CN1475107A - Normalization tissue culturing and fast breeding method of pinellia ternata - Google Patents
Normalization tissue culturing and fast breeding method of pinellia ternata Download PDFInfo
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- CN1475107A CN1475107A CNA021257256A CN02125725A CN1475107A CN 1475107 A CN1475107 A CN 1475107A CN A021257256 A CNA021257256 A CN A021257256A CN 02125725 A CN02125725 A CN 02125725A CN 1475107 A CN1475107 A CN 1475107A
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Abstract
A tissue culture method for quick large-scale reproduction of pinellia includes getting 0.3-0.6 mm stemp tip, in vitro culture to obtain plant, taking its petiole, leaf, or bud as the explant for next tissue culture, sterilizing, cutting to become segements, inoculating them to culture medium, large-scale culture of calli, inducing the growth of root and bud to form green seedlings, more than 3-5 cm, and transplanting them in sandy soil.
Description
Technical field
The present invention relates to pinellia detoxification seedling scale tissue culture and rapid propagation method.
Background technology
The tuber of pinellia is the Araeceae herbaceos perennial, and its stem tuber is a kind of important traditional Chinese medicine, and in 558 kinds of traditional Chinese medicine prescriptions, the tuber of pinellia is classified the 22nd in the 30 the highest flavor Chinese medicines of frequency of utilization as.Owing to excessively gather, the wild resource of the tuber of pinellia is exhausted.Carry out artificial cultivation and become the task of top priority.The tuber of pinellia has three kinds of natural propagation modes: by seed (often needing artificial supplementary pollination to obtain), tubercle and bulbil breeding, formed the reproduction characteristics based on vegetative propagation in evolving naturally, old friend worker cultivates general employing tubercle and bulbil as organ of multiplication.Above method is because reproduction coefficient is low, and the land for growing field crops of having limited the tuber of pinellia produces.Simultaneously in cultivation and producing and since long-term vegetative propagation cause its output and medicinal ingredient all descend (one of reason, be virus infections and constantly due to the accumulation).Breed the existing more report of tuber of pinellia seedling with tissue culture method, also have simultaneously report to show that the group training tuber of pinellia and the wild tuber of pinellia do not have essential distinction qualitatively, even the alkaloid of tissue cultivating seedling is higher than the wild tuber of pinellia, meets the requirement of the commodity tuber of pinellia, so available tissue cultivating seedling is cultivated for planting.
Tissue cultivating seedling will be used for the land for growing field crops and produce and also to need to carry out the batch production seed cultivation and new varieties are effectively protected.As for can improving of the stem tuber yield and quality of tissue cultivating seedling, then be the problem that solves by virus-free.
Therefore, in the scale group training production process of the tuber of pinellia, still need a kind of method that can solve tuber of pinellia virus-free seedling tissue-culturing rapid propagation.
Summary of the invention
The method that the purpose of this invention is to provide a kind of tuber of pinellia seedling virus-free scale quick breeding by group culture.
Method of the present invention comprises the steps:
1) gets 0.3-0.6mm stem apex cultured in vitro and obtain plant, get its petiole, blade or bulbil as next stage group training explant;
2) explant sterile-processed after, under aseptic condition on the segment inoculation medium, induce tuber of pinellia callus to generate and the organ seedling differentiation again;
3) reach more than 3～5 centimetres when test-tube plantlet growth, plant in the sandy soil.
The present invention can also adopt another kind of method, and this method comprises the steps:
1) gets 0.3-0.6mm stem apex cultured in vitro and obtain plant, get its petiole, blade or bulbil as next stage group training explant;
2) explant sterile-processed after, again under aseptic condition on the segment inoculation medium, the enlarged culture by callus forms a large amount of callus, root induction then, sprouts, and forms green seedling;
3) reach more than 3～5 centimetres when the growth of green seedling, plant in the sandy soil.
