CN102550409A - Method of tissue culture propagation for rhizome polygonati - Google Patents

Method of tissue culture propagation for rhizome polygonati Download PDF

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CN102550409A
CN102550409A CN2011104507384A CN201110450738A CN102550409A CN 102550409 A CN102550409 A CN 102550409A CN 2011104507384 A CN2011104507384 A CN 2011104507384A CN 201110450738 A CN201110450738 A CN 201110450738A CN 102550409 A CN102550409 A CN 102550409A
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culture
days
callus
observed
sealwort
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赵勇彪
祝利
杨再江
向廷军
何全慧
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CHONGQING XIUSHAN RED STAR TCM DEVELOPMENT Co Ltd
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CHONGQING XIUSHAN RED STAR TCM DEVELOPMENT Co Ltd
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Abstract

The invention provides a method of tissue culture propagation for rhizome polygonati, comprising the steps of introducing and sterilizing, tissue inducing, subculturing, and carrying out differentiation culture, strong seedling culture and rooting culture. The beneficial effects of the method are that seedlings can be produced in large amount by the tissue culture, and only seven months are needed from tuber to rooted seedling by culturing; thus production time and cost of seedlings are greatly decreased and propagation speed is increased.

Description

The sealwort tissue culture propagation method
Technical field
The present invention relates to the tissue culture propagation method in a kind of traditional Chinese medicine culture technique field, particularly a kind of sealwort.
Background technology
Sealwort is a Liliaceae Polygonatum herbaceos perennial, and sealwort is arranged, Yunnan sealwort and David's-harp etc., and its dry rhizome is used as medicine.According to the classical record of traditional medicine, sealwort belongs to rare Chinese medicine, have wide in beneficial gas, enriching kidney essence, nourishing Yin and moistening lung, the effect of the tonifying spleen of promoting the production of body fluid.Cure mainly illnesss such as dryness of the lung dry cough, body void are weak, palpitation, prolonged illness body fluid deficiency dry, diabetes, hypertension.To treatment angiocardiopathy, tuberculosis, chronic hepatitis and at antibiotic, detoxifcation, antifatigue, anti-ageing, hypoglycemic, reducing blood lipid, aspect such as antitumor better effect is arranged all; Both can be used as medicine; Can be used as health food again; Since ancient times, people sealwort as the good merchantable brand of promoting longevity, thereby being in great demand to sealwort.
For a long time, the sealwort crude drug source is main with wild collection mainly, and the culture technique of sealwort does not cause more concern.Along with market to the increase of medicinal material sealwort demand and the rapid minimizing of wild resource, gather wild sealwort and not only can not meet the need of market, destroyed ecotope and sealwort wild resource on the contrary.People begin man culture technique, the high-yield culture technique etc. of planting of the wild change of sealwort are put into practice gradually thus.The propagation technique of sealwort is mainly seminal propagation and rhizome breeding dual mode at present.
Collect because the sealwort seed is difficult, percentage of seedgermination is low, and seminal propagation, and seedling raise period is long, has increased the cultivation cost of sealwort greatly, is unfavorable for the artificial establishing in large scale of sealwort.So in current artificial cultivation, breed the breeding of main dependence rhizome, but this method reproduction coefficient is low, kind rhizome consumption is big, both uneconomical, has limited the yield potentiality of sealwort again, inconvenience is planted to manage and is promoted, thereby the expansion cultivated area is suppressed.
Summary of the invention
