CN104067938A - Tissue culture one-step seedling formation batch production method of medicinal peltate yam rhizome - Google Patents
Tissue culture one-step seedling formation batch production method of medicinal peltate yam rhizome Download PDFInfo
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Abstract
The invention discloses a tissue culture one-step seedling formation batch production method of medicinal peltate yam rhizome. The method comprises the following steps: (1) obtaining basic seedlings; (2) performing one-step seedling formation batch production, namely dividing callus blocks with buds into small pieces, and inoculating to a solid agar culture medium containing lots of MS, trace B5, MS organics, MS iron salt, 0.2-2.0mg/L of 6-BA, 0.2-0.8mg/L of KT, 0.5-5.0mg/L of paclobutrazol, 2.0-4.0mg/L of cycocel, 0.2-0.8mg/L of IAA and 0.2-0.8mg/L of NAA for performing multiplication and rooting; (3) performing test-tube plantlet transplanting; and (4) breeding field tubers. Preferential single plants are enlarged in a short time through a tissue culture technology, the time for breeding high-quality germ lines of peltate yam rhizome is greatly shortened, the research result can be converted into practical productivity at the highest speed, and high-efficiency application of modern biotechnology to industrialized yam growth is fully shown.
Description
Technical field
The invention belongs to the tissue culture technology field of medicinal dioscorea zingiberensis wright, be specifically related to a kind of medicinal dioscorea zingiberensis wright group training seedling and criticize the method that forming seedling through one step culture quantizes production.
Background technology
Yam (Dioscorea) has important medical value, and its rhizome sapogenin is the important initiation material of synthetic contraceptive and steroid hormone class medicine, occupies critical role in medical industry.In < < China Precious, Rare, Endangered protective plant register > >, being listed in national secondary gradually endangers species and is protected.Because of the highest preferred raw materials that becomes yam industrialization development of the rhizomatic Diosgenin Content of dioscorea zingiberensis wright.Dioscorea zingiberensis wright, mainly by artificial planting, for a long time owing to only planting and not selecting, causes Different Varieties to be degenerated, quality of medicinal material and production declining, and chemical cost is unstable.Adopt plant cell engineering technology and method to carry out the extensive tissue-culturing rapid propagation of dioscorea zingiberensis wright, standardized planting for the development important role that promotes pharmacy industry.
The present invention, for solving dioscorea zingiberensis wright deterioration of variety problem, improving 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 dioscorea zingiberensis wright tissue is cultivated to production technology, cultural method and condition and carry out global optimization and improvement, mainly for how shortening incubation time, improving culture efficiency, reducing and cultivate cost, to createing the best approach of dioscorea zingiberensis wright forming seedling through one step culture, the cultivating system of setting up a set of efficient low-consume amount, is converted into actual productivity with the fastest speed by achievement in research.
Summary of the invention
The dioscorea zingiberensis wright method for tissue culture that the object of this invention is to provide a kind of efficient low-consume amount, the method formula effect is good, cultivation cycle is short, easy to operate, cultivation program is simple, culture efficiency is high, and good stability, production cost are low, are a kind of method for quickly breeding that obtains a large amount of test-tube plantlets within the shortest time.And the inventive method is free from environmental pollution, be applicable to very much suitability for industrialized production, reached industrialization level.
The object of the invention is to realize in the following manner:
The method of medicinal dioscorea zingiberensis wright group training forming seedling through one step culture mass production, comprises the following steps:
(1) basic seedling obtains: selecting the stem section of good medicinal dioscorea zingiberensis wright plant and terminal bud is explant, after explant sterilization MS a large amount of+B5 trace+MS is organic+2 * MS molysite+6-BA2.0～4.0mg/L+KT0.2～0.8mg/L+ paclobutrazol, 2.0～8.0mg/L+ chlormequat, 2.0～5.0mg/L+IAA0.2～0.8mg/L+NAA0.2～0.8mg/L Solid agar culture on, synchronously induce the callus with bud;
(2) forming seedling through one step culture mass production: the band bud callus lines that step (1) is obtained is divided into fritter, be connected on MS a large amount of+B5 trace+MS is organic+breed and take root on the Solid agar culture of MS molysite+6-BA0.2～2.0mg/L+KT0.2～0.8mg/L+ paclobutrazol 0.5～5.0mg/L+ chlormequat 2.0～4.0mg/L+IAA0.2～0.8mg/L+NAA0.2～0.8mg/L;
(3) test-tube seedling transplanting: before test-tube plantlet bottle outlet is transplanted, the seedling that grows thickly that root leaf is various is divided into individual plant, the seedling of bottle outlet is first soaked 10～15 minutes with carbendazim or the mancozeb of 500～800 times, subsequently seedling is moved in the transplanting container of top glass, in temperature, it is 20～30 ℃, under the condition of humidity 80～90%, grow, when 25～30d grows young leaves, already survive;
(4) land for growing field crops stem tuber is bred: by the seedling of transplant survival, after natural conditions hardening 3～5d, transplanting is carried out stem tuber and bred to land for growing field crops.
