CN100367846C - Highly effective revulsion induction method for pinellia tuber excised tuber - Google Patents
Highly effective revulsion induction method for pinellia tuber excised tuber Download PDFInfo
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- CN100367846C CN100367846C CNB2005100497424A CN200510049742A CN100367846C CN 100367846 C CN100367846 C CN 100367846C CN B2005100497424 A CNB2005100497424 A CN B2005100497424A CN 200510049742 A CN200510049742 A CN 200510049742A CN 100367846 C CN100367846 C CN 100367846C
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Abstract
The present invention discloses a highly effective inducement method for pinellia tuber excised tubers, which uses a successive culture medium for transfer culture, a rooting culture medium for rooting culture and an excised tuber inducement culture medium for excised tuber inducement culture, wherein the successive culture medium is composed of an MS basic culture medium, cytokinin, naphthylacetic acid, agar powder and saccharose; the rooting culture medium is composed of an MS basic culture medium, naphthylacetic acid, activated carbon, agar powder and saccharose; the excised tuber inducement culture medium is composed of an MS basic culture medium, gibberellin, abscisic acid, dihydro jasmonic acid propyl ester, saccharose, paclobutrazol, cytokinin, activated carbon and agar powder. The pinellia tuber excised tubers have high stress resistance, can be directly transplanted in fields without special acclimation or limitation of production seasons, and can produced all the year. The highly effective inducement of the excised tubers is the docking technology of pinellia tuber detoxification seedling production and field production, and has higher production and application value.
Description
Technical field
The present invention relates to plant regeneration, relate in particular to a kind of abductive approach of pinellia tuber excised tuber by tissue culture technique.
Background technology
The tuber of pinellia (Pinellia.ternata. (Thumb) Breit) is the perennial perennial root herbaceous plant of Araeceae, and base portion forms stem tuber, is main depot organ and organ of multiplication.Contain plurality of active ingredients such as alkaloid, pinellin, be used as medicine have eliminating dampness and eliminating phlegm, fall that check is coughed, anti-early pregnancy, multiple pharmacological effect such as antitumor, be the distinctive a kind of traditional Chinese medicine material of China, demand is bigger.Owing to excessively gather, the wild resource of the tuber of pinellia is closely exhausted.The tuber of pinellia has three kinds of modess of reproduction: by seminal propagation, tubercle breeding and bulbil breeding have formed the reproduction characteristics based on vegetative propagation in evolving naturally, and old friend worker cultivates general employing tubercle 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.Owing to long-term vegetative propagation, virus infection also constantly is accumulated in the stem tuber simultaneously, causes its output and medicinal ingredient to descend.
Stem tuber (bulb, bulb, piece root) is a species transformation of axis, is a kind of genetic character, but its generation and growth are subjected to the influence of environment, nutritional condition to a great extent.In the plant cultured in vitro, by adding the form generation that allogenic material influences the inside plants biochemical reaction and then influences plant, induce it to form tuber in vitro (bulb, bulb, napiform root) etc., this tests successfully on various plants.Quick breeding method for tissue culture can be turned out the seedling (being virus-elimination seedlings) of sloughing virus.The research of relevant tuber of pinellia tissue culture is more, and can carry out large-scale production.But the conventional tissue-culturing rapid propagation of the tuber of pinellia was to go out tuber of pinellia seedling by tuber of pinellia tissue culture in the past, through taking root, hardening, transplanting supervisor, more loaded down with trivial details, and be subjected to the influence of transplanting survival rate, could form stem tuber after a period of time transplanting in the land for growing field crops.The pinellia detoxification test-tube plantlet is applied to domestication condition and the bookkeeping that the land for growing field crops produces needs strictness.
Summary of the invention
The abductive approach that the purpose of this invention is to provide a kind of pinellia tuber excised tuber is produced pinellia tuber excised tuber, overcomes the shortcoming that tuber of pinellia test-tube plantlet is used.
