CN104067938B - The method of medicinal Rhizome of Peltate Yam group training forming seedling through one step culture mass production - Google Patents
The method of medicinal Rhizome of Peltate Yam group training forming seedling through one step culture mass production Download PDFInfo
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Abstract
The present invention is the method for medicinal Rhizome of Peltate Yam group training forming seedling through one step culture mass production, comprises (1) basis seedling and obtains; (2) forming seedling through one step culture mass production: band bud callus lines is divided into fritter, is connected on the Solid agar culture of a large amount of organic+MS molysite+6-BA0.2 of+B5 trace+MS��2.0mg/L+KT0.2��0.8mg/L+ paclobutrazol 0.5��5.0mg/L+ choline dichloride 2.0��4.0mg/L+IAA0.2��0.8mg/L+NAA0.2��0.8mg/L of MS and carries out breeding and taking root; (3) test-tube seedling transplanting; (4) land for growing field crops stem tuber is bred. The present invention expands Superior line colony in a short time by tissue culture technique, substantially reduce the seed selection time of the excellent germline of Rhizome of Peltate Yam, with the fastest speed, achievement in research can be converted into actual productivity, fully show the high benefit application of modern biotechnology in Chinese yam industrialization grows.
Description
Technical field
The invention belongs to the tissue culture technique field of medicinal Rhizome of Peltate Yam, it is specifically related to a kind of method that medicinal Rhizome of Peltate Yam tissue cultured seedling is criticized forming seedling through one step culture and quantized to produce.
Background technology
Yam (Dioscorea) has important pharmaceutical use, and its rhizome sapogenin is the important starting raw material of synthesis contraceptive bian and steroid hormone class medicine, occupies critical role in medicine industry. " China rare endangered plants register " is listed in national two grades gradually endanger species and protected. Because the diosgenin content of Rhizome of Peltate Yam root stock is the highest and become the preferred raw materials of yam industrialization development. Rhizome of Peltate Yam mainly by artificial growth, does not select owing to only planting for a long time, causes all property of product to be degenerated, and quality of medicinal material and output decline, chemical cost is unstable. Adopt plant cell engineering techniques and methods to carry out the extensive tissue culture fast-propagation of Rhizome of Peltate Yam, for promoting, the development of pharmacy industry has vital role in the plantation that standardizes.
The present invention, for solving Rhizome of Peltate Yam deterioration of strains problem, improve root yield and quality, adopts tissue culture technique, accelerates the production application popularization of its improved seeds. The present invention mainly adopts tissue culture technique to expand Superior line colony, and Rhizome of Peltate Yam tissue culture production technique, cultural method and condition are carried out global optimization and improvement, mainly for how shortening incubation time, improving culture efficiency, reducing toxigenic capacity, to createing the best approach of Rhizome of Peltate Yam forming seedling through one step culture, set up the culture system of a set of efficient low-consume amount, with the fastest speed, achievement in research is converted into actual productivity.
Summary of the invention
It is an object of the invention to provide the Rhizome of Peltate Yam tissue culture method of a kind of efficient low-consume amount, the method formulation efficacy is good, culture cycle is short, easily operation, cultivation program is simple, culture efficiency height, good stability, production cost are low, are a kind of rapid breeding method obtaining a large amount of test-tube plantlet within the shortest time. And the inventive method is free from environmental pollution, it is applicable to very much suitability for industrialized production, has reached industrialization level.
It is an object of the invention to realize in the following manner:
The method of medicinal Rhizome of Peltate Yam group training forming seedling through one step culture mass production, comprises the following steps:
(1) basis seedling obtains: select the stem section of excellent medicinal Rhizome of Peltate Yam plant and terminal bud to be explant, after explant sterilization on a large amount of organic+2 �� MS molysite+6-BA2.0 of+B5 trace+MS��4.0mg/L+KT0.2��0.8mg/L+ paclobutrazol 2.0��8.0mg/L+ choline dichloride 2.0��5.0mg/L+IAA0.2��0.8mg/L+NAA0.2��0.8mg/L Solid agar culture of MS, synchronously induce the callus of band bud;
(2) forming seedling through one step culture mass production: band bud callus lines step (1) obtained is divided into fritter, is connected on the Solid agar culture of a large amount of organic+MS molysite+6-BA0.2 of+B5 trace+MS��2.0mg/L+KT0.2��0.8mg/L+ paclobutrazol 0.5��5.0mg/L+ choline dichloride 2.0��4.0mg/L+IAA0.2��0.8mg/L+NAA0.2��0.8mg/L of MS and carries out breeding and taking root;
(3) test-tube seedling transplanting: before test-tube plantlet bottle outlet is transplanted, the seedling that grows thickly various for root leaf is divided into single strain, the seedling of bottle outlet is first soaked 10��15 minutes with the derosal of 500��800 times or zinc manganese ethylenebisdithiocarbamate, seedling is moved in the transplanting container having top glass subsequently, it it is 20��30 DEG C in temperature, grow when humidity 80��90%, when 25��30d grows new leaf, already survive;
(4) land for growing field crops stem tuber is bred: by the seedling of transplant survival, and after natural condition hardening 3��5d, transplanting carries out stem tuber to land for growing field crops and breeds.