In step (1), explant is cut into the specification that contains 1 joint 1 bud, first with washing powder, clean up, on aseptic operating platform with aseptic washing 5～7 times, with 70% alcohol, infiltrate 30 seconds-1 minute, put into again 0.1% mercuric chloride solution sterilizing 12～18 minutes, aseptic water washing 5～6 times, standby.
The cultivation temperature of step (1) and (2) is in 24 ℃ ± 2,1000～2000lx low light environment, 10～12 hours every days illumination cultivation.
When the bud subculture that step (1) induces is cultivated, go 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 callus lines is divided into fritter by the specification of 0.5～1cm * 0.5～1cm.
When step (2) is cultivated every 25～30d subculture once.
The medium that step (1) is preferably used: MS is a large amount of+and B5 trace+MS is organic+2 * MS molysite+6-BA2.0mg/L+KT0.4mg/L+ paclobutrazol 4.0mg/L+ chlormequat 2.0mg/L+IAA0.2mg/L+NAA0.4mg/L.Or preferably use medium: MS a large amount of+B5 trace+MS is organic+2 * MS molysite+6-BA3.0mg/L+KT0.4mg/L+ paclobutrazol 4.0mg/L+ chlormequat 2.0mg/L+IAA0.4mg/L+NAA0.4mg/L;
The medium that step (2) is preferably used: MS is a large amount of+and B5 trace+MS is organic+MS molysite+6-BA0.5mg/L+KT0.2mg/L+ paclobutrazol 2.0mg/L+ chlormequat 2.0mg/L+IAA0.2mg/L+NAA0.2mg/L.Or preferably use medium: MS a large amount of+B5 trace+MS is organic+MS molysite+6-BA0.8mg/L+KT0.5mg/L+ paclobutrazol 2.0mg/L+ chlormequat 3.0mg/L+IAA0.2mg/L+NAA0.2mg/L.
Compared with prior art, beneficial effect of the present invention is in the present invention:
1, the weak point existing for prior art, the present invention has expanded Superior line colony in a short time by tissue culture technique, greatly shortened the seed selection time of the good germline of dioscorea zingiberensis wright, can achievement in research be converted into actual productivity with the fastest speed, fully show the high benefit application of modern biotechnology in Chinese yam industrialization growth.
2,, according to forefathers' research, the tissue of dioscorea zingiberensis wright is cultivated main explant one callus (induction an is cultivated) differentiation seedling (differentiation is cultivated) a plurality of cultivation programs such as (culture of rootage) of taking root that adopt and is completed, and at least needs 90d.The present invention just generation with subculture, breed and take root, a plurality of cultivation program simplifications are 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, improves propagation multiple, accelerates the breeding of basic seedling; Synchronous evoked callus of mass production stage, bud, adventive root, breed and be reduced to a step and complete with taking root, i.e. " forming seedling through one step culture " method, and forming whole plant only needed about 30 days, had saved the rootage duration of 25～30 days.
4, routine techniques is produced 1,000,000 strain dioscorea zingiberensis wright group training seedlings, according to every bottle of subculture seedling, forms the group training seedling of 15 strains for taking root, and needs to produce 140,000 bottles of group training seedlings, comprising 70,000 bottles of subculture seedlings, and 70,000 bottles of seedlings of taking root.The technology of the present invention is produced 1,000,000 strain dioscorea zingiberensis wright groups training seedlings, only needs to produce 5.5 ten thousand bottles of subcultures seedling of taking root.The present invention has not only reduced sterile working step, and has saved culturing room space, has simplified health seedling production routine, has reduced production cost, has improved production efficiency.
5, forefathers studies have shown that, the domestication of dioscorea zingiberensis wright introduces a fine variety with the research of cultured in vitro Fast-propagation and has obtained larger progress, but aseptic brachyplast difficulty is taken root, the transplanting survival rate of test-tube plantlet is very low, this two large key problem becomes the bottleneck that the extension of dioscorea zingiberensis wright group culturation rapid propagating technology is produced.The present invention not only creates forming seedling through one step culture method, and transplanting survival rate high (reaching more than 95%); Cultivation is (about 8 months) then, obtain the high-quality kind stem of 30g/ strain left and right.