The object of the present invention is to provide a kind of sealwort tissue culture propagation method, improve the expanding propagation speed of sealwort, realize the standardization and the large-scale development of sealwort cultivation.For realizing this purpose; The inventor is through overtesting; The various tissue culture prescriptions and the cultural method of breeding sealwort have successfully been studied; To tissue culture prescription and production technology, and the special habit of growth of sealwort carried out big quantity research and repetition test, obtained the culture medium prescription and the cultural method of original creation.
For solving the problems of the technologies described above, the technical scheme that the present invention adopted is the sealwort tissue culture propagation method, comprise introduce a fine variety that sterilization, tissue are induced, successive transfer culture, differentiation culture, strong seedling culture and culture of rootage, concrete grammar is following.
introduces a fine variety sterilization: the sealwort rhizome of getting the band bud; Be cut into the segment of 0.5-1.0cm by knife; Put into the liquid detergent aqueous solution and soaked 5 minutes, rinse well with running water then; Use 75% ethanol scrub surfaces again, rinse well with running water again; Get in the superclean bench then and operate, put into for 75% ethanol half a minute, the sterilization deionized water is rinsed and is washed twice; Put into 2.5% clorox again 5 minutes, the sterilization deionized water is rinsed and is washed 2 times; And put into 5% clorox 5 minutes, the sterilization deionized water is rinsed continuously and is washed 3-5 time, cuts wound necrosis part with cutter then.
Figure 947663DEST_PATH_IMAGE002
tissue is induced: the rhizome after will disinfecting is seeded on the inducing culture; Every bottle graft kind 1-3 piece; Carrying out callus induction cultivates; The rhizome incision had a small amount of callus to begin to sprout in 28-32 days; The callus of faint yellow densification occurred in 50-60 days, inductivity reaches 50%.
Figure DEST_PATH_IMAGE003A
successive transfer culture: will induce the good faint yellow granular callus of growth conditions of generation to be inoculated in the subculture medium; Every bottle graft kind 2-3 piece; Carry out successive transfer culture; Can be observed in 28-32 days has new callus to produce again in the subculture medium; 43-47 days, can be observed callus and increase 1 times than original.
Figure DEST_PATH_IMAGE003AA
differentiation culture: the good faint yellow graininess callus of growth conditions that shoot proliferation is produced is transferred on the differential medium; Every bottle graft kind 2-3 piece carries out differentiation culture, can be observed callus and begins Growth and Differentiation in 9-11 days; It is green that color turns; Can be observed the pistac indefinite bud in 19-21 days, began to produce the bud of growing thickly, bud ratio 66% in 45-50 days; 5 buds of average every callus differentiation, the average length of bud is 12mm.
strong seedling culture: the bud of growing thickly of differentiation culture is transferred to carries out strong seedling culture in the strong seedling culture base, can be observed the seedling of growing thickly that the bud of growing thickly has developed into 2.0-4.0cm in 30 days.
Figure DEST_PATH_IMAGE003AAAA
culture of rootage: the seedling of growing thickly of strong seedling culture is transferred to root induction on the root media; Can be observed shoot root in 15 days begins to sprout; Can be observed 86% seedling in 30 days all takes root; Coring average length 4cm, every young plant are on average taken root 6; But bottle outlet refining seedling is transplanted then.
In the above-mentioned sealwort tissue culture propagation method, the condition of culture in each development growth stage of being mentioned is: the artificial climate incubator, and the periodicity of illumination beginning and ending time is 8:00-20:00; Cultivation temperature day 25 ℃ of temperature; Night, temperature was 18 ℃, and cultivating humidity is 60%, and intensity of illumination is 2000-2400lx.
In the above-mentioned sealwort tissue culture propagation method, the minimal medium in each development growth stage of being mentioned is: MS+30g/L sucrose+5g/L agar powder, pH value are 5.8-6.0.