It is that successive transfer culture does not have radical bud to growing 2-5cm on subculture medium with virus-free tuber of pinellia 2-20mm grow thickly bud and not detoxification maternal plant of the same age three leaf tuber of pinellia tissue cultivating seedling; Get the growth test tube bud of the detoxification three leaf tuber of pinellia 2-5cm of successive transfer culture, be used for tuber in vitro and induce, before inducing, change the MS minimal medium earlier over to and cultivated 7-14 days; Do to shift cultivation with subculture medium; Make culture of rootage with root media; Make the tuber in vitro inducing culture with the tuber in vitro inducing culture; The medium pH value is 5.6-6.0, contains agar 4.5-5.0g/L, conventional moist heat sterilization 20-22min, in the medium that aseptic adding was sterilized behind gibberellin, abscisic acid, the n-propyl dihydro-jasmonate filtration sterilization, intensity of illumination 1000-2000lx, illumination 10-12h/d, temperature 22-28 ℃.
The weight ratio that subculture medium is formed is: MS minimal medium: the basic element of cell division: methyl: agar powder: sucrose=1000000: 0.5-1.2: 0.001-0.2: 4500-5000: 20000-30000;
The weight ratio that root media is formed is: MS minimal medium: methyl: active carbon: agar powder: sucrose=500000: 0.1-0.3: 200-600: 4500-5000: 15000-30000;
The weight ratio that the tuber in vitro inducing culture is formed is: MS minimal medium: sucrose: the basic element of cell division: paclobutrazol: active carbon: gibberellin: n-propyl dihydro-jasmonate: abscisic acid: agar powder=1000000: 15000-120000: 0.5-1.5: 0.01-12.8: 500-2500: 0.005-0.5: 0.2-20.0: 0.05-0.5: 4500-5000.
The present invention has stronger resistance, storage property, but does not need special domestication direct transplanting in the land for growing field crops, is not produced the restriction in season, but whole year production.Efficiently inducing of tuber in vitro is that the pinellia detoxification seedling is produced the docking technique of producing with the land for growing field crops, has bigger production application and is worth.Induce under the suitable condition to form tuber in vitro external form, size unanimity, it is good that synchronism takes place, and can artificially control it and grow.In addition, the tuber in vitro genesis and development is beneficial to the germ plasm resource preservation and exchanges with international, can directly be applied to produce as propagating materials, saved the breeding space of original seed, also can shorten the grown in field cycle, avoid the variety of problems in seedling long-distance transport and the storage process, have more wide application prospect.Similar with potato, pinellia tuber excised tuber forms the material that also can be used as research tuber of pinellia physiology, cytology, zymetology etc., and bigger using value is arranged.
Embodiment:
MS minimal medium prescription is: (unit: mg/L)
Stock solution I:NH
4NO
3, 33000; KNO
3, 38000; C
aCL
2.2H
2O, 8800; M
gSO
4.7H
2O, 7400; KH
2PO
4, 3400.
Stock solution II:KI, 166; H
3BO
3, 1240; M
nSO
4.4H
2O, 4460; Z
nSO
4.7H
2O, 1720; N
A2M
oO
42H
2O, 50; C
0SO
45H
2O, 5; C
0CL
26H
2O, 5.
Stock solution III:F
ESO
47H
2O, 5560; N
A2EDTA2H
2O, 7460;
Stock solution IV: inositol, 20000; Nicotinic acid, 100; Puridoxine hydrochloride, 100; Thiamine hydrochloride, 100; Glycine, 400.
Prepare 1 liter of MS medium, get 50ml stock solution I, 5ml stock solution II, 5ml stock solution III, 5ml stock solution IV.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.