The detoxication and tissue culture of the tuber of pinellia theoretically, adopts the plant of the suitably stem apex cultured in vitro acquisition of size (for example 0.3-0.6mm) just might remove virus and make the corresponding raising of yield and quality.The stem apex cultured in vitro of the tuber of pinellia can adopt following method: the wet husky vernalization of tuber of pinellia stem tuber, when treating that blade grows to 3～4cm, getting petiole sterilized 15 minutes with 0.1% mercuric chloride, aseptic water washing 5 times, the petiole segment is seeded on the solid culture medium of MS+KT0.5mg/L+NAA0.2mg/L, in certain illumination, cultivate under 25 ℃ of left and right sides conditions.Approximately cultivate after 1 month and grow many plantlets on the petiole.The stem apex (0.3-0.6mm) that strips test-tube plantlet with dissecting needle under aseptic condition adopts conventional method to cultivate, petiole, blade and the bulbil of the plantlet that obtains, and preferred petiole is re-used as the next stage group and trains and use explant, may obtain a large amount of tissue cultural seedlings of free.
The tissue culture of the tuber of pinellia can adopt for example following step to carry out:
Adopt tender petiole, blade, bulbil and the fresh stem tuber of tuber of pinellia children, alcohol-pickled 0.5～1min through 75%, again with 2% aqueous sodium hypochlorite solution sterilization 15min, or with 0.1% mercuric chloride solution sterilization 10min, under aseptic condition, use aseptic water washing then, segment under aseptic condition (piece) is seeded on the suitable culture base again, basal medium is MS, sucrose 3%, agar 0.7%, PH5.8, additional certain plant growth regulator combination induces tuber of pinellia callus to generate and the organ differentiation.25 ± 2 ℃ of cultivation temperature, light intensity 1500LX, light application time 10～12h/d.
In tuber of pinellia group training process, single use growth hormone of the plant growth regulator in the medium or basic element of cell division effect are all bad, need the two suitable collocation.Growth hormone forms callus and plays a decisive role, but the growth hormone and the basic element of cell division suitably are combined with synergistic effect.The growth of growth hormone induction root, the basic element of cell division particularly suppress the growth of root during high concentration.Suppress the growth of bud when auxin concentration is high, the basic element of cell division promotes the differentiation of bud, but concentration is also unsuitable too high.If tuber of pinellia tissue culture hormone combination is suitable, can be on a kind of medium once property acquisition test-tube plantlet (can adopt MS+2,4-D0.2mg/L+KT1.0mg/L).Also can form a large amount of callus, root induction then, sprout, form green seedling by the enlarged culture of callus.When test-tube plantlet growth reaches more than 3～5 centimetres, when growing flourishing root system, open bottle stopper, take out test-tube plantlet after 3 days, plant in the sandy soil behind the flush away root medium, avoid the direct light irradiation initial two weeks, to note keeping soil moisture, survival rate is more than 90%.
When adopting the virus-elimination seedlings of stem apex cultured in vitro acquisition, then can need not through sterilisation step, be seeded on the suitable culture base and directly carry out segment under aseptic condition (piece), carry out subsequent group training step.
The main feature of carrying out the batch production of the virus-free tuber of pinellia, the production that standardizes by the group training is as follows:
1. solve virus and cause tuber of pinellia germplasm degenerate problem, recover its quality.The virus-free tuber of pinellia not only quality is recovered, and its vitality improves, and the output of planting can improve 1.6～3 times.Thereby planting benefit improves greatly.
2. effectively solve wild tuber of pinellia streaking, outward appearance, effective ingredient and growth characteristics require to vary, and the characteristics that are not suitable for cultivating are for road, grown place medicinal material be convenient to processing in postpartum, sell the condition that provides safeguard.
3. effectively solve on the market cultivar and the under-supply present situation of seedling, form first tuber of pinellia cultivated species in the near future.Declare protection developer ground interests by the registration of finger-print and other intellectual property.
4. genetic background unanimity, efficacy component are stablized and the consistent new varieties of cultivation feature, are convenient to country and carry out trade management.