In the above-mentioned sealwort tissue culture propagation method, the prescription of each development growth stage medium of being mentioned is.
Figure DEST_PATH_IMAGE003AAAAA
inducing culture: MS+6-BA2.0mg/L+2,4-D1.0mg/L.
Figure DEST_PATH_IMAGE003AAAAAA
subculture medium: MS+6-BA2.0mg/L+2,4-D2.0mg/L.
differential medium: MS+6-BA3.0mg/L+IAA1.0mg/L.
Figure DEST_PATH_IMAGE003AAAAAAAA
strong seedling culture base: MS+6-BA2.0mg/L+IAA0.5mg/L.
Figure DEST_PATH_IMAGE003AAAAAAAAA
root media: MS+IAA0.5mg/L.
The invention has the beneficial effects as follows: stem tuber repeated multiple times sterilizing and washing in short-term is of reduced contamination, reduced the damage of bactericidal agent to explant; Through tissue culture, can produce seedling in a large number, become to take root seedling from stem tuber to cultivating, only need 7 months, shortened seedling production time and cost greatly, improved reproductive speed.
Embodiment
Below in conjunction with embodiment the present invention is described further.
At first prepare following material.
Minimal medium: MS+30g/L sucrose+5g/L agar powder, pH value are 5.8-6.0.
The artificial climate incubator: control periodicity of illumination beginning and ending time 8:00-20:00,25 ℃ of day temperature, night, temperature was 18 ℃, humidity 60%, intensity of illumination 2000-2400lx.
Inducing culture: MS+6-BA2.0mg/L+2,4-D1.0mg/L.
Subculture medium: MS+6-BA2.0mg/L+2,4-D2.0mg/L.
Differential medium: MS+6-BA3.0mg/L+IAA1.0mg/L.
Strong seedling culture base: MS+6-BA2.0mg/L+IAA0.5mg/L.
Root media: MS+IAA0.5mg/L.
The sealwort rhizome of 100 sections 0.7cm.
Begin to organize training work then.
Get the sealwort rhizome of band bud, be cut into the segment of 0.5-1.0cm by knife, put into the liquid detergent aqueous solution and soaked 5 minutes, rinse well with running water then; Use 75% ethanol scrub surfaces again, rinse well with running water again; Get in the superclean bench then and operate, put into for 75% ethanol half a minute, the sterilization deionized water is rinsed and is washed twice; Put into 2.5% clorox again 5 minutes, the sterilization deionized water is rinsed and is washed 2 times; And put into 5% clorox 5 minutes, the sterilization deionized water is rinsed continuously and is washed 2 times; Cut the downright bad part of wound with cutter then.
After the sealwort rhizome that is cut into segment disinfected; Be seeded on the inducing culture; 2 of every bottle graft kinds are carried out callus induction and are cultivated, and observing the rhizome incision after 30 days has a small amount of callus to begin to sprout; Occurred the callus of faint yellow densification in 55 days, find that through statistics inductivity reaches 50%.
The faint yellow granular callus that the growth conditions of inducing generation is good is inoculated in the subculture medium; 2 of every bottle graft kinds are carried out successive transfer culture, and can be observed in the 30th day has new callus to produce again in the subculture medium; The 45th day, observe callus and increase 1 times than original.
The good faint yellow graininess callus of growth conditions that shoot proliferation is produced is transferred on the differential medium, and 2 of every bottle graft kinds are carried out differentiation culture; Observed callus on the 10th day and begin Growth and Differentiation, it is green that color turns, and observed the pistac indefinite bud in 20 days; Observed in 45 days and begin to produce the bud of growing thickly; The 50th day statistics bud ratio 66%, 5 buds of average every callus differentiation, the average length 12mm of bud.
The bud of growing thickly of differentiation culture is transferred to carries out strong seedling culture in the strong seedling culture base, observed the seedling of growing thickly that the bud of growing thickly has developed into 2.0-4.0cm in 30 days.
The seedling of growing thickly of strong seedling culture is transferred to root induction on the root media, observed shoot root in 15 days and begin to sprout, observed to count on 86% seedling and all take root in 30 days, coring average length 4cm, every young plant are on average taken root 6.

