sterilization 8-10 min, often shake beaker in immersion process, then outwell and rise hydrogen peroxide liquid, with after aseptic water washing 4-5 time, then soak standby with sterile water;
(2) processing of material: the scale of disinfecting is blotted with aseptic filter paper, under aseptic condition, scale is also had to base portion according to upper, middle and lower section, be cut into respectively the square fritter of 3-5mm, and upper, middle and lower section also has the material of base portion to carry out respectively next step cultivation;
(3) callus induction and bud differentiation: during the explant under aseptic condition, step (2) obtained is seeded in callus inducing medium, this medium is to add the 6-BA of 0.6mg/L, the GA of 0.8mg/L in the MS conventional medium
3, the ZT of the sucrose of 80mg/L, 10 g/L agar and 0.4 mg/L; Cultivation temperature is 26 ℃, light application time 13h/d, and intensity of illumination 2200 Lx, PH is 6.5, through 10-15 days formation callus, also has part directly to differentiate little thin bud;
(4) propagation of bud: the little thin bud directly broken up out in step (3) is inoculated into to 3/4MS+ agar 6.0g/L+ NAA 0. 5mg/L+GA
30.5mg/L+ on the medium of ZT 0.2mg/L+sucrose 15g/L, cultivation temperature is 25 ℃, light application time 12h/d, intensity of illumination 2 000 Lx, PH is 6.5,1 bud of every bottle graft kind, the clove that grows thickly that will induce after 8-12 days, robust growth wherein, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out the subculture cultivation again;
(5) subculture of callus is cultivated: by the callus that forms in step (3) be inoculated into 1/2MS+6-BA2.0 mg/L+GA
30.5mg/L+ on the medium of KT 0.5mg/L+sucrose 20g/L+agar 5 g/L, cultivation temperature is 25 ℃, light application time 12h/d, and intensity of illumination 2 000 Lx, PH is 6.5, through within 15-20 days, growing clove;
(6) culture of rootage: will be inoculated in respectively one by one in following inducing culture and carry out root induction 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 ℃, periodicity of illumination 12h/d, luminous intensity is 2200lux, pH value is 6.5, high through within 20-25 days, treating that clove grows to 2-3cm, when root system is more healthy and stronger and grow 6-10 root, start the next stage;
(7) soil matrix management: regulating the soil pH value is 7.0～8.2, after mixing with 5 times of volume fine earths after Cosan is sieved, use, except regulating the soil pH value with Cosan, Cosan is better with peat composed of rotten mosses mixing application effect, perhaps use pine needle to coordinate Cosan to be improved soil, wherein pine needle, original soil, muck, Cosan are according to ratio of weight and number 2:3:1:2, perhaps in soil, apply saw not, the peat composed of rotten mosses, liver moss and Cosan carry out soil melioration, wherein saw is not, the ratio of weight and number of the peat composed of rotten mosses, liver moss and Cosan is 1:1:1:2;
Increase soil organic matter content: the field planting ditch that digs wide 50 cm, dark 40 cm, organic matter, fertilizer, original soil are improved the soil in the ratio of 1:1:1, the acid peat composed of rotten mosses of organic matter, if more than must first stacking January with sawdust, bark, become thoroughly decomposed fully after decomposing and re-use, as the employment domestic animals manure becomes thoroughly decomposed or straw leaf rot the to become thoroughly decomposed natural fertilizers of rear formation, note not mixing ash, lime alkaline matter, content reaches more than 3%～5%, to increase the permeability of soil;
Soil covers: soil can carry out after covering seedling planting, by the covering uniform fold at bed surface, wide 1 meter, thick 5～10 centimetres, cover again 2 cm thicks every year later, to keep original thickness, the new sawdust decomposed if application is not rotted, need enrich 50% nitrogenous fertilizer, the sawdust decomposed rots, nitrogen fertilizer amount should correspondingly reduce, cover the unregistered land film and can prevent soil water evaporation, control weeds, improve ground temperature, preferably the orchard application of drip irrigation facility is being arranged, covering the unregistered land film is combined with the organic phase of covering before the better or field planting of effect and kills weeds with weed killer herbicide, entirely cultivate field after field planting and sow green manure Bai Sanye, the leguminous plants such as red clover, can cover field fully after March, can increase soil fertility again,
(8) greenhouse hardening and transplanting: in order to improve survival rate, at first blake bottle was moved to greenhouse before transplanting, temperature hardening one week in the time of 20-25 ℃, first in culturing room, open the cultivation bottle cap during transplanting, the agar that removes root is rinsed in taking-up with running water, avoid damaging root, then soak 2min with 2000 times of potassium permanganate, after drying, be transplanted in the soil matrix in step (7), for keeping higher humidity, spray every day 2 times, after growing young leaves, the suitable Nitrogen Top Dressing of visual plant strain growth situation, when growing to 4-8cm, plant moved to land for growing field crops through 10-20 days when high, and the suitable processing of being shaded.
Through above-mentioned processing, the formation rate of callus reaches more than 96%, and the propagation multiple has surpassed 7.3 times, rooting rate has reached more than 98%, final transplanting survival rate has reached more than 98%, and plant strain growth is vigorous, has improved soil fertility 10.2%, disease control rate 21%.
The foregoing is only embodiments of the invention; not thereby limit the scope of the claims of the present invention; every equivalent structure or conversion of equivalent flow process that utilizes description of the present invention to do; or directly or indirectly be used in other relevant technical fields, all in like manner be included in scope of patent protection of the present invention.
