CN105815212A - Codonopsis pilosula tissue culture breeding method - Google Patents
Codonopsis pilosula tissue culture breeding method Download PDFInfo
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- CN105815212A CN105815212A CN201510008394.XA CN201510008394A CN105815212A CN 105815212 A CN105815212 A CN 105815212A CN 201510008394 A CN201510008394 A CN 201510008394A CN 105815212 A CN105815212 A CN 105815212A
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Abstract
The invention discloses a Codonopsis pilosula tissue culture breeding method. The method comprises six steps of culture by a medium, disinfection of an explant, induction of the explant, differentiation of subculture buds, rooting culture of subculture buds, hardening seedling and transplanting; plant breeding amount can be enlarged with large scale in short time, the parent excellent property is kept; asexual rapid breeding of Codonopsis pilosula is carried out by using tissue culture, material acquisition amount is little, the culture method is economic; culture condition is artificially controlled without influence of natural condition and restriction of plant growth environment, region and season time; growth period is short, reproductive rate is high, a lot of Codonopsis pilosula tissue culture plants can be bred in the short time, and the growth period of the Codonopsis pilosula is shortened.
Description
Technical field
The present invention relates to the mating system of medicinal plants, the tissue culture method for breeding of a kind of Radix Codonopsis.
Background technology
Radix Codonopsis (Codonopsispilosula (Franch.) Nannf.) ovalize or the sheet of similar round, just yellowish complexion brown or lark, tangent plane yellow-white or yellowish-brown, have crack or Flos Chrysanthemi stricture of vagina, and there are the faint yellow center of circle in central authorities.Periphery yellowish white puts yellowish-brown, has vertical wrinkle.Having special aroma, taste is micro-sweet.Raw Radix Codonopsis sheet supplementing QI for promoting the production of body fluid power victory.It is usually used in gas Tianjin two to hinder or deficiency of both QI and blood.In the woods limit, mountain region of place of production north of China height above sea level 1560-3100 rice and shrubbery.The Codonopsis plant whole world there are about 40 kinds, and China there are about 39 kinds, and medicinal have 21 kinds, 4 mutation.Radix Codonopsis is traditional tonic that China is conventional, and ancient times are top grade with the Radix Codonopsis that area, Shangdang, Shanxi produces, and have invigorating the spleen and replenishing QI, effect of strengthening spleen and tonifying lung.Modern study, Radix Codonopsis has enhancing immunity, expansion blood vessel, blood pressure lowering, improves the effect such as microcirculation, enhancing hemopoietic function.In addition the leukopenia caused chemotherapy radiotherapy has castering action
Codonopsis's perennial prehensile draft.Radix Codonopsis stem is about 1.5 meters.Herb has milk and special foul smell.Root meat is loose, elongated cylindrical.Leaf is to raw or alternate, avette to wide avette hair.Xia Qiu is bloomed, corolla mitriform, and pistac has purple dot.Capsule cone, seed is tiny.The nice and cool weather of Radix Codonopsis happiness gentleness.Radix Codonopsis root is containing saponin, saccharide and Alkaloid.Pharmacological testing proves have increase erythrocyte and the effect of hematochrome.In medical for strengthening by means of tonics medicine, function invigorating the spleen and replenishing QI, nourishing blood to promote the production of body fluid.Cure mainly that insufficiency of vital energy and blood, overstrain be weak, anorexia and loose stool, blood deficiency and yellow complexion, have blood in stool, the disease such as metrorrhagia.But the breeding of Radix Codonopsis has been used up traditional implantation methods, its efficiency is low, and in order to overcome the deficiency in existing Radix Codonopsis artificial cultivation technique, the present invention provides the mating system of a kind of Radix Codonopsis tissue culture, the emergence rate of Radix Codonopsis can not only be improved, and cultivation period can be shortened.
Summary of the invention
It is an object of the invention to be to use tissue culture method for breeding, solve the problem during Radix Codonopsis planting process utilizes seminal propagation, to improve the fertility of Radix Codonopsis.