Explant is cut into the specification containing 1 joint 1 bud by step (1), first clean clean with washing powder, with aseptic washing 5��7 times on aseptic operating platform, 30 seconds-1 minute is infiltrated with 70% alcohol, put into the mercuric chloride solution sterilizing 12��18 minutes of 0.1% again, aseptic water washing 5��6 times, for subsequent use.
The culture temperature of step (1) and (2) is in 24 DEG C �� 2,1000��2000lx low light environment, and every day 10��12, h light was cultivated.
During the bud succeeding transfer culture that step (1) induces, going old stem to stay sprouting, new leaf stem section or terminal bud are divided into 1 joint 1 bud again and proceed in identical inducing culture, every 25��30d subculture once.
Step (2) is that by the specification of 0.5��1cm �� 0.5��1cm, callus lines is divided into fritter.
When step (2) is cultivated every 25��30d subculture once.
A large amount of+B5 trace+MS organic+2 �� MS molysite+6-BA2.0mg/L+KT0.4mg/L+ paclobutrazol 4.0mg/L+ choline dichloride 2.0mg/L+IAA0.2mg/L+NAA0.4mg/L of the substratum that step (1) preferably uses: MS. Or preferably use substratum: a large amount of+B5 trace+MS organic+2 �� MS molysite+6-BA3.0mg/L+KT0.4mg/L+ paclobutrazol 4.0mg/L+ choline dichloride 2.0mg/L+IAA0.4mg/L+NAA0.4mg/L of MS;
A large amount of+B5 trace+MS organic+MS molysite+6-BA0.5mg/L+KT0.2mg/L+ paclobutrazol 2.0mg/L+ choline dichloride the 2.0mg/L+IAA0.2mg/L+NAA0.2mg/L of the substratum that step (2) preferably uses: MS. Or preferably use substratum: a large amount of+B5 trace+MS organic+MS molysite+6-BA0.8mg/L+KT0.5mg/L+ paclobutrazol 2.0mg/L+ choline dichloride 3.0mg/L+IAA0.2mg/L+NAA0.2mg/L of MS.
Compared with prior art, the useful effect of the present invention is in the present invention:
1, the weak point existed for prior art, the present invention expands Superior line colony in a short time by tissue culture technique, substantially reduce the seed selection time of the excellent germline of Rhizome of Peltate Yam, with the fastest speed, achievement in research can be converted into actual productivity, fully show the high benefit application of modern biotechnology in Chinese yam industrialization grows.
2, according to the research of forefathers, the tissue culture of Rhizome of Peltate Yam mainly adopts explant one callus (inducing culture) to break up seedling (differentiation culture) multiple cultivation programs such as (root culture) of taking root and completes, and at least needs 90d. The present invention by just generation and subculture, breed and take root, multiple cultivation program simplification is that a step completes.
3, during large-scale industrialized production, the present invention designs cultural method according to need of production, and first synchronous callus induction and bud, suppress the formation of root, it is to increase proliferation times, accelerate the breeding of basis seedling;Mass production stage synchronous evoked callus, bud, adventive root, breed and be reduced to a step complete with taking root, be i.e. " forming seedling through one step culture " method, forms whole plant and only needs about 30 days, has saved the rootage duration of 25��30 days.
4, routine techniques produces 1,000,000 strain Rhizome of Peltate Yam tissue cultured seedling, forms the tissue cultured seedling of 15 strains for taking root according to every bottle of Regenerated plant, need to produce 140,000 bottles of tissue cultured seedling, comprising 70,000 bottles of Regenerated plant, and 70,000 bottles of seedlings of taking root. The technology of the present invention produces 1,000,000 strain Rhizome of Peltate Yam tissue cultured seedling, only needs to produce 5.5 ten thousand bottles of subcultures and takes root seedling. The present invention not only reduces aseptic technique step, and has saved culturing room space, simplify health seedling production sequence, reduce production cost, it is to increase production efficiency.