5. the batch production planningization production of drug germchit is to carry out key technology and the guarantee that the GAP medicinal material is produced.
6. in the tuber of pinellia planting process, topmost limiting factor, except that virus caused the germplasm decline, southern blight and other rhizome portion disease caused its output to descend about 30%.In the tuber of pinellia group training process, can also screen variant, obtain disease-resistant variety.
7. by the directed mutagenesis in group training stage, can improve the content and the yield of effective ingredient, further improve medical material quanlity.
8. set up culture plant cell (fermenting and producing) system in the group training stage, be expected to obtain the directly technical system of production effective ingredient of the tuber of pinellia " fermentation method ".
The present invention adopts following non-limiting embodiment to be further described.
Embodiment 1 tuber of pinellia virus-free seedling production method
Tuber of pinellia stem tuber, is got petiole and was sterilized 15 minutes with 0.1% mercuric chloride when treating that blade grows to 3～4cm with wet husky vernalization, aseptic water washing 5 times, the petiole segment is seeded on the solid culture medium of MS+KT0.5mg/L+NAA0.2mg/L, in certain illumination, cultivates under 25 ℃ of left and right sides conditions.Approximately cultivate after 1 month and grow many plantlets on the petiole.The stem apex (0.3-0.6mm) that strips test-tube plantlet with dissecting needle under aseptic condition adopts conventional method to cultivate, and petiole, blade and the bulbil of the plantlet that obtains is re-used as the explant that the next stage group is trained usefulness, may obtain a large amount of tissue cultural seedlings of free.
Embodiment 2 tuber of pinellia tissue culture quick propagation culturing methods
Employing is according to petiole, blade and the bulbil of the plantlet of method cultivation among the embodiment 1, segment is seeded on the suitable culture base under aseptic condition, basal medium is MS, sucrose 3%, agar 0.7%, PH5.8, additional plant growth regulator combination (MS+2,4-D0.2mg/L+KT1.0mg/L), induce tuber of pinellia callus to generate and the organ differentiation.25 ± 2 ℃ of cultivation temperature, light intensity 1500LX, light application time 10～12h/d.When test-tube plantlet growth reaches more than 3～5 centimetres, when growing flourishing root system, open bottle stopper, take out test-tube plantlet after 3 days, plant in the sandy soil behind the flush away root medium, avoid the direct light irradiation initial two weeks, to note keeping soil moisture, survival rate is more than 90%.
Embodiment 3 tuber of pinellia tissue culture quick propagation culturing methods
Adopt tender petiole, blade, bulbil and the fresh stem tuber of tuber of pinellia children, alcohol-pickled 0.5～1min through 75%, again with 2% aqueous sodium hypochlorite solution sterilization 15min, or with 0.1% mercuric chloride solution sterilization 10min, under aseptic condition, use aseptic water washing then, segment under aseptic condition (piece) is seeded on the suitable culture base again, basal medium is MS, sucrose 3%, agar 0.7%, PH5.8, additional plant growth regulator combination (MS+2,4-D0.2mg/L+KT1.0mg/L), induce tuber of pinellia callus to generate and the organ differentiation.25 ± 2 ℃ of cultivation temperature, light intensity 1500LX, light application time 10～12h/d.When test-tube plantlet growth reaches more than 3～5 centimetres, when growing flourishing root system, open bottle stopper, take out test-tube plantlet after 3 days, plant in the sandy soil behind the flush away root medium, avoid the direct light irradiation initial two weeks, to note keeping soil moisture, survival rate is more than 90%.