Specific embodiment:
Embodiment 1
Detoxification tuber of pinellia 2mm grow thickly bud and not detoxification maternal plant of the same age three leaf tuber of pinellia tissue cultivating seedling, successive transfer culture does not have radical bud to growing 2cm on subculture medium; Get the detoxic seedling of 2cm, changing the MS minimal medium before inducing earlier over to cultivated 8 days, cultivate with root media, the root media proportion of composing is: MS minimal medium: methyl: active carbon: agar powder: sucrose=500000: 0.1: 200: 4500: 15000, the tuber in vitro inducing culture is carried out with the tuber in vitro inducing culture in the back, the tuber in vitro inducing culture is: MS minimal medium: sucrose: the basic element of cell division: paclobutrazol: active carbon: gibberellin: n-propyl dihydro-jasmonate: abscisic acid: agar powder=1000000: 30000: 0.5: 5.0: 500: 0.005: 0.2: 0.05: 4800, check weighing after 40 days, single stem tuber heavily are 0.653 gram.The medium pH value is 5.6, contains agar 4.5g/L, conventional moist heat sterilization 20min, and in the medium that aseptic adding was sterilized behind gibberellin, abscisic acid, the n-propyl dihydro-jasmonate filtration sterilization, intensity of illumination 1000lx, illumination 10h/d, 22 ℃ of temperature.
Embodiment 2
Detoxification tuber of pinellia 3mm grow thickly bud and not detoxification maternal plant of the same age three leaf tuber of pinellia tissue cultivating seedling, successive transfer culture does not have radical bud to growing 3cm on subculture medium; Get the detoxic seedling of 3cm, changing the MS minimal medium before inducing earlier over to cultivated 7 days, cultivate with root media, the root media proportion of composing is: MS minimal medium: methyl: active carbon: agar powder: sucrose=500000: 0.3: 600: 5000: 30000, the tuber in vitro inducing culture is carried out with the tuber in vitro inducing culture in the back, the tuber in vitro inducing culture is: MS minimal medium: sucrose: the basic element of cell division: paclobutrazol: active carbon: gibberellin: n-propyl dihydro-jasmonate: abscisic acid: agar powder=1000000: 15000: 1.0: 0.05: 2500: 0.05: 2.0: 0.5: 5000, check weighing after 40 days, single stem tuber heavily are 0.776 gram.The medium pH value is 5.8, contains agar 4.7g/L, conventional moist heat sterilization 21min, and in the medium that aseptic adding was sterilized behind gibberellin, abscisic acid, the n-propyl dihydro-jasmonate filtration sterilization, intensity of illumination 1200lx, illumination 11h/d, 23 ℃ of temperature.
Embodiment 3
Detoxification tuber of pinellia 10mm grow thickly bud and not detoxification maternal plant of the same age three leaf tuber of pinellia tissue cultivating seedling, successive transfer culture does not have radical bud to growing 4cm on subculture medium; Get the detoxic seedling of 4cm, changing the MS minimal medium before inducing earlier over to cultivated 10 days, cultivate with root media, the root media proportion of composing is: MS minimal medium: methyl: active carbon: agar powder: sucrose=500000: 0.2: 300: 4800: 25000, the tuber in vitro inducing culture is carried out with the tuber in vitro inducing culture in the back, the tuber in vitro inducing culture is: MS minimal medium: sucrose: the basic element of cell division: paclobutrazol: active carbon: gibberellin: n-propyl dihydro-jasmonate: abscisic acid: agar powder=1000000: 20000: 1.5: 0.5: 2500: 0.5: 20.0: 5: 4500, check weighing after 40 days, single stem tuber heavily are 0.556 gram.The medium pH value is 6.0, contains agar 5.0g/L, conventional moist heat sterilization 22min, and in the medium that aseptic adding was sterilized behind gibberellin, abscisic acid, the n-propyl dihydro-jasmonate filtration sterilization, intensity of illumination 2000lx, illumination 12h/d, 28 ℃ of temperature.
