CN103283601B - Method for rapidly propagating Hupeh fritillary by utilizing tissue culture technology - Google Patents
Method for rapidly propagating Hupeh fritillary by utilizing tissue culture technology Download PDFInfo
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Abstract
The invention discloses a method for rapidly propagating Hupeh fritillary by utilizing a tissue culture technology. The method comprises the steps of selecting and sterilizing a material, processing the material, inducing callus, differentiating buds, proliferating the buds, performing primary culture of the callus, performing rooting culture, performing greenhouse seedling hardening and transplanting and the like. After the treatments, the formation rate of the callus reaches over 94 percent; the proliferation rate exceeds 6.3 times; the rooting rate reaches over 96 percent; the final transplanting survival rate reaches over 94 percent; and moreover, the plants grow vigorously. In addition, in a culture process, the propagation of the buds and the propagation of the callus are treated separately, so that the utilization rate of the material is increased, and the survival rates of the buds and the callus are increased.
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, plate shellfish, 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 bulb breeding as main, also available scale and seminal propagation, carry out after 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, breed and also grow slowly, need 4-5 could grow the size to commodity bulb, growth cycle is long, therefore its area under cultivation and output are all very restricted, owing at present the demand of the bulb of fritillary being increased considerably, also when the river rises the boat goes up thereupon for price, so breeding fast Hupeh Fritillary Bulb tool has very important significance, to improving people's income, there is very high value.
Through 20 years of researches, Hupeh Fritillary Bulb has become the second largest main flow bulb of fritillary commodity that are only second to fritillaria thunbergii in the bulb of fritillary at present, utilizes tissue cultivation and fast breeding technique can improve reproduction coefficient, expands 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 method that can utilize tissue culture technique Fast-propagation Hupeh Fritillary Bulb.
For solving the problems of the technologies described above, the technical scheme that the present invention adopts is:
A method of utilizing tissue culture technique Fast-propagation Hupeh Fritillary Bulb, comprises the following steps:
(1) selection of explant 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 with running water flowing water, rinse 2-3 time, then with using filter paper suck dry moisture after 300 times of immersion bubble 20-30 min of 50% rifampin, then by the strip off in layer of bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, on superclean bench, first use alcohol-pickled sterilization 30 s of 75 %, 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, in immersion process, often shake beaker, 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 portion, be cut into respectively the square fritter of 3-5mm, and upper, middle and lower portion also has the material of base portion to carry out respectively next step cultivation.
(3) callus induction and bud differentiation: the explant under aseptic condition, step (2) being obtained is seeded in callus inducing medium, and this medium is in MS conventional medium, to have added the 6-BA of 0.6mg/L, the GA of 0.8mg/L
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)
the propagation of bud: the little thin bud directly breaking up out in step (3) is inoculated into 3/4MS+ agar 6.0g/L+ NAA 0. 5mg/L+GA
3on the medium of 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, and 1 bud of every bottle graft kind, will induce the clove that grows thickly after 8-12 days, robust growth wherein, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out subculture cultivation again.
(5) subculture of callus is cultivated: the callus forming in step (3) is inoculated into 1/2MS+6-BA2.0 mg/L+GA
3on the medium of 0.5mg/L+ 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: carry out root induction in following inducing culture by being inoculated in respectively 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 ℃, 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, before transplanting, first blake bottle is moved to greenhouse, temperature hardening one week in the time of 20-25 ℃, transplant in Shi Xian culturing room and open cultivation bottle cap, the agar that removes root is rinsed in taking-up with running water, avoid damaging root, then with 2000 times of potassium permanganate, soak 2min, after drying, be transplanted to 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, depending on the suitable Nitrogen Top Dressing of plant strain growth situation, through 10-20 days, when growing to 4-8cm, plant moves to land for growing field crops when high, and the suitable processing of shading.
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) before explant is disinfected, first by 300 times of immersions of 50% rifampin, steep 20-30 min, sterilization is more thorough like this, and the formation rate of callus is improved;
(3) it is different that the upper, middle and lower portion of Hupeh Fritillary Bulb also has the differentiation capability of base portion, also has the material of base portion to carry out respectively next step upper, middle and lower portion and cultivate, 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.
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.
A method of utilizing tissue culture technique Fast-propagation Hupeh Fritillary Bulb, comprises the following steps:
(1) selection of explant 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 with running water flowing water, rinse 2 times, then after steeping 20 min by 300 times of immersions of 50% rifampin, use filter paper suck dry moisture, then by the strip off in layer of bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, on superclean bench, first use alcohol-pickled sterilization 30 s of 75 %, 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, in immersion process, often shake beaker, 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 portion, be cut into respectively the square fritter of 3-5mm, and upper, middle and lower portion also has the material of base portion to carry out respectively next step cultivation;
(3) callus induction and bud differentiation: the explant under aseptic condition, step (2) being obtained is seeded in callus inducing medium, and this medium is in MS conventional medium, to have added the 6-BA of 0.6mg/L, the GA of 0.8mg/L
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)
the propagation of bud: the little thin bud directly breaking up out in step (3) is inoculated into 3/4MS+ agar 6.0g/L+ NAA 0. 5mg/L+GA
3on the medium of 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, and 1 bud of every bottle graft kind, will induce the clove that grows thickly after 8-12 days, robust growth wherein, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out subculture cultivation again;
(5) subculture of callus is cultivated: by the callus forming in step (3) be inoculated into 1/2MS+6-BA2.0 mg/L+GA
3on the medium of 0.5mg/L+ 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: carry out root induction in following inducing culture by being inoculated in respectively 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 ℃, 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, before transplanting, first blake bottle is moved to greenhouse, temperature hardening one week in the time of 20-25 ℃, transplant in Shi Xian culturing room and open cultivation bottle cap, the agar that removes root is rinsed in taking-up with running water, avoid damaging root, then with 2000 times of potassium permanganate, soak 2min, after drying, be transplanted to 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, depending on the suitable Nitrogen Top Dressing of plant strain growth situation, through 10-20 days, when growing to 4-8cm, plant moves to land for growing field crops when high, and the suitable processing of shading.
