CN101161056A - A method for evoking yili caladium polyploid under culture in vitro - Google Patents
A method for evoking yili caladium polyploid under culture in vitro Download PDFInfo
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Abstract
The invention relates to a polyploid inducement method of Fritillaria pallidiflora Schrenk in vitro culture, which uses fresh bulb to be induced to produce adventitious buds. Inducement treatment is applied through cultivating in MS liquid medium with different density of colchicine to produce Fritillaria pallidiflora Schrenk polyploid plant. Bulb yield and medicinal components content of the plant are improved by changing chromosome ploidy to produce plant with good characteristics. Therefore, the problem of variety degeneration of Fritillaria pallidiflora Schrenk in a long-term course of Fritillaria pallidiflora Schrenk planting is overcomed. The method has the advantages that Fritillaria pallidiflora Schrenk polyploid can be obtained quickly with effectively improved content of active components and a shorter planting cycle.
Description
Technical field
The present invention relates to medicinal plant breeding field, specifically, relate to a kind of Fritillaria pallidiflora Schrenk multiploid induction method.
Background technology
Fritillaria pallidiflora Schrenk (Fritillaria Pallidiflora Schrenk), Liliaceae (Liliaceae) Fritillaria (Fri tillaria L.) herbaceos perennial, being used as medicine with bulb, is important simply conventional Chinese medicine, has the effect of clearing heat and moistening lung, preventing phlegm from forming and stopping coughing.Because the Fritillaria pallidiflora Schrenk alkaloids content is higher relatively in the similar bulb of fritillary, and premunition is strong, and price of medicinal material is relatively low, in recent years people's attention extremely.All there is plantation on Fritillaria pallidiflora Schrenk ground in Xinjiang reaches, has become a kind of important Chinese herbal medicine and economic extra earning crop.Yet, tame Fritillaria pallidiflora Schrenk exists that reproduction coefficient is low, the problem of quality deterioration, and wild Fritillaria pallidiflora Schrenk is excavated for a long time in a large number, and distribution area reduces day by day, the destruction Of resources is serious, and the research of therefore seeking Fritillaria pallidiflora Schrenk new germ plasm resource more and more is subject to people's attention.
The Fritillaria pallidiflora Schrenk main component is different steroid alkaloid and glycosides thereof, mainly comprise D, the imperialine of the two hydrogen cis of E ring and new steroid alkaloid-sinpeinine etc., D wherein, the two hydrogen cis imperialines of E ring belong to western veratrine (cevine groups), be one of main alkaloid in the Siberian fritillary bulb, especially (imperialine-3 β-D-glucoside) is main component (Yi Jianglan, wheat Diny carry the research overview Xinjiang traditional Chinese medicine 2,002 4 of Siberian fritillary bulb total alkaloids) with imperialine (imperialine) and imperialine glycosides.Up to the present, being separated to monosomic alkali from the Fritillaria pallidiflora Schrenk total alkaloids has: imperialine (Imperialine), the imperialine glycosides (imperialine-3 β-D-glucoside), bulb of fritillary suffering (Peimisine), she is shellfish alkali glycosides A (Yibeinoside A), her shellfish alkali glycosides B (Yibeinoside B), her shellfish alkali glycosides C (Yibeinoside C), yibeissine (Yibeisine).
The antibechic of Fritillaria pallidiflora Schrenk total alkaloids, eliminate the phlegm and the research aspect of relievining asthma, as tested object, under 10mg/kg dosage, duodenal administration draws to cough to electro photoluminescence cat superior laryngeal nerve and has significant antitussive effect Wang Linhui etc. with mouse, cavy, cat and rat.Continuous gastric infusion 5d, the Fritillaria pallidiflora Schrenk total alkaloids is 40,20, under the dosage of 10mg/kg mouse ammoniacal liquor drawn to cough to have significant antitussive effect; 20, under the dosage of 10mg/kg the cavy citric acid drawn to cough and have significant antitussive effect, can significantly increase rat capillary expectoration amount; 40, having the effect of the phenol red excretion amount of remarkable increase mouse under the dosage of 20mg/kg, to acetylcholine. the cavy asthma due to the histamine has significant antiasthmatic effect.The result shows that the Fritillaria pallidiflora Schrenk total alkaloids has significant antibechic, eliminates the phlegm and antiasthmatic effect laboratory animal.The clearing heat and moistening lung of this and the traditional Chinese medical science, the effect of preventing phlegm from forming and stopping coughing match (Wang Linhui, Ji Hui, Wang Changli, He Qian, journal 200 334 172-176 of pharmacodynamic study China Medicine University of Li Ping Fritillaria pallidiflora Schrenk total alkaloids).