Embodiment 4 tuber of pinellia tissue culture quick propagation culturing methods
Adopt tender petiole, blade, bulbil and the fresh stem tuber of tuber of pinellia children, alcohol-pickled 0.5～1min through 75%, again with 2% aqueous sodium hypochlorite solution sterilization 15min, or with the 0.1% mercuric chloride solution 10min that sterilizes, under aseptic condition, use aseptic water washing then, segment under aseptic condition (piece) is seeded on the suitable culture base again, and basal medium is MS, sucrose 3%, 1.0mg/L6-BA, agar 0.7%, PH5.8, the enlarged culture of evoked callus forms a large amount of callus.The callus that obtains is cut into uniform Xiao Cong, transfer to MS basal medium (sucrose 3%, agar 0.7%, PH5.8) on, (MS+2 4-D0.2mg/L+KT1.0mg/L), induces tuber of pinellia callus to generate and the organ differentiation in additional plant growth regulator combination.25 ± 2 ℃ of cultivation temperature, light intensity 1500LX, light application time 10～12h/d.When test-tube plantlet growth reaches more than 3～5 centimetres, when growing flourishing root system, open bottle stopper, take out test-tube plantlet after 3 days, plant in the sandy soil behind the flush away root medium, avoid the direct light irradiation initial two weeks, to note keeping soil moisture, survival rate is more than 90%.
Claims (8)
1, a kind of method of tuber of pinellia scale virus-elimination seedlings tissue-culturing rapid propagation is characterized by:
1) gets 0.3-0.6mm stem apex cultured in vitro and obtain plant, get its petiole, blade or bulbil as next stage group training explant;
2) sterile-processed, under aseptic condition, on the segment inoculation medium, induce tuber of pinellia callus to generate and the organ seedling differentiation again;
3) reach more than 3～5 centimetres when test-tube plantlet growth, plant in the sandy soil.
2, according to the process of claim 1 wherein, described disinfecting is alcohol-pickled 0.5～1min through 75%, again with 2% aqueous sodium hypochlorite solution sterilization 15min, or with the 0.1% mercuric chloride solution 10min that sterilizes.
3, according to arbitrary method of claim 1 or 2, wherein, described basal medium is MS, sucrose 3%, and agar 0.7%, PH5.8, additional plant growth regulator is combined as MS+2,4-D0.2mg/L+KT 1.0mg/L.
4, according to arbitrary method of claim 1 to 3, wherein, induce the condition that tuber of pinellia callus generates and organ breaks up to be 25 ± 2 ℃ of cultivation temperature, light intensity 1500LX, light application time 10～12h/d.
5, a kind of method of tuber of pinellia scale virus-elimination seedlings tissue-culturing rapid propagation is characterized in that it comprises the steps:
1) gets 0.3-0.6mm stem apex cultured in vitro and obtain plant, get its petiole, blade or bulbil as next stage group training explant;
2) sterile-processed, again under aseptic condition on the segment inoculation medium, the enlarged culture by callus forms a large amount of callus, root induction then, sprouts, and forms green seedling;
3) reach more than 3～5 centimetres when the growth of green seedling, plant in the sandy soil.
6, according to the method for claim 5, wherein, described disinfecting is alcohol-pickled 0.5～1min through 75%, again with 2% aqueous sodium hypochlorite solution sterilization 15min, or with the 0.1% mercuric chloride solution 10min that sterilizes.
7, according to the method for claim 6, wherein, described basal medium is MS, sucrose 3%, and agar 0.7%, PH5.8,
8, according to arbitrary method of claim 5 to 7, wherein, induce the condition that tuber of pinellia callus generates and organ breaks up to be 25 ± 2 ℃ of cultivation temperature, light intensity 1500LX, light application time 10～12h/d.