5, forefathers study proof, the domestication of Rhizome of Peltate Yam introduces a fine variety the research bred fast with isolated culture and achieves bigger progress, but aseptic brachyplast difficulty is taken root, the transplanting survival rate of test-tube plantlet is very low, this two big key problem becomes the bottleneck that the extension of Rhizome of Peltate Yam group culturation rapid propagating technology is produced. The present invention not only creates forming seedling through one step culture method, and transplanting survival rate height (reaching more than 95%); Cultivation then (about 8 months), obtains the high-quality kind stem about 30g/ strain.
Accompanying drawing explanation
Fig. 1 is Rhizome of Peltate Yam stem section and the aseptic seedling growing state of terminal bud induction;
Fig. 2 is the synchronous callus induction of Rhizome of Peltate Yam first culture and bud growing state;
Fig. 3 is the synchronous callus induction of Rhizome of Peltate Yam succeeding transfer culture, bud and root growth situation;
Fig. 4 is the Rhizome of Peltate Yam test-tube plantlet growth situation of transplant survival;
Fig. 5 is for cultivating single strain kind Stem nematode situation of (about 8 months) Rhizome of Peltate Yam test-tube plantlet then.
Embodiment
It is intended to illustrate further the present invention below in conjunction with embodiment, and unrestricted the present invention.
Embodiment 1, tissue-culturing quick-propagation Rhizome of Peltate Yam
One, substratum preparation and sterilizing
First culture base is used for basis seedling breeding: a large amount of+B5 trace+MS organic+2 �� MS molysite+6-BA2.0mg/L+KT0.4mg/L+ paclobutrazol (PP of MS333) 4.0mg/L+ choline dichloride (CCC) 2.0mg/L+IAA0.2mg/L+NAA0.4mg/L;
Subculture medium is used for a large amount of+B5 trace+MS organic+MS molysite+6-BA0.5mg/L+KT0.2mg/L+ paclobutrazol (PP of forming seedling through one step culture mass production: MS333) 2.0mg/L+ choline dichloride (CCC) 2.0mg/L+IAA0.2mg/L+NAA0.2mg/L;
At above substratum 121 DEG C, sterilizing 20 minutes.
Two, basis seedling breeding
Get Rhizome of Peltate Yam (salviahispanicaL) terminal bud and stem section, first clean clean with washing powder, with aseptic washing 5��7 times on aseptic operating platform, 1 minute is infiltrated with 70% alcohol, put into the mercuric chloride solution sterilizing 12��18 minutes of 0.1% again, aseptic water washing 5��6 times, terminal bud and axillalry bud are proceeded in being equipped with on first culture base triangular flask (capacity 50 milliliters), each triangular flask explant number is 3��5, cultivate under following culture condition: about 24 DEG C, every day 10 h light, intensity of illumination is about 1000��2000lx; Terminal bud inoculates 300, and axillalry bud inoculates 320. Cultivating about 4 weeks, obtain the aseptic explant that 320 form 2-5 joint and about 1cm size callus block, synchronous callus induction and bud growing state are as shown in Figure 1. During succeeding transfer culture, callus being divided into fritter, new leaf stem section or terminal bud are divided into 1 joint 1 bud again and proceed in first culture base, and every 30d subculture once, in each subculture, every block callus increases 4-6 doubly, and synchronous callus induction and bud growing state are as shown in Figure 2.This step needs the formation suppressing root, in case the breeding of impact basis seedling. According to industrial scale, the basic seedling that breeding is sufficient, then carry out mass production.
Three, forming seedling through one step culture mass production
320 strains got in above-mentioned 320 strains carry out successive propagation, and concrete grammar is as follows: the callus and the buds that obtain step 2 are divided into fritter by the specification of 0.5cm �� 0.5cm, are seeded in subculture medium and cultivate, the same step 2 of culture condition; After cultivating 4 weeks, the clump bud of every block callus all grows up to the high bud of growing thickly of 2-6cm, goes out the whole plant of 3-7 bar root from stem minister Duan Ji simultaneously.