Polyploid plant can be satisfied the requirement that medicinal material is produced preferably because chromosome doubling often has the huge property of root, stem and leaf, and promptly the output of root, stem and leaf is higher; On the other hand, the variation of plant chromosome ploidy also tends to cause the variation of secondary metabolite content, might obtain the high medicinal plant of active constituent content.The resistance that while polyploid plant annidation reaches adverse circumstance also strengthens (Sandra De Schepper et al.Somatic polyploidpetals:regeneration offers new roads for breeding Belgian potazaleas Plant Cell, Tissue and Organ Culture 76:183-188 to some extent; M.Lotfi et al.Production of haploid and doubled haploid plantsof melon (Cucumis melo L.) for use in breeding or multiple virusresistance Plant Cell Rep (2003) 21:1121-1128; M.J.KermaniOryzalin-induced chromosome doubling in Rosa and its effect onplant morphology and pollen viabil ity Theor Appl Genet (2003) 107:1195-1200).
The breeding research of Fritillaria plant polyploid is less, and Wang Zhian etc. utilize colchicine processing Zhejiang shellfish seed to advance tetraploid breeding research (Wang Zhian is permitted again the research preliminary study Zhejiang Agricultural Univ journal 1,991 17 (1) 89～92 that China induces the fritillaria thunbergii polyploid).Wang Qiang etc. carry out tetraploid induction (Wang Qiang with colchicine to Bulbus Fritillariae Cirrhosae (Fritillaria cirrhosa D.Don) callus, the langley fine jade, the research Wuhan botany research 2002,20 (6) of Fu Hua dragon colchicine-induced Bulbus Fritillariae Cirrhosae (Fritillariacirrhosa D.Don) callus polyploid: 449-452).
In the Fritillaria pallidiflora Schrenk breeding, the present invention is in conjunction with Siberian fritillary bulb bulb stripping and slicing indefinite bud rapid induction and multiplication technique, utilize colchicine to handle indefinite bud and make chromosome doubling, induce and produce polyploid Fritillaria pallidiflora Schrenk plant, make plant produce merit by changing ploidy, overcome Fritillaria pallidiflora Schrenk and the deterioration of variety problem in long-term planting, occurs to improve the content of its bulb output and medicinal ingredient.This improves bulb of fritillary plantation income to enlarging the peculiar rare economic class crops planting area in Xinjiang, and it is significant to promote the conventional Chinese medicine development and use.
Summary of the invention
The object of the present invention is to provide a kind of method of under cultured in vitro, inducing the Fritillaria pallidiflora Schrenk polyploid, this method utilizes fresh bulb stripping and slicing to induce the generation indefinite bud, induce processing by the MS liquid nutrient medium that contains the variable concentrations colchicine, induce and produce polyploid Fritillaria pallidiflora Schrenk plant, make plant produce merit by changing ploidy to improve the content of its bulb output and medicinal ingredient, Screening and Identification by polyploid obtains the polyploid Fritillaria pallidiflora Schrenk, overcomes Fritillaria pallidiflora Schrenk and the deterioration of variety problem occurs in long-term planting.The method can obtain the polyploid Fritillaria pallidiflora Schrenk fast, effectively improves the content of active component, shortens crop cycle.