Accompanying drawing explanation
Fig. 1 is the Aseptic Seedling Growth situation of dioscorea zingiberensis wright stem section and terminal bud induction;
Fig. 2 is the just synchronous callus induction of culture and bud growing state of dioscorea zingiberensis wright;
Fig. 3 is that dioscorea zingiberensis wright subculture is cultivated synchronous callus induction, bud and root growth situation;
Fig. 4 is the dioscorea zingiberensis wright test-tube plantlet growth situation of transplant survival;
Fig. 5 is for cultivating the individual plant kind stem growing state of (about 8 months) dioscorea zingiberensis wright test-tube plantlet then.
Embodiment
Below in conjunction with embodiment, be intended to further illustrate the present invention, and unrestricted the present invention.
Embodiment 1, tissue-culturing quick-propagation dioscorea zingiberensis wright
One, medium preparation and sterilizing
Just culture base for basic seedling breeding: MS a large amount of+B5 trace+MS is organic+2 * MS molysite+6-BA2.0mg/L+KT0.4mg/L+ paclobutrazol (PP
333) 4.0mg/L+ chlormequat (CCC) 2.0mg/L+IAA0.2mg/L+NAA0.4mg/L;
Subculture medium for forming seedling through one step culture mass production: MS a large amount of+B5 trace+MS is organic+MS molysite+6-BA0.5mg/L+KT0.2mg/L+ paclobutrazol (PP
333) 2.0mg/L+ chlormequat (CCC) 2.0mg/L+IAA0.2mg/L+NAA0.2mg/L;
At 121 ℃ of above medium, sterilizing 20 minutes.
Two, basic seedling breeding
Get dioscorea zingiberensis wright (salvia hispanica L) terminal bud and stem section, first with washing powder, clean up, on aseptic operating platform with aseptic washing 5～7 times, with 70% alcohol, infiltrate 1 minute, put into again 0.1% mercuric chloride solution sterilizing 12～18 minutes, aseptic water washing 5～6 times, terminal bud and axillalry bud are proceeded to and be equipped with just on culture base in triangular flask (50 milliliters of capacity), each triangular flask explant number is 3～5, under following condition of culture, cultivate: 24 ℃ of left and right, illumination 10 hours every days, intensity of illumination is 1000～2000lx left and right; 300 of terminal bud inoculations, 320 of axillalry bud inoculations.Cultivate about 4 weeks, obtain 320 aseptic explants that form 2-5 joint and the big or small callus piece in 1cm left and right, synchronous callus induction and bud growing state are as shown in Figure 1.When subculture is cultivated, callus is 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 callus increases 4-6 doubly, and synchronous callus induction and bud growing state are as shown in Figure 2.This step need to suppress the formation of root, in case affect the breeding of basic seedling.According to production scale, breed sufficient basic seedling, then carry out mass production.
Three, forming seedling through one step culture mass production
320 strains of getting in above-mentioned 320 strains are carried out successive propagation, and concrete grammar is as follows: callus and bud that step 2 is obtained are divided into fritter by the specification of 0.5cm * 0.5cm, are seeded in subculture medium and cultivate, the same step 2 of condition of culture; Cultivate after 4 weeks, the clump bud of every callus all grows up to the Multiple Buds that 2-6cm is high, goes out the whole plant of 3-7 bar root from stem segment base minister simultaneously.
This callus more according to the method described above every 4 weeks subcultures once, altogether subculture is 5 times.In each subculture, every callus increases 4-6 doubly, and callus surface all grows the Multiple Buds that 2-6cm is high, goes out the whole plant of 3-7 bar root from stem segment base minister simultaneously.The test-tube plantlet growth situation that synchronous propagation is 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 that root leaf is various is divided into individual plant, the seedling of bottle outlet is first soaked 15 minutes with carbendazim or the mancozeb of 500～800 times, subsequently seedling is moved in the transplanting container of top glass, in temperature, it is 20～30 ℃, under the condition of humidity 80～90%, grow, when 25～30d grows young leaves, already survive;
Five, land for growing field crops stem tuber is bred:
By the seedling of transplant survival, after natural conditions hardening 3～5d, transplanting is carried out stem tuber and is bred to land for growing field crops.