Claims (4)

1. sealwort tissue culture propagation method, comprise introduce a fine variety that sterilization, tissue are induced, successive transfer culture, differentiation culture, strong seedling culture and culture of rootage, it is characterized in that concrete grammar is following:
Figure 548975DEST_PATH_IMAGE001
introduces a fine variety sterilization: the sealwort rhizome of getting the band bud; Be cut into the segment of 0.5-1.0cm by knife; Put into the liquid detergent aqueous solution and soaked 5 minutes, rinse well with running water then; Use 75% ethanol scrub surfaces again, rinse well with running water again; Get in the superclean bench then and operate, put into for 75% ethanol half a minute, the sterilization deionized water is rinsed and is washed twice; Put into 2.5% clorox again 5 minutes, the sterilization deionized water is rinsed and is washed 2 times; And put into 5% clorox 5 minutes, the sterilization deionized water is rinsed continuously and is washed 3-5 time, cuts wound necrosis part with cutter then;
Figure 926604DEST_PATH_IMAGE002
tissue is induced: the rhizome after will disinfecting is seeded on the inducing culture; Every bottle graft kind 1-3 piece; Carrying out callus induction cultivates; The rhizome incision had a small amount of callus to begin to sprout in 28-32 days, occurred the callus of faint yellow densification in 50-60 days;
Figure 40054DEST_PATH_IMAGE003
successive transfer culture: will induce the good faint yellow granular callus of growth conditions of generation to be inoculated in the subculture medium; Every bottle graft kind 2-3 piece; Carry out successive transfer culture; Can be observed in 28-32 days has new callus to produce again in the subculture medium; 43-47 days, can be observed callus and increase 1 times than original;
Figure 496443DEST_PATH_IMAGE004
differentiation culture: the good faint yellow graininess callus of growth conditions that shoot proliferation is produced is transferred on the differential medium; Every bottle graft kind 2-3 piece; Carry out differentiation culture; Can be observed callus in 9-11 days and begin Growth and Differentiation; It is green that color turns, and can be observed the pistac indefinite bud in 19-21 days, began to produce the bud of growing thickly in 45-50 days; 5 buds of average every callus differentiation, the average length of bud is 12mm;
Figure 720751DEST_PATH_IMAGE005
strong seedling culture: the bud of growing thickly of differentiation culture is transferred to carries out strong seedling culture in the strong seedling culture base, can be observed the seedling of growing thickly that the bud of growing thickly has developed into 2.0-4.0cm in 30 days;
Figure 188772DEST_PATH_IMAGE006
culture of rootage: the seedling of growing thickly of strong seedling culture is transferred to root induction on the root media; Can be observed shoot root in 15 days begins to sprout; Can be observed 86% seedling in 30 days all takes root; Coring average length 4cm, every young plant are on average taken root 6; But bottle outlet refining seedling is transplanted then.
2. sealwort tissue culture propagation method according to claim 1; It is characterized in that the condition of culture of organizing each development growth stage in the training process is: the artificial climate incubator; The periodicity of illumination beginning and ending time is 8:00-20:00, cultivation temperature day 25 ℃ of temperature, night, temperature was 18 ℃; Cultivating humidity is 60%, and intensity of illumination is 2000-2400lx.
3. sealwort tissue culture propagation method according to claim 1, it is characterized in that the minimal medium of organizing each development growth stage in the training process is: MS+30g/L sucrose+5g/L agar powder, pH value are 5.8-6.0.
4. sealwort tissue culture propagation method according to claim 1, the prescription that it is characterized in that organizing each development growth stage medium in the training process is:
Figure 4282DEST_PATH_IMAGE001
inducing culture: MS+6-BA2.0mg/L+2,4-D1.0mg/L;
subculture medium: MS+6-BA2.0mg/L+2,4-D2.0mg/L;
Figure 851332DEST_PATH_IMAGE003
differential medium: MS+6-BA3.0mg/L+IAA1.0mg/L;
Figure 32915DEST_PATH_IMAGE004
strong seedling culture base: MS+6-BA2.0mg/L+IAA0.5mg/L;
Figure 753746DEST_PATH_IMAGE005
root media: MS+IAA0.5mg/L.
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102771395A (en) * 2012-08-13 2012-11-14 安徽农业大学 Tissue culture method for Polygonaturm sibiricum Redoute
CN103535281A (en) * 2013-11-01 2014-01-29 重庆文理学院 Tissue culture medium of sealwort roots and stems and in-vitro regeneration method
CN103858769A (en) * 2014-04-03 2014-06-18 张旺凡 Rapid rhizoma polygonati propagation technology method
CN104472353A (en) * 2014-11-21 2015-04-01 广西中医药大学 Method for establishing rapid polygonatum sibiricum reproduction system
CN105532448A (en) * 2015-12-03 2016-05-04 普洱市玉林林业开发有限公司 Polygonatum kingianum tissue culture method
CN106134996A (en) * 2016-06-30 2016-11-23 恩施州源惠科技开发有限公司 A kind of method of sealwort once-seedling forming
CN108476982A (en) * 2018-04-14 2018-09-04 湖北襄草源生态农业科技有限公司 A kind of method of sterile rootage breeding in sealwort test tube
CN109169286A (en) * 2018-10-17 2019-01-11 重庆市药物种植研究所 A kind of polygonatum cyrtonema method for tissue culture
CN110741937A (en) * 2019-11-27 2020-02-04 绍兴文理学院 Rapid propagation method of polygonatum sibiricum
CN112806259A (en) * 2021-01-20 2021-05-18 云南帅豪农牧科技有限公司 High-disease-resistance polygonatum sibiricum tissue culture seedling method