This callus more according to the method described above every 4 weeks subcultures once, altogether subculture 5 times. In each subculture, every block callus increases 4-6 doubly, and callus surface all grows the high bud of growing thickly of 2-6cm, goes out the whole plant of 3-7 bar root from stem minister Duan Ji simultaneously. Synchronously breed the test-tube plantlet growth situation taken root as shown in Figure 3;
Four, test-tube seedling transplanting:
Before test-tube plantlet bottle outlet is transplanted, the seedling that grows thickly various for root leaf is divided into single strain, the seedling of bottle outlet first derosal or zinc manganese ethylenebisdithiocarbamate with 500��800 times soak 15 minutes, seedling is moved in the transplanting container having top glass subsequently, it it is 20��30 DEG C in temperature, grow when humidity 80��90%, when 25��30d grows new leaf, already survive;
Five, land for growing field crops stem tuber is bred:
By the seedling of transplant survival, after natural condition hardening 3��5d, transplanting carries out stem tuber to land for growing field crops and breeds.
Rhizome of Peltate Yam tissue culture propagation provided by the present invention, terminal bud or stem section are explant, draw materials easily, and quantity is big, genetic stability height, the advantages such as well-grown. In first culture process, not only terminal bud or axillary bud growth speed are fast, and energy synchronously evoked callus and bud, and breeding coefficient is big. The mass production stage, adopt the method for synchronous callus induction, bud, adventive root, simplify health seedling production sequence, routine techniques produces 1,000,000 strain Rhizome of Peltate Yam tissue cultured seedling, the tissue cultured seedling of 15 strains for taking root is formed according to every bottle of Regenerated plant, 140,000 bottles of tissue cultured seedling need to be produced, comprising 70,000 bottles of Regenerated plant, 70,000 bottles of seedlings of taking root. The technology of the present invention produces 1,000,000 strain Rhizome of Peltate Yam tissue cultured seedling, only needs to produce 5.5 ten thousand bottles of subcultures and takes root seedling. The present invention not only reduces aseptic technique step, and has saved culturing room space, reduce production cost, it is to increase production efficiency. And the aseptic brachyplast difficulty of Rhizome of Peltate Yam is taken root, the transplanting survival rate of test-tube plantlet is very low, this two big key problem becomes the bottleneck that the extension of Rhizome of Peltate Yam group culturation rapid propagating technology is produced. The present invention not only creates forming seedling through one step culture method, and transplanting survival rate height (reaching more than 95%); Cultivation then (about 8 months), obtains the high-quality kind stem about 30g/ strain.
Embodiment 2, tissue-culturing quick-propagation Rhizome of Peltate Yam
One, substratum preparation and sterilizing
A large amount of+B5 trace+MS organic+2 �� MS molysite+6-BA3.0mg/L+KT0.4mg/L+ paclobutrazol (PP of first culture base: MS333) 4.0mg/L+ choline dichloride (CCC) 2.0mg/L+IAA0.4mg/L+NAA0.4mg/L; A large amount of+B5 trace+MS organic+MS molysite+6-BA0.8mg/L+KT0.5mg/L+ paclobutrazol (the PP of subculture medium: MS333) 2.0mg/L+ choline dichloride (CCC) 3.0mg/L+IAA0.2mg/L+NAA0.2mg/L;
At above substratum 121 DEG C, sterilizing 20 minutes.
Getting Rhizome of Peltate Yam (salviahispanicaL) terminal bud or stem section, elementary operation method is consistent with embodiment 1;Effect and embodiment 1 are more or less the same.
Embodiment 3
A large amount of organic+2 �� MS molysite+6-BA2.0 of+B5 trace+MS��4.0mg/L+KT0.2��0.8mg/L+ paclobutrazol 2.0��8.0mg/L+ choline dichloride 2.0��5.0mg/L+IAA0.2��0.8mg/L+NAA0.2��0.8mg/L Solid agar culture of first culture base: MS;
The Solid agar culture of a large amount of organic+MS molysite+6-BA0.2 of+B5 trace+MS��2.0mg/L+KT0.2��0.8mg/L+ paclobutrazol 0.5��5.0mg/L+ choline dichloride 2.0��4.0mg/L+IAA0.2��0.8mg/L+NAA0.2��0.8mg/L of subculture medium: MS.
Adopt above-mentioned culture medium prescription, and in described formula range, other elementary operation method is consistent with embodiment 1.
