CN101999317B - Multiploid bulb induction method with firtillaria cirrhosa scale leaf as explant - Google Patents
Multiploid bulb induction method with firtillaria cirrhosa scale leaf as explant Download PDFInfo
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- CN101999317B CN101999317B CN2009101643394A CN200910164339A CN101999317B CN 101999317 B CN101999317 B CN 101999317B CN 2009101643394 A CN2009101643394 A CN 2009101643394A CN 200910164339 A CN200910164339 A CN 200910164339A CN 101999317 B CN101999317 B CN 101999317B
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Abstract
The invention discloses a multiploid bulb induction method with a firtillaria cirrhosa scale leaf as an explant. The innovation of the invention lies in that a used material is a close callus with a certain differentiation characteristic, which is obtained through a predifferentiation culture process on the basis of generating a callus through induction of the scale leaf and has. The material is cut into small blocks and is soaked into a colchicine solution to carry out multiploid mutagenesis treatment, thereby greatly lightening damage of the colchicine solution to a processing material and enhancing the inductivity of the firtillaria cirrhosa multiploid bulb.
Description
Technical field
The present invention relates to a kind of abductive approach of polyploid bulb, particularly relating to a kind of is the polyploid bulb abductive approach of explant with leaf roll bulb of fritillary scaly leaf.
Background technology
The Bulbus Fritillariae Cirrhosae medicinal material is the dry bulb of liliaceous plant leaf roll bulb of fritillary F.cirrhosa, dark violet bulb of fritillary F.unibracteata, Gansu bulb of fritillary F.przew alskii Maxim. and Bulbus Fritillariae cirrhosae F.delavayiFranch.; For one of famous birth canal ground, river medicinal material, be used as medicine with bulb.Leaf roll bulb of fritillary plant requires harsh to growing environment, it likes cold cloudy weather conditions, have cold-resistant, happiness is wet, happiness covers, be afraid of the characteristic of high temperature.Temperature reaches 30 ℃ or ground temperature and surpasses 25 ℃, and plant will wither, and can not survive in the area low at height above sea level, that temperature is high, so the leaf roll bulb of fritillary could normal growth at the height of height above sea level 3500m-4500m.The leaf roll bulb of fritillary does not have excavating of plan blindness in addition in recent years owing to receive the restriction of growth conditions, and its natural resources is exhausted day by day, estimates in nearly decades, be difficult to alleviate yet.The tissue culture technique growth cycle is short; Reproduction rate is high; Broken away from the adverse effect of the four seasons, circadian variation and disastrous weather in the Nature; Very favourable to plant growing, be convenient to stably carry out cultivating and producing, so the application organizes cultured method carry out Bulbus Fritillariae Cirrhosae fast breeding be to solve one of effective ways of Bulbus Fritillariae Cirrhosae herb resource shortage problem.
The polyploid medicinal plant is because chromosomal doubling; Make plant organ show the notable feature of " giantism "; Thereby can improve the biological yield of plant medicinal part through the multiploid induction technology; Simultaneously because the medicinal plant chromosome number doubles, make that also normal dliploid of content of gene regulation active chemical increases on the chromosome, the content of its active chemical of polyploid medicinal material of therefore cultivating also obviously increases.
" research of colchicine-induced Bulbus Fritillariae Cirrhosae callus polyploid " (Wuhan botany research 2002.20 (6): 449～452) disclose two kinds of colchicine processing methods in the literary composition that Wang Qiang etc. deliver; A kind of is that colchicine adds the medium facture; Another kind is a colchicine solution immersion treatment method; The conclusion that they draw is: colchicine solution immersion treatment method is more direct to the injury of callus, and it is good not as colchicine adding medium facture that it doubles effect.In fact,, can alleviate the injury of colchicine solution widely to callus as long as the callus that produces is suitably handled, its POLYPLOID INDUCEMENT rate far above mentioned in the literary composition 70%.
Summary of the invention
The object of the present invention is to provide a kind of is the polyploid bulb abductive approach of explant with leaf roll bulb of fritillary scaly leaf; This method is owing to the cultivation that the callus to new generation has carried out presorting is handled; Make colchicine solution alleviate widely, thereby improved its POLYPLOID INDUCEMENT rate the callus injury effect after handling.