Embodiment 4
Detoxification tuber of pinellia 20mm grow thickly bud and not detoxification maternal plant of the same age three leaf tuber of pinellia tissue cultivating seedling, successive transfer culture does not have radical bud to growing 4cm on subculture medium; Get the detoxic seedling of 4cm, changing the MS minimal medium before inducing earlier over to cultivated 8 days, cultivate with root media, the root media proportion of composing is: MS minimal medium: methyl: active carbon: agar powder: sucrose=500000: 0.15: 400: 4900: 29000, the tuber in vitro inducing culture is carried out with the tuber in vitro inducing culture in the back, the tuber in vitro inducing culture is: MS minimal medium: sucrose: the basic element of cell division: paclobutrazol: active carbon: gibberellin: n-propyl dihydro-jasmonate: abscisic acid: agar powder=1000000: 70000: 1.0: 0.5: 1000: 0.05: 2.0: 0.5: 50000, check weighing after 40 days, single stem tuber heavily are 0.852 gram.The medium pH value is 5.9, contains agar 4.9g/L, conventional moist heat sterilization 21min, and in the medium that aseptic adding was sterilized behind gibberellin, abscisic acid, the n-propyl dihydro-jasmonate filtration sterilization, intensity of illumination 1800lx, illumination 12h/d, 27 ℃ of temperature.
Embodiment 5
Detoxification tuber of pinellia 19mm grow thickly bud and not detoxification maternal plant of the same age three leaf tuber of pinellia tissue cultivating seedling, successive transfer culture does not have radical bud to growing 4cm on subculture medium; Get the detoxic seedling of 4cm, changing the MS minimal medium before inducing earlier over to cultivated 13 days, cultivate with root media, the root media proportion of composing is: MS minimal medium: methyl: active carbon: agar powder: sucrose=500000: 0.25: 500: 4200: 12000, the tuber in vitro inducing culture is carried out with the tuber in vitro inducing culture in the back, the tuber in vitro inducing culture is: MS minimal medium: sucrose: the basic element of cell division: paclobutrazol: active carbon: gibberellin: n-propyl dihydro-jasmonate: abscisic acid: agar powder=1000000: 110000: 1.5: 5.0: 25000: 0.5: 0.2: 5: 4500, check weighing after 40 days, single stem tuber heavily are 0.539 gram.The medium pH value is 6.0, contains agar 4.9g/L, conventional moist heat sterilization 22min, and in the medium that aseptic adding was sterilized behind gibberellin, abscisic acid, the n-propyl dihydro-jasmonate filtration sterilization, intensity of illumination 1700lx, illumination 11.5h/d, 26.5 ℃ of temperature.
Embodiment 6
Detoxification tuber of pinellia 15mm grow thickly bud and not detoxification maternal plant of the same age three leaf tuber of pinellia tissue cultivating seedling, successive transfer culture does not have radical bud to growing 5cm on subculture medium; Get the detoxic seedling of 5cm, changing the MS minimal medium before inducing earlier over to cultivated 12 days, cultivate with root media, the root media proportion of composing is: MS minimal medium: methyl: active carbon: agar powder: sucrose=500000: 0.27: 240: 4800: 28000, the tuber in vitro inducing culture is carried out with the tuber in vitro inducing culture in the back, the tuber in vitro inducing culture is: MS minimal medium: sucrose: the basic element of cell division: paclobutrazol: active carbon: gibberellin: n-propyl dihydro-jasmonate: abscisic acid: agar powder=1000000: 16000: 1.4: 0.05: 1000: 0.005: 20.0: 0.05: 4700, check weighing after 40 days, single stem tuber heavily are 0.622 gram.The medium pH value is 5.8, contains agar 5.0g/L, conventional moist heat sterilization 20min, and in the medium that aseptic adding was sterilized behind gibberellin, abscisic acid, the n-propyl dihydro-jasmonate filtration sterilization, intensity of illumination 1900lx, illumination 12h/d, 28 ℃ of temperature.
Embodiment 7
Detoxification tuber of pinellia 17mm grow thickly bud and not detoxification maternal plant of the same age three leaf tuber of pinellia tissue cultivating seedling, successive transfer culture does not have radical bud to growing 4.5cm on subculture medium; Get the detoxic seedling of 4.5cm, changing the MS minimal medium before inducing earlier over to cultivated 7 days, cultivate with root media, the root media proportion of composing is: MS minimal medium: methyl: active carbon: agar powder: sucrose=500000: 0.22: 350: 4480: 17000, the tuber in vitro inducing culture is carried out with the tuber in vitro inducing culture in the back, the tuber in vitro inducing culture is: MS minimal medium: sucrose: the basic element of cell division: paclobutrazol: active carbon: gibberellin: n-propyl dihydro-jasmonate: abscisic acid: agar powder=1000000: 90000: 1.0: 0.05: 2500: 0.5: 0.2: 5: 4900, check weighing after 40 days, single stem tuber heavily are 0.563 gram.The medium pH value is 5.6, contains agar 4.6g/L, conventional moist heat sterilization 20min, and in the medium that aseptic adding was sterilized behind gibberellin, abscisic acid, the n-propyl dihydro-jasmonate filtration sterilization, intensity of illumination 1500lx, illumination 12h/d, 23 ℃ of temperature.