Claims (1)
1.
a kind of growth-promoting cultivation method of Hupeh Fritillary Bulb, is characterized in that, comprises the following steps:
(1) selection of explant with disinfect: at 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 with running water flowing water, rinse 2-3 time, then with after 300 times of immersion bubble 20-30 min of 50% rifampin, using the filter paper suck dry moisture, then the strip off in layer by the bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, first use alcohol-pickled sterilization 30 s of 75 % on superclean bench, remove alcohol, by rinsed with sterile water 3-4 time, again with the 2% liquor natrii hypochloritis 10min that sterilizes, aseptic water washing 2-3 time,
add rapidly 3 % hydrogen peroxide dipping sterilization 8-10 min, often shake beaker in immersion process, then outwell and rise hydrogen peroxide liquid, with after aseptic water washing 4-5 time, then soak standby with sterile water;
(2) processing of material: the scale of disinfecting is blotted with aseptic filter paper, under aseptic condition, scale is also had to base portion according to upper, middle and lower section, be cut into respectively the square fritter of 3-5mm, and upper, middle and lower section also has the material of base portion to carry out respectively next step cultivation;
(3) callus induction and bud differentiation: during the explant under aseptic condition, step (2) obtained is seeded in callus inducing medium, this medium is to add the 6-BA of 0.6mg/L, the GA3 of 0.8mg/L in the MS conventional medium, the ZT of the sucrose of 80mg/L, 10 g/L agar and 0.4 mg/L; Cultivation temperature is 26 ℃, light application time 13h/d, and intensity of illumination 2200 Lx, PH is 6.5, through 10-15 days formation callus, also has part directly to differentiate little thin bud;
(4) propagation of bud: the little thin bud directly broken up out in step (3) is inoculated on the medium of 3/4MS+ agar 6.0g/L+ NAA 0. 5mg/L+GA3 0.5mg/L+ ZT 0.2mg/L+sucrose 15g/L, cultivation temperature is 25 ℃, light application time 12h/d, intensity of illumination 2 000 Lx, PH is 6.5,1 bud of every bottle graft kind, the clove that grows thickly that to induce after 8-12 days, robust growth wherein, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out the subculture cultivation again;
(5) subculture of callus is cultivated: by the callus that forms in step (3) be inoculated on the medium of 1/2MS+6-BA2.0 mg/L+GA3 0.5mg/L+ KT 0.5mg/L+sucrose 20g/L+agar 5 g/L, cultivation temperature is 25 ℃, light application time 12h/d, intensity of illumination 2 000 Lx, PH is 6.5, through within 15-20 days, growing clove;
(6) culture of rootage: will be inoculated in respectively one by one in following inducing culture and carry out root induction 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 ℃, periodicity of illumination 12h/d, luminous intensity is 2200lux, pH value is 6.5, high through within 20-25 days, treating that clove grows to 2-3cm, when root system is more healthy and stronger and grow 6-10 root, start the next stage;
(7) soil matrix management: regulating the soil pH value is 7.0～8.2, after mixing with 5 times of volume fine earths after Cosan is sieved, use, except regulating the soil pH value with Cosan, Cosan is better with peat composed of rotten mosses mixing application effect, perhaps use pine needle to coordinate Cosan to be improved soil, wherein pine needle, original soil, muck, Cosan are according to ratio of weight and number 2:3:1:2, perhaps in soil, apply saw not, the peat composed of rotten mosses, liver moss and Cosan carry out soil melioration, wherein saw is not, the ratio of weight and number of the peat composed of rotten mosses, liver moss and Cosan is 1:1:1:2;
increase soil organic matter content: the field planting ditch that digs wide 50 cm, dark 40 cm, organic matter, fertilizer, original soil are improved the soil in the ratio of 1:1:1, the acid peat composed of rotten mosses of organic matter, if more than must first stacking January with sawdust, bark, become thoroughly decomposed fully after decomposing and re-use, as the employment domestic animals manure becomes thoroughly decomposed or straw leaf rot the to become thoroughly decomposed natural fertilizers of rear formation, note not mixing ash, lime alkaline matter, content reaches more than 3%～5%, to increase the permeability of soil;
soil covers: soil can carry out after covering seedling planting, by the covering uniform fold at bed surface, wide 1 meter, thick 5～10 centimetres, cover again 2 cm thicks every year later, to keep original thickness, the new sawdust decomposed if application is not rotted, need enrich 50% nitrogenous fertilizer, the sawdust decomposed rots, nitrogen fertilizer amount should correspondingly reduce, cover the unregistered land film and can prevent soil water evaporation, control weeds, improve ground temperature, preferably the orchard application of drip irrigation facility is being arranged, covering the unregistered land film is combined with the organic phase of covering before the better or field planting of effect and kills weeds with weed killer herbicide, entirely cultivate field after field planting and sow green manure Bai Sanye, the leguminous plants such as red clover, can cover field fully after March, can increase soil fertility again,
(8) greenhouse hardening and transplanting: in order to improve survival rate, at first blake bottle was moved to greenhouse before transplanting, temperature hardening one week in the time of 20-25 ℃, first in culturing room, open the cultivation bottle cap during transplanting, the agar that removes root is rinsed in taking-up with running water, avoid damaging root, then soak 2min with 2000 times of potassium permanganate, after drying, be transplanted in the soil matrix in step (7), for keeping higher humidity, spray every day 2 times, after growing young leaves, the suitable Nitrogen Top Dressing of visual plant strain growth situation, when growing to 4-8cm, plant moved to land for growing field crops through 10-20 days when high, and the suitable processing of being shaded.
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| CN111296211A (en) * | 2020-03-12 | 2020-06-19 | 重庆市药物种植研究所 | Rapid propagation method of fritillaria taipaiensis bulbs |
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