Invent described a kind of method of under cultured in vitro, inducing the Fritillaria pallidiflora Schrenk polyploid, follow these steps to carry out:
A, the fresh bulb of Fritillaria pallidiflora Schrenk is clean surperficial with the running water flushing, big bulb airing 12-24h under the sun, clove airing 8-12h, then bulb is adopted 75% alcohol time 30s or 0.1-1% potassium permanganate solution processing 1h or 84 medicining liquid dipping 20min to carry out surface sterilization on superclean bench, handle 5-10min with 0.1% mercuric chloride 6-10min or 3-5% aqueous sodium hypochlorite solution then, handle 30-60min, aseptic water washing 2-3 time with 15% hydrogen peroxide again;
B, the bulb after handling among the step a is cut into 0.1-0.2cm
3Size inserts adventitious bud inducing and propagation solid culture medium MS+KT 0.5-1.0mg/L+NAA 0.1-0.4mg/L, 20 ± 1 ℃ of temperature, and illumination 2000lx, the time, 18h/d cultivated;
C, Fritillaria pallidiflora Schrenk indefinite bud among the step b is cut into simple bud, is immersed in and contains colchicine 50-1000mg/L, in the liquid MS medium of 2% dimethyl sulfoxide (DMSO), soak 12-48h and carry out POLYPLOID INDUCEMENT;
D, Fritillaria pallidiflora Schrenk indefinite bud among the step b is cut into simple bud, is immersed in the MS liquid nutrient medium that does not add colchicine, placing rotating speed is to soak on the 100r/min shaking table, soaks 12-48h;
E, step c and d simple bud used aseptic water washing 2-3 time respectively after, change over to after 15-25min dries on aseptic filter paper among adventitious bud inducing that step b do not contain colchicine and dimethyl sulfoxide (DMSO) and the propagation solid culture medium MS+KT 0.5-1.0mg/L+NAA 0.1-0.4mg/L and cultivate;
F, the screening of Fritillaria pallidiflora Schrenk polyploid: the screening of polyploid Fritillaria pallidiflora Schrenk is determined doubtful strain according to dliploid and colchicine processing back indefinite bud in width of blade, leaf area, Stomacal guard cell size and density, doubtful strain is changed in the 1/2 MS+NAA0.5-1.0mg/L root media take root;
G, Fritillaria pallidiflora Schrenk polyploid are identified: get the doubtful strain tender tip of a root of children and young tender indefinite bud tissue, under 4 ℃, through oxine preliminary treatment 4-6h or saturated paracide aqueous solution preliminary treatment 5-10h or 20% essential wind oil preliminary treatment 3-7h or 0.1% colchicine preliminary treatment 6-8h, the Kano fixer is 12-18h fixedly, 60 ℃ of 8-10min that dissociate of 1-2mol/L hydrochloric acid, the pinkish red dyeing liquor dyeing of improvement phenol 5-10min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud chromosome, determines polyploid plant.
Surface sterilization described in the step a is preferably 75% alcohol or 1% potassium permanganate solution, uses 0.1% mercuric chloride 6-10min then, handles 30-60min with 15% hydrogen peroxide again.
Surface sterilization the best described in the step a is 75% alcohol, uses 0.1% mercuric chloride 6-10min then, handles 30-60min with 15% hydrogen peroxide again.
Colchicine 200-800mg/L among the step c, soak time 12-24h.
Colchicine 400mg/L among the step c, soak time 24h.
The preferred 0.002mol/L oxine of preliminary treatment described in the step g or the saturated paracide aqueous solution.
Preliminary treatment the best described in the step g is the 0.002mol/L oxine.
Fritillaria pallidiflora Schrenk multiploid induction method advantage of the present invention is:
The selection of induced material.Fritillaria pallidiflora Schrenk is a perennial herb, the annual aerial growth time had only about 60 days, then underground bulb just changes dormant state over to, and it is short that the indefinite bud that utilizes bulb to induce can overcome the bulb of fritillary aerial growth phase as mutant materials, the shortcoming that polyploid breeding is subject to seasonal restrictions.Indefinite bud growth is vigorous in the tissue culture, and cell division is active to be utilized colchicine to handle indefinite bud can to guarantee efficiency of inducing mutation, reduce the workload of screening.
The selection of abductive approach.The present invention is the colchicine-induced indefinite bud chromosome polyploidization that matrix is added variable concentrations with the liquid MS medium of additional 2% dimethyl sulfoxide (DMSO), is beneficial to colchicine and infiltrates through indefinite bud performance mutagenesis.Adopt washing after the mutagenic treatment or be immersed in that a period of time discharges the colchicine that is adsorbed on the surface in the sterile water, reduced the toxic action of colchicine, can improve the survival rate of indefinite bud after the mutagenesis tender tissue.