To achieve these goals, the present invention includes following technical step
Prepared by first step culture medium: select MS culture medium as minimal medium, 6-benzyl aminopurine (6-BA), naphthalene acetic acid (NAA), gibberellins, zeatin (ZT) and 3-indolyl acetic acid (IAA) prepare three kinds of culture medium respectively as regulation and control hormone: (1) just division culture medium A:MS+6-BA0.1～0.6mg/L+ gibberellins 1～8mg/L, (2) subculture division culture medium B:MS+6-BA0.3～1.8mg/L+ZT0.1～1.5mg/L, (3) root media C:MS+NAA0.1～0.6mg/L+IAA0.1～0.6mg/L.Culture medium A and B are all added 5% sucrose and 1% agar, culture medium C adds 4% sucrose and 0.5% agar, NaOH and HCl with 1～2mgl/L regulates pH value to 6.0, and by culture medium A, B and C in high-pressure steam sterilizing pan 0.1～0.15MPa, 120～125 DEG C of sterilizings 15～20min；
The sterilization of the outer implant of second step: select the tender leaf that Radix Codonopsis has just been sprouted as outer implant, 30～60min are rinsed with tap water, with 75% alcohol disinfecting 4min on aseptic operating platform, with aseptic water washing 3～4 times, again with mercuric chloride that concentration is 0.2% sterilization 7～15min, finally with aseptic water washing 3～4 times；
The induction of the outer implant of the 3rd step: the outer implant of Radix Codonopsis step (2) sterilized is seeded on first division culture medium A, puts 1～3 outer implant for every bottle in inoculation bottle, is placed in cultivation indoor cultivation 45 days, and outer implant grows adventitious bud；
The differentiation of the 4th step subculture bud: the outer implant just broken up in step (3) transferred in subculture division culture medium B, is placed in cultivation indoor cultivation 30 days, and outer implant differentiates clump bud；
Taking root of 5th step subculture bud: by highly transferring in root media C for about 2.0cm, healthy and strong subculture simple bud, carrying out root culture, after 30～40 days, root length reaches 4～5cm；
6th step seedling exercising and transplanting: inoculation bottle bottleneck is opened culturing room's inscattering light lower refining seedling 5～7 days, and periodicity of illumination is 16h/d, and intensity of illumination is 2000～2400Lux, and culturing room's temperature is 23～25 DEG C；Take out the tissue cultured seedling in bottle afterwards, the agar of root is cleaned with tap water, the carbendazim being diluted with distilled water into 500～600 times again soaks tissue cultured seedling root 20～30min, transplants to the culturing pot of fertile soil substrate, illumination 16h/d, intensity of illumination is 2000～2400Lux, daytime 23～25 DEG C, evening 15～18 DEG C, keep humidity 40～60%, it is transplanted in booth after 30～40 days, survival rate more than 95%.
Described breeding fertile soil is that fertile soil is neutralized to pH value is 4.8-5.8, then adds fertilizer one trace elements and major elements in 100 parts of fertile soil, and its weight portion is as follows:
Ammonium salt 15-65 part potash fertilizer 30-55 part
Trace element magnesium 30-55 part trace elements iron 3-6 part
Trace copper 3-6 part trace element zinc 0.5-1.5 part
Trace element manganese 0.3-0.5 part boric acid 0.5-0.8 part shale ashes 300-500 part.
Described ammonium salt is at least one in ammonium phosphate, ammonium nitrate, ammonium molybdate.
Beneficial effects of the present invention: the present invention utilizes tissue culture technique can expand the breeding amount of plant at short notice on a large scale, and makes the merit of parent be preserved；Utilize tissue culture to carry out the asexual Fast-propagation of Radix Codonopsis, draw materials few, cultivate economy；Can manual control condition of culture, not by effect of natural conditions, jumped out plant growth environment, region and the restriction of time in season；Growth cycle is short, and breeding potential is high, can breed out substantial amounts of Radix Codonopsis tissue cultured seedling in the short period of time, shorten the growth cycle of Radix Codonopsis.