For achieving the above object, the solution that the present invention adopts comprises the following steps:
(1) scaly leaf of the fresh bulb of the selection leaf roll bulb of fritillary is as explant;
(2) strip anosis and the scaly leaf that has no mechanical damage in the above-mentioned bulb internal layer; Clean the back with detergent liquid earlier and wash 25～30min down in flowing water; Use 2.0% liquor natrii hypochloritis's rinsing, 12～15min again, after cleaning and blot water with clear water, put it into the 7～10min that sterilizes in 0.1% mercuric chloride solution; Use aseptic water washing then 2～3 times; Blot the moisture on surface again with aseptic filter paper, carry out inducing culture in the medium with its access evoked callus at last, the dark cultivation that the initial stage of inducing adopted 12～15 days;
(3) new life who (2) step is obtained, the loose callus of quality insert presorting medium MS+6-BA2.0mgL
-1+ NAA0.0～0.3mgL
-1The middle cultivation obtains tight callus;
(4) the tight callus of the leaf roll bulb of fritillary that (3) step is obtained is cut into small pieces, and is soaked in the colchicine solution of filtration sterilization, handles the back with aseptic washing 2～3 times, blot water with aseptic filter paper again after, access does not contain the MS+6-BA1.0mgL of colchicine
-1+ NAA0.0～0.3mgL
-1Carrying out the induction polyploid bulb in the differential medium cultivates;
Above-mentioned all culture medium preparation pH values are 5.6～6.5, sucrose 30～50gL
-1, agar 6.0～7.0gL
-1
It is 1000～1800lx that above-mentioned condition of culture is 15～24 ℃ of temperature, illumination every day 8～16 hours, intensity of illumination.
The medium of the evoked callus that is adopted in above-mentioned (2) step is MS+6-BA0.1～2.0mgL
-1+ IAA0.4～1.5mgL
-1+ sucrose 30～50gL
-1+ agar 6.0～7.0gL
-1Or MS+6-BA2.0mgL
-1+ NAA0.4～1.0mgL
-1+ sucrose 30～50gL
-1+ agar 6.0～7.0gL
-1
The colchicine concentration that is adopted in above-mentioned (4) step is 0.01～0.22%, and the processing time is 6～72h.
The present invention adopts colchicine solution immersion process induction polyploid bulb.It is to be induced by scaly leaf on the basis that produces callus that its innovation is in employed material; The tight callus that certain differentiating characteristic is arranged that obtains through presorting incubation again; When adopting the colchicine solution immersion treatment; Can alleviate the injury of soup greatly, thereby improve survival rate and the POLYPLOID INDUCEMENT rate of handling material, reach as high as 80.3% through the inductivity of testing the polyploid bulb to material.