Description of drawings
Fig. 1 is the bulb exploded chart of evoking adventive bud of the present invention
The indefinite bud bud point diagram that Fig. 2 produces for bulb stripping and slicing differentiation of the present invention
Fig. 3 is the simple bud figure of induction polyploid of the present invention
Fig. 4 is dliploid of the present invention (left side) and polyploid (right side) phenotype comparison diagram
Fig. 5 is dliploid of the present invention (A) and polyploid (B) pore comparison diagram
Fig. 6 is dliploid chromosome (A) 2n=2x=24 of the present invention and tetraploid chromosomes (B) 2n=4x=48 figure
Embodiment
Embodiment 1
A, the fresh bulb of Fritillaria pallidiflora Schrenk is clean surperficial with the running water flushing, big bulb airing 12h under the sun, clove airing 8h, then bulb is used volume ratio 75% alcohol surface sterilization 30s on superclean bench, quality percent by volume 0.1% mercuric chloride sterilization 6min, 15% hydrogen peroxide is handled 30min, aseptic water washing 2 times;
B, the bulb after handling among the step a is cut into 0.1-0.2cm
3Size inserts adventitious bud inducing and propagation solid culture medium MS+KT 1.0mg/L+NAA 0.3mg/L, 20 ± 1 ℃ of temperature, and illumination 2000lx, the time, 18h/d cultivated;
C, after 40 days, each bulb stripping and slicing can grow 5-10 indefinite bud among the step b, and these indefinite buds are cut into simple bud, gets 40 simple buds at random and is immersed in and contains the 50mg/L colchicine, in the liquid MS medium of 2% dimethyl sulfoxide (DMSO), placing rotating speed is to soak 12h on the 100r/min shaking table;
D, get 40 of the simple buds that the Fritillaria pallidiflora Schrenk indefinite bud is cut among the step b at random, be immersed in the MS liquid nutrient medium that does not add colchicine, placing rotating speed is to soak 12h in contrast on the 100r/min shaking table;
E, step c and d simple bud used aseptic water washing 2 times respectively after, change over to after 15min dries on aseptic filter paper among adventitious bud inducing that step b do not contain colchicine and dimethyl sulfoxide (DMSO) and the propagation solid culture medium MS+KT 0.5mg/L+NAA 0.1mg/L and cultivate;
F, the screening of Fritillaria pallidiflora Schrenk polyploid: the screening of polyploid Fritillaria pallidiflora Schrenk is determined doubtful strain according to dliploid and colchicine processing back indefinite bud in width of blade, leaf area, Stomacal guard cell size and density, doubtful strain is changed in the 1/2 MS+NAA0.5mg/L root media take root;
G, Fritillaria pallidiflora Schrenk polyploid are identified: after 3 weeks, the part indefinite bud growth that colchicine was handled is fast, blade area is average bigger more than 50% than control group, get the doubtful strain tender tip of a root of children and young tender indefinite bud tissue, through 0.002mol/L oxine preliminary treatment 4h, the Kano fixer is 18h fixedly, 60 ℃ of 8min that dissociate of 1mol/L hydrochloric acid, the pinkish red dyeing liquor dyeing of improvement phenol 5min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud chromosome, determines polyploid plant.Each indefinite bud of taking root is got four tips of a root, the tender indefinite bud bud point of children, 20 divisions of each tip of a root statistics phase cell, 20 divisions of indefinite bud statistics phase cell chromosome number.Handle 12h indefinite bud survival rate through the 50mg/L colchicine and can reach 100%, the polyploid mutagenesis rate can reach 2%.Induced mutation rate: polyploid bud number/(handling bud number-dead bud number).