Embodiment 8
Detoxification tuber of pinellia 16mm grow thickly bud and not detoxification maternal plant of the same age three leaf tuber of pinellia tissue cultivating seedling, successive transfer culture does not have radical bud to growing 2.5cm on subculture medium; Get the detoxic seedling of 2.5cm, changing the MS minimal medium before inducing earlier over to cultivated 14 days, cultivate with root media, the root media proportion of composing is: MS minimal medium: methyl: active carbon: agar powder: sucrose=500000: 0.11: 210: 4100: 16000, the tuber in vitro inducing culture is carried out with the tuber in vitro inducing culture in the back, the tuber in vitro inducing culture is: MS minimal medium: sucrose: the basic element of cell division: paclobutrazol: active carbon: gibberellin: n-propyl dihydro-jasmonate: abscisic acid: agar powder=1000000: 120000: 1.5: 12.8: 580: 0.05: 20: 0.05: 4800, check weighing after 40 days, single stem tuber heavily are 0.596 gram.The medium pH value is 6.0, contains agar 4.9g/L, conventional moist heat sterilization 21min, and in the medium that aseptic adding was sterilized behind gibberellin, abscisic acid, the n-propyl dihydro-jasmonate filtration sterilization, intensity of illumination 1800lx, illumination 10.5h/d, 24 ℃ of temperature.
Embodiment 9
Detoxification tuber of pinellia 14mm grow thickly bud and not detoxification maternal plant of the same age three leaf tuber of pinellia tissue cultivating seedling, successive transfer culture does not have radical bud to growing 3.5cm on subculture medium; Get the detoxic seedling of 3.5cm, changing the MS minimal medium before inducing earlier over to cultivated 9 days, cultivate with root media, the root media proportion of composing is: MS minimal medium: methyl: active carbon: agar powder: sucrose=500000: 0.29: 580: 4600: 24000, the tuber in vitro inducing culture is carried out with the tuber in vitro inducing culture in the back, the tuber in vitro inducing culture is: MS minimal medium: sucrose: the basic element of cell division: paclobutrazol: active carbon: gibberellin: n-propyl dihydro-jasmonate: abscisic acid: agar powder=1000000: 110000: 0.8: 11.0: 2400: 0.005: 20.0: 0.5: 5.0, check weighing after 40 days, single stem tuber heavily are 0.560 gram.The medium pH value is 5.7, contains agar 5.0g/L, conventional moist heat sterilization 20min, and in the medium that aseptic adding was sterilized behind gibberellin, abscisic acid, the n-propyl dihydro-jasmonate filtration sterilization, intensity of illumination 1600lx, illumination 12h/d, 28 ℃ of temperature.
Reference examples 1
In the time of with the inducing culture tuber in vitro, the hardening of uncapping when changing root media to the long 1～2cm of root over to 2～3 days with detoxic seedling with the onesize high 2cm of the detoxic seedling of inducing culture tuber in vitro, transplant in the seedling-cultivating tray that sterile-processed peat soil+perlite+vermiculite (volume ratio 5: 1: 3) is housed, put the domestication chamber.Transplant after 10 days in the process insect protected greenhouse of disinfecting.Detoxic seedling transplants that to take the average fresh weight of stem tuber weighing after 60 days be 0.515g, and average diameter is 1.224cm, although to compare growth time long with tuber in vitro, its diameter, fresh weight are still obviously less than normal than tuber in vitro.