Detailed description of the invention
Below in conjunction with instantiation, the invention will be further described.
Example 1, the tissue culture method for breeding of Radix Codonopsis, comprise the following steps:
Prepared by first step culture medium: select MS culture medium as minimal medium, 6-benzyl aminopurine (6-BA), naphthalene acetic acid (NAA), gibberellins, zeatin (ZT) and 3-indolyl acetic acid (IAA) prepare three kinds of culture medium respectively as regulation and control hormone: (1) just division culture medium A:MS+6-BA0.1～0.6mg/L+ gibberellins 1～8mg/L, (2) subculture division culture medium B:MS+6-BA0.3～1.8mg/L+ZT0.1～1.5mg/L, (3) root media C:MS+NAA0.1～0.6mg/L+IAA0.1～0.6mg/L.Culture medium A and B are all added 5% sucrose and 1% agar, culture medium C adds 4% sucrose and 0.5% agar, NaOH and HCl with 1～2mgl/L regulates pH value to 6.0, and by culture medium A, B and C in high-pressure steam sterilizing pan 0.1～0.15MPa, 120～125 DEG C of sterilizings 15～20min；
The sterilization of the outer implant of second step: select the tender leaf that Radix Codonopsis has just been sprouted as outer implant, 30～60min are rinsed with tap water, with 75% alcohol disinfecting 4min on aseptic operating platform, with aseptic water washing 3～4 times, again with mercuric chloride that concentration is 0.2% sterilization 7～15min, finally with aseptic water washing 3～4 times；
The induction of the outer implant of the 3rd step: the outer implant of Radix Codonopsis step (2) sterilized is seeded on first division culture medium A, puts 1～3 outer implant for every bottle in inoculation bottle, is placed in cultivation indoor cultivation 45 days, and outer implant grows adventitious bud；
The differentiation of the 4th step subculture bud: the outer implant just broken up in step (3) transferred in subculture division culture medium B, is placed in cultivation indoor cultivation 30 days, and outer implant differentiates clump bud；
Taking root of 5th step subculture bud: by highly transferring in root media C for about 2.0cm, healthy and strong subculture simple bud, carrying out root culture, after 30～40 days, root length reaches 4～5cm；
6th step seedling exercising and transplanting: inoculation bottle bottleneck is opened culturing room's inscattering light lower refining seedling 5～7 days, and periodicity of illumination is 16h/d, and intensity of illumination is 2000～2400Lux, and culturing room's temperature is 23～25 DEG C；Take out the tissue cultured seedling in bottle afterwards, the agar of root is cleaned with tap water, the carbendazim being diluted with distilled water into 500～600 times again soaks tissue cultured seedling root 20～30min, transplants to the culturing pot of fertile soil substrate, illumination 16h/d, intensity of illumination is 2000～2400Lux, daytime 23～25 DEG C, evening 15～18 DEG C, keep humidity 40～60%, it is transplanted in booth after 30～40 days, survival rate more than 95%.
Described breeding fertile soil is that fertile soil is neutralized to pH value is 4.8-5.8, then adds fertilizer one trace elements and major elements in 100 parts of fertile soil, and its weight portion is as follows:
Ammonium salt 15-65 part potash fertilizer 30-55 part
Trace element magnesium 30-55 part trace elements iron 3-6 part
Trace copper 3-6 part trace element zinc 0.5-1.5 part
Trace element manganese 0.3-0.5 part boric acid 0.5-0.8 part shale ashes 300-500 part.
Described ammonium salt is at least one in ammonium phosphate, ammonium nitrate, ammonium molybdate.