Dioscorea zingiberensis wright tissue culture propagation provided by the present invention, the advantage such as terminal bud or stem section are explant, draw materials easily, and quantity is large, and genetic stability is high, well-grown.In first culture process, not only terminal bud or axillary bud growth speed are fast, and the synchronous evoked callus of energy and bud, and reproduction coefficient is large.The mass production stage, adopt the method for synchronous callus induction, bud, adventive root, simplified health seedling production routine, routine techniques is produced 1,000,000 strain dioscorea zingiberensis wright group training seedlings, according to every bottle of subculture seedling, form the group training seedling of 15 strains for taking root, need to produce 140,000 bottles of group training seedlings, comprising 70,000 bottles of subculture seedlings, 70,000 bottles of seedlings of taking root.The technology of the present invention is produced 1,000,000 strain dioscorea zingiberensis wright groups training seedlings, only needs to produce 5.5 ten thousand bottles of subcultures seedling of taking root.The present invention has not only reduced sterile working step, and has saved culturing room space, has reduced production cost, has improved production efficiency.And the aseptic brachyplast difficulty of dioscorea zingiberensis wright is taken root, the transplanting survival rate of test-tube plantlet is very low, this two large key problem becomes the bottleneck that the extension of dioscorea zingiberensis wright group culturation rapid propagating technology is produced.The present invention not only creates forming seedling through one step culture method, and transplanting survival rate high (reaching more than 95%); Cultivation is (about 8 months) then, obtain the high-quality kind stem of 30g/ strain left and right.
Embodiment 2, tissue-culturing quick-propagation dioscorea zingiberensis wright
One, medium preparation and sterilizing
Just culture base: MS a large amount of+B5 trace+MS is organic+2 * MS molysite+6-BA3.0mg/L+KT0.4mg/L+ paclobutrazol (PP
333) 4.0mg/L+ chlormequat (CCC) 2.0mg/L+IAA0.4mg/L+NAA0.4mg/L; Subculture medium: MS is a large amount of+and B5 trace+MS is organic+MS molysite+6-BA0.8mg/L+KT0.5mg/L+ paclobutrazol (PP
333) 2.0mg/L+ chlormequat (CCC) 3.0mg/L+IAA0.2mg/L+NAA0.2mg/L;
At 121 ℃ of above medium, sterilizing 20 minutes.
Get dioscorea zingiberensis wright (salvia hispanica L) terminal bud or stem section, basic operation method is consistent with embodiment 1; Effect and embodiment 1 are more or less the same.
Embodiment 3
Just culture base: MS a large amount of+B5 trace+MS is organic+2 * MS molysite+6-BA2.0～4.0mg/L+KT0.2～0.8mg/L+ paclobutrazol, 2.0～8.0mg/L+ chlormequat, 2.0～5.0mg/L+IAA0.2～0.8mg/L+NAA0.2～0.8mg/L Solid agar culture;
Subculture medium: MS is a large amount of+and B5 trace+MS is organic+Solid agar culture of MS molysite+6-BA0.2～2.0mg/L+KT0.2～0.8mg/L+ paclobutrazol 0.5～5.0mg/L+ chlormequat 2.0～4.0mg/L+IAA0.2～0.8mg/L+NAA0.2～0.8mg/L.
Adopt above-mentioned culture medium prescription, and in described formula range, other basic operation method is consistent with embodiment 1.
Claims (10)
1. the method for medicinal dioscorea zingiberensis wright group training forming seedling through one step culture mass production, is characterized in that, comprises the following steps:
(1) basic seedling obtains: selecting the stem section of good medicinal dioscorea zingiberensis wright plant and terminal bud is explant, after explant sterilization MS a large amount of+B5 trace+MS is organic+2 * MS molysite+6-BA2.0～4.0mg/L+KT0.2～0.8mg/L+ paclobutrazol, 2.0～8.0mg/L+ chlormequat, 2.0～5.0mg/L+IAA0.2～0.8mg/L+NAA0.2～0.8mg/L Solid agar culture on, synchronously induce the callus with bud;
(2) forming seedling through one step culture mass production: the band bud callus lines that step (1) is obtained is divided into fritter, be connected on MS a large amount of+B5 trace+MS is organic+breed and take root on the Solid agar culture of MS molysite+6-BA0.2～2.0mg/L+KT0.2～0.8mg/L+ paclobutrazol 0.5～5.0mg/L+ chlormequat 2.0～4.0mg/L+IAA0.2～0.8mg/L+NAA0.2～0.8mg/L;
(3) test-tube seedling transplanting: before test-tube plantlet bottle outlet is transplanted, the seedling that grows thickly that root leaf is various is divided into individual plant, the seedling of bottle outlet is first soaked 10～15 minutes with carbendazim or the mancozeb of 500～800 times, subsequently seedling is moved in the transplanting container of top glass, in temperature, it is 20～30 ℃, under the condition of humidity 80～90%, grow, when 25～30d grows young leaves, already survive;
(4) land for growing field crops stem tuber is bred: by the seedling of transplant survival, after natural conditions hardening 3～5d, transplanting is carried out stem tuber and bred to land for growing field crops.