Embodiment 2
A, the fresh bulb of Fritillaria pallidiflora Schrenk is clean surperficial with the running water flushing, big bulb airing 18h under the sun, clove airing 10h, then bulb is handled 1h with 1% potassium permanganate solution on superclean bench, 3% aqueous sodium hypochlorite solution is handled 10min, 15% hydrogen peroxide is handled 45min, aseptic water washing 3 times;
B, the bulb after handling among the step a is cut into 0.1-0.2cm
3Size inserts adventitious bud inducing and propagation solid culture medium MS+KT 0.5mg/L+NAA 0.1mg/L, 20 ± 1 ℃ of temperature, and illumination 2000lx, the time, 18h/d cultivated;
C, after 40 days, each bulb stripping and slicing can grow 5-10 indefinite bud among the step b, and these indefinite buds are cut into simple bud, gets 40 simple buds at random and is immersed in and contains colchicine 400mg/L, in the liquid MS medium of 2% dimethyl sulfoxide (DMSO), placing rotating speed is to soak 24h on the 100r/min shaking table;
D, in addition get 40 of the simple buds that the Fritillaria pallidiflora Schrenk indefinite bud is cut among the step b at random, be immersed in the MS liquid nutrient medium that does not add colchicine, placing rotating speed is to soak 24h in contrast on the 100r/min shaking table;
E, step c and d simple bud used aseptic water washing 3 times respectively after, change over to after 20min dries on aseptic filter paper among adventitious bud inducing that step b do not contain colchicine and dimethyl sulfoxide (DMSO) and the propagation solid culture medium MS+KT 1.0mg/L+NAA 0.3mg/L and cultivate;
F, the screening of Fritillaria pallidiflora Schrenk polyploid: the screening of polyploid Fritillaria pallidiflora Schrenk is determined doubtful strain according to dliploid and colchicine processing back indefinite bud in width of blade, leaf area, Stomacal guard cell size and density, doubtful strain is changed in the 1/2 MS+NAA 1.0mg/L root media take root;
G, Fritillaria pallidiflora Schrenk polyploid are identified: after 3 weeks, the part indefinite bud growth of handling is fast, blade area is average bigger more than 50% than control group, get the doubtful strain tender tip of a root of children and young tender indefinite bud bud point, through saturated paracide aqueous solution preliminary treatment 8h, the Kano fixer is 18h fixedly, 60 ℃ of 10min that dissociate of 1mol/L hydrochloric acid, the pinkish red dyeing liquor dyeing of improvement phenol 8min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud chromosome, determines polyploid plant.Each indefinite bud of taking root is got four tips of a root, the tender indefinite bud bud point of children, 20 divisions of each tip of a root statistics phase cell, 20 divisions of indefinite bud statistics phase cell chromosome number.Handle 24h indefinite bud survival rate through colchicine 400mg/L and can reach 75%, the polyploid mutagenesis rate can reach 33%.Induced mutation rate: polyploid bud number/(handling bud number-dead bud number)
Embodiment 3
A, the fresh bulb of Fritillaria pallidiflora Schrenk is clean surperficial with the running water flushing, big bulb airing 24h under the sun, clove airing 12h, then with bulb on superclean bench with 84 medicining liquid dipping 20min, 0.1% mercuric chloride sterilization 10min, 15% hydrogen peroxide is handled 60min, aseptic water washing 3 times;
B, the bulb after handling among the step a is cut into 0.1-0.2cm
3Size inserts adventitious bud inducing and propagation solid culture medium MS+KT 0.8mg/L+NAA 0.4mg/L, 20 ± 1 ℃ of temperature, and illumination 2000lx, the time, 18h/d cultivated;
C, after 40 days, each bulb stripping and slicing can grow 5-10 indefinite bud among the step b, and these indefinite buds are cut into simple bud, gets 40 simple buds at random and is immersed in and contains colchicine 1000mg/L, in the liquid MS medium of 2% dimethyl sulfoxide (DMSO), placing rotating speed is to soak 48h on the 100r/min shaking table;
D, in addition get 40 of the simple buds that the Fritillaria pallidiflora Schrenk indefinite bud is cut among the step b at random, be immersed in the MS liquid nutrient medium that does not add colchicine, placing rotating speed is to soak 48h in contrast on the 100r/min shaking table;
E, step c and d simple bud used aseptic water washing 3 times respectively after, change over to after 25min dries on aseptic filter paper among adventitious bud inducing that step b do not contain colchicine and dimethyl sulfoxide (DMSO) and the propagation solid culture medium MS+KT 0.8mg/L+NAA0.4mg/L and cultivate;