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| CNA021257256A CN1475107A (en) | 2002-08-14 | 2002-08-14 | Normalization tissue culturing and fast breeding method of pinellia ternata |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100382679C (en) * | 2005-12-30 | 2008-04-23 | 浙江省农业科学院 | Pinellia detoxification, tissue culture and quick propagation method |
| CN102160523A (en) * | 2010-02-23 | 2011-08-24 | 中国医学科学院医药生物技术研究所 | Method for producing pinellia ternate embryoid and tissue culture seedling |
| CN102228005A (en) * | 2011-05-24 | 2011-11-02 | 汉中植物研究所 | Pinellia ternate tissue culture one-step speciation method |
| CN101595844B (en) * | 2009-06-29 | 2012-05-02 | 西南大学 | Method for quickly propagating transgenic tuber pinellia by adopting micro adventitious bud technology |
| CN101720670B (en) * | 2009-12-17 | 2012-07-04 | 昆明理工大学 | Rapid breeding method for pinellia tuber tissue culture |
| CN102687630A (en) * | 2012-06-14 | 2012-09-26 | 四川新荷花中药饮片股份有限公司 | Storage method and application of pinellia ternata seed stems |
| CN102919122A (en) * | 2012-10-19 | 2013-02-13 | 遵义市龙驰生物科技有限公司 | High-efficiency method for inducing pinellia in vitro bulb |
| CN103858765A (en) * | 2014-03-25 | 2014-06-18 | 江苏农牧科技职业学院 | Simple and rapid seedling growing method of Tai Pinellia ternate |
| CN105123328A (en) * | 2015-07-31 | 2015-12-09 | 河南科技大学 | Pinellia ternate seedling tissue culturing, hardening and domesticating substrate and preparation and use method for same |
| CN108668904A (en) * | 2018-07-09 | 2018-10-19 | 苏州枫彩生态科技集团有限公司 | A kind of processing method of drought tuber of pinellia explant |
| CN111202007A (en) * | 2020-04-15 | 2020-05-29 | 广西壮族自治区农业科学院 | Method for improving proliferation coefficient of pinellia ternata |
-
2002
- 2002-08-14 CN CNA021257256A patent/CN1475107A/en active Pending
Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100382679C (en) * | 2005-12-30 | 2008-04-23 | 浙江省农业科学院 | Pinellia detoxification, tissue culture and quick propagation method |
| CN101595844B (en) * | 2009-06-29 | 2012-05-02 | 西南大学 | Method for quickly propagating transgenic tuber pinellia by adopting micro adventitious bud technology |
| CN101720670B (en) * | 2009-12-17 | 2012-07-04 | 昆明理工大学 | Rapid breeding method for pinellia tuber tissue culture |
| CN102160523B (en) * | 2010-02-23 | 2012-10-24 | 中国医学科学院医药生物技术研究所 | Method for producing pinellia ternate embryoid and tissue culture seedling |
| CN102160523A (en) * | 2010-02-23 | 2011-08-24 | 中国医学科学院医药生物技术研究所 | Method for producing pinellia ternate embryoid and tissue culture seedling |
| CN102228005A (en) * | 2011-05-24 | 2011-11-02 | 汉中植物研究所 | Pinellia ternate tissue culture one-step speciation method |
| CN102687630A (en) * | 2012-06-14 | 2012-09-26 | 四川新荷花中药饮片股份有限公司 | Storage method and application of pinellia ternata seed stems |
| CN102919122A (en) * | 2012-10-19 | 2013-02-13 | 遵义市龙驰生物科技有限公司 | High-efficiency method for inducing pinellia in vitro bulb |
| CN102919122B (en) * | 2012-10-19 | 2014-07-16 | 遵义市龙驰生物科技有限公司 | High-efficiency method for inducing pinellia in vitro bulb |
| CN103858765A (en) * | 2014-03-25 | 2014-06-18 | 江苏农牧科技职业学院 | Simple and rapid seedling growing method of Tai Pinellia ternate |
| CN105123328A (en) * | 2015-07-31 | 2015-12-09 | 河南科技大学 | Pinellia ternate seedling tissue culturing, hardening and domesticating substrate and preparation and use method for same |
| CN108668904A (en) * | 2018-07-09 | 2018-10-19 | 苏州枫彩生态科技集团有限公司 | A kind of processing method of drought tuber of pinellia explant |
| CN111202007A (en) * | 2020-04-15 | 2020-05-29 | 广西壮族自治区农业科学院 | Method for improving proliferation coefficient of pinellia ternata |
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