Embodiment
Embodiment 1
(1) obtains the tight callus of the leaf roll bulb of fritillary
A. be chosen in the kangding and roll over the fresh bulb of the leaf roll bulb of fritillary that the base plantation is fostered in the life of many hills, strip the scaly leaf that internal layer is anosis and have no mechanical damage, clean back flushing 30min under flowing water with detergent liquid; Use 2% liquor natrii hypochloritis's rinsing 15min again; With clear water clean blot water after, the scaly leaf after will handling on the superclean bench is put into the 0.1% mercuric chloride solution 9min that sterilizes, aseptic water washing 3 times; Blot surface water with aseptic filter paper again, at last it is inserted MS+6-BA1mgL
-1+ IAA0.5mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Medium under 18～22 ℃ of temperature, illumination every day 8～16 hours, intensity of illumination are the condition of culture of 1200lx, carry out inducing culture, the dark cultivation that the initial stage of inducing carried out earlier 14 days;
B. the loose callus of new life, quality of above-mentioned acquisition is transferred into MS+6-BA2.0mgL
-1+ NAA0.2mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Presorting medium in be under the condition of culture of 1200lx in 18～22 ℃ of temperature, illumination every day 8～16 hours, intensity of illumination, cultivate 40d and can obtain tight callus;
(2) the polyploid bulb induces
The tight callus of the leaf roll bulb of fritillary that obtains is cut into small pieces is put in that filtration sterilization concentration is 1500mg.L
-1Colchicine solution in immersion treatment 24h, give a baby a bath on the third day after its birth time with sterile water then, blot water with aseptic filter paper again, insert the MS+6-BA1.0mgL that does not contain colchicine at last
-1+ NAA0.3mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Carry out polyploid bulb differentiation culture in the medium, the inductivity of its polyploid bulb is 80.3%;
(3) morphological feature of polyploid bulb
Compare with control group dliploid bulb, significantly variation takes place in the polyploid bulb that after colchicine-induced is handled, produces on form, show that volume obviously increases, and scaly leaf plumpness parcel closely is difficult for separately, and color is also significantly deepened than the dliploid bulb; And along with the prolongation of incubation time, the seedling phenomenon can not take place significantly to take out in the polyploid bulb, and pore obviously increases in the microexamination scaly leaf epidermal cell, but the stoma number in the unit are reduces;
(4) chromosome number is measured
Get and induce bulb heart bud point that group just differentiated with control group in 0.20% colchicine solution about preliminary treatment 2.5h, washing afterwards with the fixing 4h of methyl alcohol-glacial acetic acid (3: 1) fixer, uses mixed enzyme (cellulase and pectase respectively account for 2.5%) in 25 ℃ of insulating boxs, to handle 3h again; Remove enzyme liquid, with distillation washing 3 times, each 2min; After removing distilled water; The preparation cell suspension is used the sessile drop method film-making, and dry back is observed with Giemsa dyeing back; The result shows; The chromosome number 2n=2x=24 of control group dliploid bulb cell, and the leaf roll bulb of fritillary bulb cell chromosome number behind the process multiploid induction is 2n=4x=48, this shows that the multiploid induction of leaf roll bulb of fritillary bulb is successful;
(5) Determination of Total Alkaloid.
With the Peininine is standard items (purchasing in Chinese biological goods calibrating institute); The total alkaloid content that the employing ultraviolet spectrophotometry records through the polyploid bulb of induction culturing is 0.249%, is higher than the content of commercially available leaf roll bulb of fritillary bulb medicinal material Bulbus Fritillariae Cirrhosae 0.068% far away.
Claims (1)
1. one kind is the polyploid bulb abductive approach of explant with leaf roll bulb of fritillary scaly leaf, it is characterized in that comprising the following steps:
(1) scaly leaf of selecting the fresh bulb of the leaf roll bulb of fritillary is as explant;
(2) strip anosis and the scaly leaf that has no mechanical damage in the above-mentioned bulb internal layer; Earlier clean the back in flowing water flushing 25～30min down, use 2.0% liquor natrii hypochloritis's rinsing, 12～15min again, after cleaning and blot water with clear water with detergent liquid; Put it into the 7～10min that sterilizes in 0.1% mercuric chloride solution; Use aseptic water washing then 2～3 times, blot the moisture on surface again with aseptic filter paper, at last it is inserted the MS+6-BA1mgL of evoked callus