F, the screening of Fritillaria pallidiflora Schrenk polyploid: the screening of polyploid Fritillaria pallidiflora Schrenk is determined doubtful strain according to dliploid and colchicine processing back indefinite bud in width of blade, leaf area, Stomacal guard cell size and density, doubtful strain is changed in the 1/2 MS+NAA 1.0mg/L root media take root;
G, Fritillaria pallidiflora Schrenk polyploid are identified: after 3 weeks, the part indefinite bud growth of handling is fast, blade area is average bigger more than 50% than control group, get the doubtful strain tender tip of a root of children and young tender indefinite bud bud point, through 20% essential wind oil preliminary treatment 5h, the Kano fixer is 18h fixedly, 60 ℃ of 10min that dissociate of 1mol/L hydrochloric acid, the pinkish red dyeing liquor dyeing of improvement phenol 10min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud chromosome, determines polyploid plant.Each indefinite bud of taking root is got four tips of a root, the tender indefinite bud bud point of children, 20 divisions of each tip of a root statistics phase cell, 20 divisions of indefinite bud statistics phase cell chromosome number.Handle 48h indefinite bud survival rate through colchicine 1000mg/L and can reach 45%, the polyploid mutagenesis rate can reach 9.1%.Induced mutation rate: polyploid bud number/(handling bud number-dead bud number)
Embodiment 4
A, the fresh bulb of Fritillaria pallidiflora Schrenk is clean surperficial with the running water flushing, big bulb airing 18h under the sun, clove airing 10h, then with bulb on superclean bench with 75% alcohol surface sterilization 30s, 0.1% mercuric chloride sterilization 10min, 15% hydrogen peroxide is handled 60min, aseptic water washing 2 times;
B, the bulb after handling among the step a is cut into 0.1-0.2cm
3Size inserts adventitious bud inducing and propagation solid culture medium MS+KT 0.5mg/L+NAA 0.2mg/L, 20 ± 1 ℃ of temperature, and illumination 2000lx, the time, 18h/d cultivated;
C, Fritillaria pallidiflora Schrenk indefinite bud among the step b is cut into simple bud, gets 40 simple buds at random and be immersed in and contain colchicine 400mg/L, in the liquid MS medium of 2% dimethyl sulfoxide (DMSO), placing rotating speed is to soak 36h on the 100r/min shaking table;
D, Fritillaria pallidiflora Schrenk indefinite bud among the step b is cut into simple bud, gets 40 simple buds at random and be immersed in the MS liquid nutrient medium that does not add colchicine, placing rotating speed is to soak 36h in contrast on the 100r/min shaking table;
E, step c and d simple bud used aseptic water washing 2-3 time respectively after, change over to after 18min dries on aseptic filter paper among adventitious bud inducing that step b do not contain colchicine and dimethyl sulfoxide (DMSO) and the propagation solid culture medium MS+KT 0.5mg/L+NAA 0.2mg/L and cultivate;
F, the screening of Fritillaria pallidiflora Schrenk polyploid: the screening of polyploid Fritillaria pallidiflora Schrenk is determined doubtful strain according to dliploid and colchicine processing back indefinite bud in width of blade, leaf area, Stomacal guard cell size and density, doubtful strain is changed in the 1/2 MS+NAA0.2mg/L root media take root;
G, Fritillaria pallidiflora Schrenk polyploid are identified: get the doubtful strain tender tip of a root of children and young tender indefinite bud tissue, through 0.1% colchicine preliminary treatment 8h, the Kano fixer is 12h fixedly, 60 ℃ of 8min that dissociate of hydrochloric acid, the pinkish red dyeing liquor dyeing of improvement phenol 5min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud chromosome, determines polyploid plant.Each indefinite bud of taking root is got four tips of a root, the tender indefinite bud bud point of children, 20 divisions of each tip of a root statistics phase cell, 20 divisions of indefinite bud statistics phase cell chromosome number.Handle 36h indefinite bud survival rate through colchicine 400mg/L and can reach 57.5%, the polyploid mutagenesis rate can reach 21.7%.Induced mutation rate: polyploid bud number/(handling bud number-dead bud number)
Embodiment 5
A, the fresh bulb of Fritillaria pallidiflora Schrenk is clean surperficial with the running water flushing, big bulb airing 24h under the sun, clove airing 12h, then bulb is handled 1h with 0.1% potassium permanganate solution on superclean bench, 0.1% mercuric chloride sterilization 8min, 15% hydrogen peroxide is handled 45min, aseptic water washing 3 times;