2. medicinal dioscorea zingiberensis wright group according to claim 1 is trained the method for forming seedling through one step culture mass production, it is characterized in that, in step (1), explant is cut into the specification that contains 1 joint 1 bud, first with washing powder, clean up, on aseptic operating platform, with aseptic washing 5～7 times, with 70% alcohol, infiltrate 30 seconds-1 minute, then put into 0.1% mercuric chloride solution sterilizing 12～18 minutes, aseptic water washing 5～6 times, standby.
3. medicinal dioscorea zingiberensis wright group according to claim 1 is trained the method for forming seedling through one step culture mass production, it is characterized in that, the cultivation temperature of step (1) and (2) is in 24 ℃ ± 2,1000～2000lx low light environment, 10～12 hours every days illumination cultivation.
4. medicinal dioscorea zingiberensis wright group according to claim 1 is trained the method for forming seedling through one step culture mass production, it is characterized in that, when the bud subculture that step (1) induces is cultivated, go 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.
5. the method for medicinal dioscorea zingiberensis wright group training forming seedling through one step culture according to claim 1 mass production, is characterized in that, step (2) is that callus lines is divided into fritter by the specification of 0.5～1cm * 0.5～1cm.
6. the method for medicinal dioscorea zingiberensis wright group training forming seedling through one step culture according to claim 1 mass production, is characterized in that, when step (2) is cultivated every 25～30d subculture once.
7. medicinal dioscorea zingiberensis wright group according to claim 1 is trained the method for forming seedling through one step culture mass production, it is characterized in that, the medium that step (1) is used: MS is a large amount of+and B5 trace+MS is organic+2 * MS molysite+6-BA2.0mg/L+KT0.4mg/L+ paclobutrazol 4.0mg/L+ chlormequat 2.0mg/L+IAA0.2mg/L+NAA0.4mg/L.
8. medicinal dioscorea zingiberensis wright group according to claim 1 is trained the method for forming seedling through one step culture mass production, it is characterized in that, the medium that step (1) is used: MS is a large amount of+and B5 trace+MS is organic+2 * MS molysite+6-BA3.0mg/L+KT0.4mg/L+ paclobutrazol 4.0mg/L+ chlormequat 2.0mg/L+IAA0.4mg/L+NAA0.4mg/L;
9. medicinal dioscorea zingiberensis wright group according to claim 1 is trained the method for forming seedling through one step culture mass production, it is characterized in that, the medium that step (2) is used: MS is a large amount of+and B5 trace+MS is organic+MS molysite+6-BA0.5mg/L+KT0.2mg/L+ paclobutrazol 2.0mg/L+ chlormequat 2.0mg/L+IAA0.2mg/L+NAA0.2mg/L.
10. medicinal dioscorea zingiberensis wright group according to claim 1 is trained the method for forming seedling through one step culture mass production, it is characterized in that, the medium that step (2) is used: MS is a large amount of+and B5 trace+MS is organic+MS molysite+6-BA0.8mg/L+KT0.5mg/L+ paclobutrazol 2.0mg/L+ chlormequat 3.0mg/L+IAA0.2mg/L+NAA0.2mg/L.
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| CN104904601A (en) * | 2015-06-11 | 2015-09-16 | 中国科学院亚热带农业生态研究所 | Rooting method of sterile short shoot of medicinal peltate yam rhizome tissue culture seedling |
| CN106472318A (en) * | 2016-10-20 | 2017-03-08 | 中国科学院亚热带农业生态研究所 | A kind of method of medicinal Rhizoma Dioscoreae Zingiberensiss mass production |
| CN109644871A (en) * | 2019-01-21 | 2019-04-19 | 花垣县精虹生物科技发展有限公司 | A method of suitable for a variety of orchidaceae medicinal plant mass productions |
| 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 |
| CN116114603A (en) * | 2023-03-06 | 2023-05-16 | 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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