-1+ IAA0.5mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Carry out inducing culture in the medium;
(3) callus that the new life who (2) step is obtained, quality are loose inserts presorting medium MS+6-BA2.0mgL
-1+ NAA0.2mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1The tight callus of middle acquisition;
(4) the tight callus of the leaf roll bulb of fritillary that (3) step is obtained is cut into small pieces, and being soaked in, filtration sterilization concentration is 1500mgL
-1Colchicine solution in immersion treatment 24h, handle the back with aseptic washing 2～3 times, blot water with aseptic filter paper again after, access does not contain the MS+6-BA 1.0mgL of colchicine
-1+ NAA0.3mgL
-1+ sucrose 30gL
-1+ agar 6.5gL
-1Carry out inducing culture in the differential medium and obtain the polyploid bulb;
Above-mentioned all culture medium preparation pH values are 5.6～6.5;
Above-mentioned condition of culture adopted 12～15 days the dark cultivation except the evoked callus cultivation initial stage, and all the other are 15～24 ℃ of temperature, illumination every day 8～16 hours, intensity of illumination is 1000～1800lx.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103238525A (en) * | 2013-05-31 | 2013-08-14 | 常熟市佳盛农业科技发展有限公司 | Method for breeding fritillary bulb by tissue culture technique |
| CN103238524A (en) * | 2013-05-31 | 2013-08-14 | 常熟市佳盛农业科技发展有限公司 | Method for rapidly breeding fritillaria pallidiflora by using tissue culture technique |
| CN103283600A (en) * | 2013-05-31 | 2013-09-11 | 常熟市佳盛农业科技发展有限公司 | Method for rapidly propagating dark purple fritillary by utilizing tissue culture technology |
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| CN103283590B (en) * | 2012-03-01 | 2015-11-18 | 湖北省农科院中药材研究所 | A kind of production method of Hupeh Fritillary Bulb kind ball |
| CN103444526B (en) * | 2013-08-16 | 2015-11-25 | 顾祥茂 | A kind of growth-promoting cultivation method of Fritillaria unibracteata |
| CN103444527B (en) * | 2013-08-16 | 2016-04-27 | 苏州仁成生物科技有限公司 | A kind of growth-promoting cultivation method of Hupeh Fritillary Bulb |
| CN103598099B (en) * | 2013-11-27 | 2015-04-29 | 成都大学 | Cultural method for bulbus fritillariae cirrhosae multiploid callus capable of inhibiting brown stain |
| CN111492976B (en) * | 2020-05-18 | 2022-09-13 | 成都大学 | Method for culturing fleshy straight roots of bulbus fritillariae cirrhosae |
| CN116998407B (en) * | 2023-09-28 | 2023-12-05 | 西南林业大学 | Method for detoxification and rapid propagation of fritillaria fusiformis and application |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101161056A (en) * | 2007-09-28 | 2008-04-16 | 中国科学院新疆理化技术研究所 | A method for evoking yili caladium polyploid under culture in vitro |
-
2009
- 2009-09-03 CN CN2009101643394A patent/CN101999317B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101161056A (en) * | 2007-09-28 | 2008-04-16 | 中国科学院新疆理化技术研究所 | A method for evoking yili caladium polyploid under culture in vitro |
Non-Patent Citations (3)
| Title |
|---|
| 刘传军，等.离体培养条件下伊犁贝母四倍体诱导及筛选.《时珍国医国药》.2008,第19卷(第12期),2849-2851. * |
| 王强,等.秋水仙素诱导川贝母(Fritillaria cirrhosa D. Don)愈伤组织多倍体的研究.《武汉植物学研究》.2002,第20卷(第6期), * |
| 耿茂林，等.贝母组织培养研究进展.《淮北煤炭师范学院学报(自然科学版)》.2007,第28卷(第1期),38-42. * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103238525A (en) * | 2013-05-31 | 2013-08-14 | 常熟市佳盛农业科技发展有限公司 | Method for breeding fritillary bulb by tissue culture technique |
| CN103238524A (en) * | 2013-05-31 | 2013-08-14 | 常熟市佳盛农业科技发展有限公司 | Method for rapidly breeding fritillaria pallidiflora by using tissue culture technique |
| CN103283600A (en) * | 2013-05-31 | 2013-09-11 | 常熟市佳盛农业科技发展有限公司 | Method for rapidly propagating dark purple fritillary by utilizing tissue culture technology |
| CN103283600B (en) * | 2013-05-31 | 2014-10-15 | 太仓云联信息科技有限公司 | Method for rapidly propagating dark purple fritillary by utilizing tissue culture technology |
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Application publication date: 20110406 Assignee: Sichuan Enwei Pharmacy Co., Ltd. Assignor: Xue Yongxin Contract record no.: 2013510000030 Denomination of invention: Multiploid bulb induction method with firtillaria cirrhosa scale leaf as explant Granted publication date: 20120808 License type: Exclusive License Record date: 20130520 |
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