Claims (5)
1. the method for medicinal Rhizome of Peltate Yam group training forming seedling through one step culture mass production, it is characterised in that, comprise the following steps:
(1) basis seedling obtains: select the stem section of excellent medicinal Rhizome of Peltate Yam plant and terminal bud to be explant, at a large amount of+B5 trace+MS organic+2 �� MS molysite+6-BA2.0mg/L+KT0.4mg/L+ paclobutrazol 4.0mg/L+ choline dichloride 2.0mg/L+IAA0.2mg/L+NAA0.4mg/L of MS after explant sterilization; Or on the Solid agar culture of a large amount of+B5 trace+MS organic+2 �� MS molysite+6-BA3.0mg/L+KT0.4mg/L+ paclobutrazol 4.0mg/L+ choline dichloride 2.0mg/L+IAA0.4mg/L+NAA0.4mg/L of MS, synchronously induce the callus of band bud;
(2) forming seedling through one step culture mass production: band bud callus lines step (1) obtained is divided into fritter, is connected on a large amount of+B5 trace+MS organic+MS molysite+6-BA0.5mg/L+KT0.2mg/L+ paclobutrazol 2.0mg/L+ choline dichloride 2.0mg/L+IAA0.2mg/L+NAA0.2mg/L of MS; Or the Solid agar culture of a large amount of+B5 trace+MS organic+MS molysite+6-BA0.8mg/L+KT0.5mg/L+ paclobutrazol 2.0mg/L+ choline dichloride 3.0mg/L+IAA0.2mg/L+NAA0.2mg/L of MS carries out breeding and taking root;
(3) test-tube seedling transplanting: before test-tube plantlet bottle outlet is transplanted, the seedling that grows thickly various for root leaf is divided into single strain, the seedling of bottle outlet is first soaked 10��15 minutes with the derosal of 500��800 times or zinc manganese ethylenebisdithiocarbamate, seedling is moved in the transplanting container having top glass subsequently, it it is 20��30 DEG C in temperature, grow when humidity 80��90%, when 25��30d grows new leaf, already survive;
(4) land for growing field crops stem tuber is bred: by the seedling of transplant survival, and after natural condition hardening 3��5d, transplanting carries out stem tuber to land for growing field crops and breeds;
The culture temperature of step (1) and (2) is in 24 DEG C �� 2,1000��2000lx low light environment, and every day 10��12, h light was cultivated.
2. the method for medicinal Rhizome of Peltate Yam group according to claim 1 training forming seedling through one step culture mass production, it is characterized in that, explant is cut into the specification containing 1 joint 1 bud by step (1), first clean clean with washing powder, with aseptic washing 5��7 times on aseptic operating platform, infiltrate 30 seconds-1 minute with 70% alcohol, then put into the mercuric chloride solution sterilizing 12��18 minutes of 0.1%, aseptic water washing 5��6 times, for subsequent use.
3. the method for medicinal Rhizome of Peltate Yam group according to claim 1 training forming seedling through one step culture mass production, it is characterized in that, during the bud succeeding transfer culture that step (1) induces, old stem is gone to stay sprouting, new leaf stem section or terminal bud are divided into 1 joint 1 bud again and proceed in identical substratum, every 25��30d subculture once.
4. the method for medicinal Rhizome of Peltate Yam group according to claim 1 training forming seedling through one step culture mass production, it is characterised in that, step (2) is that by the specification of 0.5��1cm �� 0.5��1cm, callus lines is divided into fritter.
5. the method for medicinal Rhizome of Peltate Yam group according to claim 1 training forming seedling through one step culture mass production, it is characterised in that, when step (2) is cultivated every 25��30d subculture once.
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| CN106472318B (en) * | 2016-10-20 | 2018-08-10 | 中国科学院亚热带农业生态研究所 | A kind of method of medicinal dioscorea zingiberensis wright mass production |
| CN109644871B (en) * | 2019-01-21 | 2022-01-11 | 花垣县精虹生物科技发展有限公司 | Batch production method suitable for various orchidaceous medicinal plants |
| CN116114603B (en) * | 2023-03-06 | 2024-05-24 | 贵州省生物技术研究所(贵州省生物技术重点实验室、贵州省马铃薯研究所、贵州省食品加工研究所) | Proliferation induction rapid propagation method of double-cell yam |
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Address after: 410125 No. 644 Yuanda Second Road, Changsha City, Hunan Province Co-patentee after: Kunming Pharmaceutical Group Chongqing Wuling Pharmaceutical Co. Ltd. Patentee after: Institute of Subtropical Agriculture, Chinese Academy of Sciences Address before: 410125 No. 644 Yuanda Second Road, Changsha City, Hunan Province Co-patentee before: Chongqing Huafang Wulingshan Pharmaceutical Co., Ltd. Patentee before: Institute of Subtropical Agriculture, Chinese Academy of Sciences |
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