Field-transplanting method after example 2, Radix Codonopsis tissue culture method for breeding nursery
(1) selection of land.Radix Codonopsis nursery selects the Huo Eryin district, tableland, river of height above sea level 2200～2500m, hillside fields require the gradient≤15 ° nursery to require that the soil is porous is fertile, soil layer is deep, draining is good, irrigation is convenient.Farming crops for rotation are optimal with beans and wheat crops, avoid and root class medical material continuous cropping.The plot that perennial root ruderal is many is also unfit to do Radix Codonopsis nursery.
(2), deep ploughing fertilizer.During whole ground, distinct methods should be used according to different plot feature.Plains region nursery, front stubble preferably Semen Maydis, millet, Rhizoma Solani tuber osi, should in winter the previous year plough sun the earth which has been ploughed up, deep ploughing leveling, bedding；Turning over before broadcasting 1 time again, execute people's base manure (compost, barnyard manure) 1500～3000kg for every mu, execute people for every mu and become pesticide-clay mixture 50kg by 40% phoxim emulsifiable concentrate 500g mixing, with good conditionsi can execute more than biogas slurry 500kg or biogas residue 200kg by mu, rake carefully flattens bedding.Mountain area begins to rehearse ditch by domatic in 7～August, and base manure made by every mu of ground manuring or compost 2500～5000kg, uniformly spreads in people ground, then deep ploughs 1 time, flattens bedding.
(3), level land bedding.Depending on physical features, the most in easy flights, the relatively flat ground of physical features can be made flat furrow or high furrow, and the steeper ground of the gradient must be made high furrow.Furrow wide 100～120cm, furrow spacing 30cm, furrow high 15～20cm, depending on furrow length is because of physical features, the bedding of north-south, level land, ground, gentle slope is along equal pitch contour bedding.Furrow surrounding begins to rehearse ditch.Also wide 25～the micro-ridge of 35cm or the big ridge of wide 50～60cm can be made.If nursery on cohesive soil, then must be when the time of the year when autumn changes into winter, soil deeptillage 30cm, the fine sand that people has sieved is mixed in the clay routed up, consumption is about the 1/3 of clay amount, then makees base manure with cattle, horsehit, one layer of oxen and horses excrement, one layer of soil having mixed sand, respectively spreading two-layer by several times, bedding of raking is transplanted again.
(4), Radix Codonopsis seedling management) shelter from heat or light.The growth of seedling of Radix Codonopsis tissue culture propagation is thin and delicate, is afraid of drought, is afraid of flood, Keep away from sun, happiness cool place；No matter when spring, summer, autumn are sowed, and will shelter from heat or light.Should intertill when height of seedling reaches l0 DEG C of m and hoe up weeds, and point throw off several times and to cover grass, make seedling that sunlight to be adapted to gradually, dried up by burning sun with the too many seedling of tamper-evident grass.All can throw off covering grass when 15 DEG C of m of height of seedling.
(5), fill the draining Radix Codonopsis seeding stage and Seedling Stage furrow face keeps humidity, in order to emerging.Seedling Stage is suitably poured water according to natural conditions such as area, soil properties or sprinkles water, but can not water by flood, thrusts seedling in order to avoid causing.Seedling can be poured water after growing up less, does not topdresses, and excess moisture easily causes too much branch and leaf excessive growth, and seedling stage, suitably arid was conducive to joining the elongation growth of root.Should water in time during soil drought.With good conditionsi can water before soil is freeze-up 1 time permeable, in order to surviving the winter.Rainy season pays special attention to draining, prevents rotten seedling rot, causes seedling dead.
(6) weed with a hoe, nursery duty to be accomplished is cut weeds, and prevents farmland running to weeds.See that grass just pulls out with broadcasting sowing, loosen the soil to drilling and weeding is carried out simultaneously.Note suitable thinning during height of seedling 5～7 DEG C of m, keep spacing in the rows 1～3 DEG C of m, remove a part of overstocked weak Seedling by several times.If it is live, during 15 DEG C of about m of height of seedling, by spacing in the rows 3～5 DEG C of m final singlings.Loosen the soil the most shallow, it is to avoid hinder root.Uprooting weed cloudy day to be selected or morning, carry out at dusk.