B, the bulb after handling among the step a is cut into 0.1-0.2cm
3Size inserts adventitious bud inducing and propagation solid culture medium MS+KT 1.0mg/L+NAA 0.4mg/L, 20 ± 1 ℃ of temperature, and illumination 2000lx, the time, 18h/d cultivated;
C, Fritillaria pallidiflora Schrenk indefinite bud among the step b is cut into simple bud, gets 40 simple buds at random and be immersed in and contain colchicine 800mg/L, in the liquid MS medium of 2% dimethyl sulfoxide (DMSO), placing rotating speed is to soak 48h on the 100r/min shaking table;
D, Fritillaria pallidiflora Schrenk indefinite bud among the step b is cut into simple bud, gets 40 simple buds at random and be immersed in the MS liquid nutrient medium that does not add colchicine, placing rotating speed is to soak 36h in contrast on the 100r/min shaking table;
E, step c and d simple bud used aseptic water washing 2-3 time respectively after, change over to after 15-25min dries on aseptic filter paper among adventitious bud inducing that step b do not contain colchicine and dimethyl sulfoxide (DMSO) and the propagation solid culture medium MS+KT 1.0mg/L+NAA 0.4mg/L and cultivate;
F, the screening of Fritillaria pallidiflora Schrenk polyploid: the screening of polyploid Fritillaria pallidiflora Schrenk is determined doubtful strain according to dliploid and colchicine processing back indefinite bud in width of blade, leaf area, Stomacal guard cell size and density, doubtful strain is changed in the 1/2 MS+NAA1.0mg/L root media take root;
G, Fritillaria pallidiflora Schrenk polyploid are identified: get the doubtful strain tender tip of a root of children and young tender indefinite bud tissue, through 0.002mol/L oxine preliminary treatment 6h, the Kano fixer is 18h fixedly, 60 ℃ of 9min that dissociate of hydrochloric acid, the pinkish red dyeing liquor dyeing of improvement phenol 10min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud chromosome, determines polyploid plant.Each indefinite bud of taking root is got four tips of a root, the tender indefinite bud bud point of children, 20 divisions of each tip of a root statistics phase cell, 20 divisions of indefinite bud statistics phase cell chromosome number.Handle 48h through colchicine 800mg/L, the polyploid mutagenesis rate can reach 11%.Induced mutation rate: polyploid bud number/(handling bud number-dead bud number)
Embodiment 6
A, the fresh bulb of Fritillaria pallidiflora Schrenk is clean surperficial with the running water flushing, big bulb airing 12h under the sun, clove airing 8h, then with bulb on superclean bench with 75% alcohol surface sterilization 30s, 0.1% mercuric chloride sterilization 6min, 15% hydrogen peroxide is handled 30min, aseptic water washing 2 times;
B, the bulb after handling among the step a is cut into 0.1-0.2cm
3Size inserts adventitious bud inducing and propagation solid culture medium MS+KT 0.8mg/L+NAA 0.3mg/L, 20 ± 1 ℃ of temperature, and illumination 2000lx, the time, 18h/d cultivated;
C, Fritillaria pallidiflora Schrenk indefinite bud among the step b is cut into simple bud, gets 40 simple buds at random and be immersed in and contain colchicine 200mg/L, in the liquid MS medium of 2% dimethyl sulfoxide (DMSO), placing rotating speed is to soak 48h on the 100r/min shaking table;
D, Fritillaria pallidiflora Schrenk indefinite bud among the step b is cut into simple bud, gets 40 simple buds at random and be immersed in the MS liquid nutrient medium that does not add colchicine, placing rotating speed is to soak 36h in contrast on the 100r/min shaking table;
E, step c and d simple bud used aseptic water washing 2-3 time respectively after, change over to after 25min dries on aseptic filter paper among adventitious bud inducing that step b do not contain colchicine and dimethyl sulfoxide (DMSO) and the propagation solid culture medium MS+KT 0.8mg/L+NAA 0.3mg/L and cultivate;
F, the screening of Fritillaria pallidiflora Schrenk polyploid: the screening of polyploid Fritillaria pallidiflora Schrenk is determined doubtful strain according to dliploid and colchicine processing back indefinite bud in width of blade, leaf area, Stomacal guard cell size and density, doubtful strain is changed in the 1/2 MS+NAA0.8mg/L root media take root;