Through above-mentioned processing, the formation rate of callus reaches 94%, and 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 method of utilizing tissue culture technique Fast-propagation Hupeh Fritillary Bulb, comprises the following steps:
(1) selection of explant 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 with running water flowing water, rinse 3 times, then after steeping 30 min by 300 times of immersions of 50% rifampin, use filter paper suck dry moisture, then by the strip off in layer of bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, on superclean bench, first use alcohol-pickled sterilization 30 s of 75 %, 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, in immersion process, often shake beaker, 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 portion, be cut into respectively the square fritter of 3-5mm, and upper, middle and lower portion also has the material of base portion to carry out respectively next step cultivation.
(3) callus induction and bud differentiation: the explant under aseptic condition, step (2) being obtained is seeded in callus inducing medium, and this medium is in MS conventional medium, to have added the 6-BA of 0.6mg/L, the GA of 0.8mg/L
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)
the propagation of bud: the little thin bud directly breaking up out in step (3) is inoculated into 3/4MS+ agar 6.0g/L+ NAA 0. 5mg/L+GA
3on the medium of 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, and 1 bud of every bottle graft kind, will induce the clove that grows thickly after 8-12 days, robust growth wherein, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out subculture cultivation again.
(5) subculture of callus is cultivated: the callus forming in step (3) is inoculated into 1/2MS+6-BA2.0 mg/L+GA
3on the medium of 0.5mg/L+ 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: carry out root induction in following inducing culture by being inoculated in respectively 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 ℃, 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, before transplanting, first blake bottle is moved to greenhouse, temperature hardening one week in the time of 20-25 ℃, transplant in Shi Xian culturing room and open cultivation bottle cap, the agar that removes root is rinsed in taking-up with running water, avoid damaging root, then with 2000 times of potassium permanganate, soak 2min, after drying, be transplanted to 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, through 10-20 days, when growing to 4-8cm, plant moves to land for growing field crops when high, and the suitable processing of shading.
Through above-mentioned processing, the formation rate of callus reaches 95%, and propagation multiple has reached 6.5 times, and rooting rate has reached 96%, and final transplanting survival rate has reached 98%, and plant strain growth is vigorous.
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 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 method of utilizing tissue culture technique Fast-propagation Hupeh Fritillary Bulb, is characterized in that, comprises the following steps:
(1) selection of explant 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 with running water flowing water, rinse 2-3 time, then with using filter paper suck dry moisture after 300 times of immersion bubble 20-30 min of 50% rifampin, then by the strip off in layer of bulb ball, then the scale of strip off is transferred in the beaker of autoclave sterilization, on superclean bench, first use alcohol-pickled sterilization 30 s of 75 %, 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, in immersion process, often shake beaker, then outwell and rise hydrogen peroxide liquid, with after aseptic water washing 4-5 time, with sterile water, soak standby again,
(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 portion, be cut into respectively the square fritter of 3-5mm, and upper, middle and lower portion also has the material of base portion to carry out respectively next step cultivation;
(3) callus induction and bud differentiation: the explant under aseptic condition, step (2) being obtained is seeded in callus inducing medium, and this medium is in MS conventional medium, to have added the 6-BA of 0.6mg/L, the GA of 0.8mg/L
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 breaking up out in step (3) is inoculated into 3/4MS+ agar 6.0g/L+ NAA 0. 5mg/L+GA
3on the medium of 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, and 1 bud of every bottle graft kind, will induce the clove that grows thickly after 8-12 days, robust growth wherein, the clove that grows fine are forwarded to root media, and remaining little simple bud carries out subculture cultivation again;
(5) subculture of callus is cultivated: by the callus forming in step (3) be inoculated into 1/2MS+6-BA2.0 mg/L+GA
3on the medium of 0.5mg/L+ 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: carry out root induction in following inducing culture by being inoculated in respectively 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 ℃, 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, before transplanting, first blake bottle is moved to greenhouse, temperature hardening one week in the time of 20-25 ℃, transplant in Shi Xian culturing room and open cultivation bottle cap, the agar that removes root is rinsed in taking-up with running water, avoid damaging root, then with 2000 times of potassium permanganate, soak 2min, after drying, be transplanted to 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, depending on the suitable Nitrogen Top Dressing of plant strain growth situation, through 10-20 days, when growing to 4-8cm, plant moves to land for growing field crops when high, and the suitable processing of shading.
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| CN107996404A (en) * | 2017-12-26 | 2018-05-08 | 丽江市古城区秋成种养殖有限公司 | A kind of tendril-leaved fritillary bulb method for tissue culture |
| 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 |
| CN117158321A (en) * | 2023-10-20 | 2023-12-05 | 江苏北环生物科技有限公司 | Tissue culture propagation-expanding explant treatment and disinfection method for fritillaria thunbergii |
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| CN101213937B (en) * | 2007-12-28 | 2010-09-08 | 浙江省农业科学院 | Method for cultivating detoxification tissue culture bulb of fritillaria thunbergii |
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