Reference examples 2
In the time of with the inducing culture tuber in vitro, the hardening of uncapping when changing root media to the long 1～2cm of root over to 2～3 days with detoxic seedling with the onesize high 5cm of the detoxic seedling of inducing culture tuber in vitro, transplant in the seedling-cultivating tray that sterile-processed peat soil+perlite+vermiculite (volume ratio 5: 1: 3) is housed, put the domestication chamber.Transplant after 10 days in the process insect protected greenhouse of disinfecting.Detoxic seedling transplants that to take the average fresh weight of stem tuber weighing after 60 days be 0.520g, and average diameter is 1.230cm, although to compare growth time long with tuber in vitro, its diameter, fresh weight are still obviously less than normal than tuber in vitro.
Claims (1)
1. the abductive approach of a pinellia tuber excised tuber is characterized in that: detoxification tuber of pinellia 2-20mm grow thickly bud and not detoxification maternal plant of the same age three leaf tuber of pinellia tissue cultivating seedling, and successive transfer culture does not have radical bud to growing 2-5cm on subculture medium; Get the growth test tube bud of the detoxification three leaf tuber of pinellia 2-5cm of successive transfer culture, be used for tuber in vitro and induce, before inducing, change the MS minimal medium earlier over to and cultivated 7-14 days; Do to shift cultivation with subculture medium; Make culture of rootage with root media; Make the tuber in vitro inducing culture with the tuber in vitro inducing culture; The medium pH value is 5.6-6.0, contain agar 4.5-5.0g/L, conventional moist heat sterilization 20-22min, gibberellin, abscisic acid, in the medium that aseptic adding was sterilized behind the n-propyl dihydro-jasmonate filtration sterilization, intensity of illumination 1000-2000 lx, illumination 10-12h/d, temperature 22-28 ℃, the weight ratio that subculture medium is formed is: MS minimal medium: the basic element of cell division: methyl: agar powder: sucrose=1000000: 0.5-1.2: 0.001-0.2: 4500-5000: 20000-30000, the weight ratio that root media is formed is: MS minimal medium: methyl: active carbon: agar powder: sucrose=500000: 0.1-0.3: 200-600: 4500-5000: 15000-30000, the weight ratio that the tuber in vitro inducing culture is formed is: MS minimal medium: sucrose: the basic element of cell division: paclobutrazol: active carbon: gibberellin: n-propyl dihydro-jasmonate: abscisic acid: agar powder=1000000: 15000-120000: 0.5-1.5: 0.01-12.8: 500-2500: 0.005-0.5: 0.2-20.0:0.05-0.5: 4500-50
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| CN101491214B (en) * | 2009-03-11 | 2011-03-23 | 华中农业大学 | Pinellia tuber artificial seed stem production method |
| CN102150624A (en) * | 2011-04-29 | 2011-08-17 | 南京工业大学 | Tissue culture and rapid propagation method for pinellia tuber plant |
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| CN100382679C (en) * | 2005-12-30 | 2008-04-23 | 浙江省农业科学院 | Pinellia detoxification, tissue culture and quick propagation method |
| CN101147466B (en) * | 2006-09-22 | 2010-10-06 | 中国农业科学院蔬菜花卉研究所 | Minitype ginger seedling tissue culture fast propagating culture medium and tissue culture fast propagating method |
| CN101248760B (en) * | 2008-04-03 | 2010-06-09 | 浙江大学 | Cultivation method of rhizoma corydalis stem tuber |
| CN101278646B (en) * | 2008-05-26 | 2010-08-18 | 福建农林大学 | Method for producing sterilized miniascape |
| CN102228005A (en) * | 2011-05-24 | 2011-11-02 | 汉中植物研究所 | Pinellia ternate tissue culture one-step speciation method |
| 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 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101491214B (en) * | 2009-03-11 | 2011-03-23 | 华中农业大学 | Pinellia tuber artificial seed stem production method |
| CN102150624A (en) * | 2011-04-29 | 2011-08-17 | 南京工业大学 | Tissue culture and rapid propagation method for pinellia tuber plant |
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