G, Fritillaria pallidiflora Schrenk polyploid are identified: get the doubtful strain tender tip of a root of children and young tender indefinite bud tissue, through 0.002mol/L oxine preliminary treatment 5h, the Kano fixer is 12h fixedly, 60 ℃ of 8min that dissociate of hydrochloric acid, the pinkish red dyeing liquor dyeing of improvement phenol 10min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud chromosome, determines polyploid plant.Each indefinite bud of taking root is got four tips of a root, the tender indefinite bud bud point of children, 20 divisions of each tip of a root statistics phase cell, 20 divisions of indefinite bud statistics phase cell chromosome number.Handle 48h through colchicine 200mg/L, the polyploid mutagenesis rate can reach 15%.Induced mutation rate: polyploid bud number/(handling bud number-dead bud number)
Claims (7)
1. method of inducing the Fritillaria pallidiflora Schrenk polyploid under cultured in vitro is characterized in that following these steps to carrying out:
A, the fresh bulb of Fritillaria pallidiflora Schrenk is clean surperficial with the running water flushing, big bulb airing 12-24h under the sun, clove airing 8-12h, then bulb is adopted 75% alcohol time 30s or 0.1-1% potassium permanganate solution processing 1h or 84 medicining liquid dipping 20min to carry out surface sterilization on superclean bench, handle 5-10min with 0.1% mercuric chloride 6-10min or 3-5% aqueous sodium hypochlorite solution then, handle 30-60min, aseptic water washing 2-3 time with 15% hydrogen peroxide again;
B, the bulb after handling among the step a is cut into 0.1-0.2cm
3Size inserts adventitious bud inducing and propagation solid culture medium MS+KT 0.5-1.0mg/L+NAA 0.1-0.4mg/L, 20 ± 1 ℃ of temperature, and illumination 2000lx, the time, 18h/d cultivated;
C, Fritillaria pallidiflora Schrenk indefinite bud among the step b is cut into simple bud, is immersed in and contains colchicine 50-1000mg/L, in the liquid MS medium of 2% dimethyl sulfoxide (DMSO), soak 12-48h and carry out POLYPLOID INDUCEMENT;
D, Fritillaria pallidiflora Schrenk indefinite bud among the step b is cut into simple bud, is immersed in the MS liquid nutrient medium that does not add colchicine, placing rotating speed is to soak on the 100r/min shaking table, soaks 12-48h;
E, step c and d simple bud used aseptic water washing 2-3 time respectively after, change over to after 15-25min dries on aseptic filter paper among adventitious bud inducing that step b do not contain colchicine and dimethyl sulfoxide (DMSO) and the propagation solid culture medium MS+KT 0.5-1.0mg/L+NAA 0.1-0.4mg/L and cultivate;
F, the screening of Fritillaria pallidiflora Schrenk polyploid: the screening of polyploid Fritillaria pallidiflora Schrenk is determined doubtful strain according to dliploid and colchicine processing back indefinite bud in width of blade, leaf area, Stomacal guard cell size and density, doubtful strain is changed in the 1/2 MS+NAA0.5-1.0mg/L root media take root;
G, Fritillaria pallidiflora Schrenk polyploid are identified: get the doubtful strain tender tip of a root of children and young tender indefinite bud tissue, under 4 ℃, through oxine preliminary treatment 4-6h or saturated paracide aqueous solution preliminary treatment 5-10h or 20% essential wind oil preliminary treatment 3-7h or 0.1% colchicine preliminary treatment 6-8h, the Kano fixer is 12-18h fixedly, 60 ℃ of 8-10min that dissociate of 1-2mol/L hydrochloric acid, the pinkish red dyeing liquor dyeing of improvement phenol 5-10min, compressing tablet is observed the statistics tip of a root, young tender indefinite bud chromosome, determines polyploid plant.
2. method according to claim 1 is characterized in that the surface sterilization described in the step a is preferably 75% alcohol or 1% potassium permanganate solution, uses 0.1% mercuric chloride 6-10min then, handles 30-60min with 15% hydrogen peroxide again.
3. method according to claim 1 is characterized in that the surface sterilization the best described in the step a is 75% alcohol, uses 0.1% mercuric chloride 6-10min then, handles 30-60min with 15% hydrogen peroxide again.
4. method according to claim 1 is characterized in that the colchicine 200-800mg/L among the step c, soak time 12-24h.
5. method according to claim 1 is characterized in that the colchicine 400mg/L among the step c, soak time 24h.
6. method according to claim 1 is characterized in that the preferred 0.002mol/L oxine of the preliminary treatment described in the step g or the saturated paracide aqueous solution.
7. method according to claim 1 is characterized in that the preliminary treatment the best described in the step g is the 0.002mol/L oxine.
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