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CN1557143A (en) * 2004-01-13 2004-12-29 四川大学 Polygonatum cyrtonema Hua regeneration system establishment method

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102771395B (en) * 2012-08-13 2014-04-16 安徽农业大学 Tissue culture method for Polygonaturm sibiricum Redoute
CN102771395A (en) * 2012-08-13 2012-11-14 安徽农业大学 Tissue culture method for Polygonaturm sibiricum Redoute
CN103535281B (en) * 2013-11-01 2015-06-17 重庆文理学院 Tissue culture medium of sealwort roots and stems and in-vitro regeneration method
CN103535281A (en) * 2013-11-01 2014-01-29 重庆文理学院 Tissue culture medium of sealwort roots and stems and in-vitro regeneration method
CN103858769A (en) * 2014-04-03 2014-06-18 张旺凡 Rapid rhizoma polygonati propagation technology method
CN103858769B (en) * 2014-04-03 2015-06-03 张旺凡 Rapid rhizoma polygonati propagation technology method
CN104472353B (en) * 2014-11-21 2016-08-17 广西中医药大学 A kind of set up the method that Rhizoma Polygonati breeds system soon
CN104472353A (en) * 2014-11-21 2015-04-01 广西中医药大学 Method for establishing rapid polygonatum sibiricum reproduction system
CN105532448A (en) * 2015-12-03 2016-05-04 普洱市玉林林业开发有限公司 Polygonatum kingianum tissue culture method
CN105532448B (en) * 2015-12-03 2017-12-22 普洱市玉林林业开发有限公司 A kind of method of P. kingianum tissue cultures
CN106134996A (en) * 2016-06-30 2016-11-23 恩施州源惠科技开发有限公司 A kind of method of sealwort once-seedling forming
CN106134996B (en) * 2016-06-30 2018-06-29 恩施州源惠科技开发有限公司 A kind of method of sealwort once-seedling forming
CN108476982A (en) * 2018-04-14 2018-09-04 湖北襄草源生态农业科技有限公司 A kind of method of sterile rootage breeding in sealwort test tube
CN109169286A (en) * 2018-10-17 2019-01-11 重庆市药物种植研究所 A kind of polygonatum cyrtonema method for tissue culture
CN109169286B (en) * 2018-10-17 2021-06-18 重庆市药物种植研究所 Polygonatum cyrtonema tissue culture method
CN110741937A (en) * 2019-11-27 2020-02-04 绍兴文理学院 Rapid propagation method of polygonatum sibiricum
CN110741937B (en) * 2019-11-27 2021-02-19 绍兴文理学院 Rapid propagation method of polygonatum sibiricum
CN112806259A (en) * 2021-01-20 2021-05-18 云南帅豪农牧科技有限公司 High-disease-resistance polygonatum sibiricum tissue culture seedling method

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Application publication date: 20120711