(7), built management Radix Codonopsis built uses for reference the agricultural sciences principle of solid cultivation, built is used to help plants stems climing, upright growth, substantially improve field ventilation and penetrating light situation, make effective photosynthesis enlarged areas of Radix Codonopsis, effectively strain number increases, Basic Seedling survival rate improves, 24.9% can be increased compared with the single fresh weight of traditional cultivation respectively by the upright built of Radix Codonopsis and horizontal built, 21.6%, effectively strain number can increase by 20.6% respectively, 11.3%, planting percent can increase by 12.2% respectively, 6.7%, fresh ginseng yield increases respectively to 456kg/ mu from 306kg/ mu, 417kg/ mu, it is respectively increased 48.8% and 36.0%, effect of increasing production is obvious.Upright built and level set in brickwork susceptible gene relatively traditional cultivation reduce by 45.5% and 27.3% respectively.
Claims (6)
1. the tissue culture method for breeding of a Radix Codonopsis, it is characterized in that: by culture medium culturing, the sterilization of outer implant, the induction of outer implant, the differentiation of subculture bud, the root culture of subculture bud, seedling exercising and transplant six steps in fertile soil, manual control condition of culture, not by effect of natural conditions, expand the breeding amount of plant the most on a large scale, improve Radix Codonopsis cultivation benefit.
The tissue culture method for breeding of a kind of Radix Codonopsis the most as claimed in claim 1, described culture medium is to select MS culture medium to prepare just division culture medium A: subculture division culture medium B, root media C as minimal medium, 6-benzyl aminopurine (6-BA), naphthalene acetic acid (NAA), gibberellins, zeatin (ZT) and 3-indolyl acetic acid (IAA) respectively as regulation and control hormone.
The tissue culture method for breeding of a kind of Radix Codonopsis the most as claimed in claim 1, the tender leaf that the sterilization selection Radix Codonopsis of described outer implant has just been sprouted is as outer implant.
The tissue culture method for breeding of a kind of Radix Codonopsis the most as claimed in claim 1, the induction sterilization of described outer implant is that the outer implant of Radix Codonopsis is seeded on first division culture medium A, putting 1～3 outer implant for every bottle in inoculation bottle, be placed in cultivation indoor cultivation 45 days, outer implant grows adventitious bud.
The tissue culture method for breeding of a kind of Radix Codonopsis the most as claimed in claim 1, the differentiation of described subculture bud: be that the outer implant just broken up is transferred in subculture division culture medium B, it is placed in cultivation indoor cultivation 30 days, outer implant differentiates clump bud.
The tissue culture method for breeding of a kind of Radix Codonopsis the most as claimed in claim 1, taking root of described subculture bud is highly will to transfer in root media C for about 2.0cm, healthy and strong subculture simple bud, carry out root culture, and after 30～40 days, root length reaches 4～5cm；
Described seedling exercising and transplanting be: inoculation bottle bottleneck is opened culturing room's inscattering light lower refining seedling 5～7 days, and periodicity of illumination is 16h/d, and intensity of illumination is 2000～2400Lux, and culturing room's temperature is 23～25 DEG C；Take out the tissue cultured seedling in bottle afterwards, the agar of root is cleaned with tap water, the carbendazim being diluted with distilled water into 500～600 times again soaks tissue cultured seedling root 20～30min, transplants to the culturing pot of fertile soil substrate, illumination 16h/d, intensity of illumination is 2000～2400Lux, daytime 23～25 DEG C, evening 15～18 DEG C, keep humidity 40～60%, it is transplanted in booth after 30～40 days, survival rate more than 95%.
Described breeding fertile soil is that fertile soil is neutralized to pH value is 4.8-5.8, then adds fertilizer one trace elements and major elements in 100 parts of fertile soil, and its weight portion is as follows:
Ammonium salt 15-65 part potash fertilizer 30-55 part
Trace element magnesium 30-55 part trace elements iron 3-6 part
Trace copper 3-6 part trace element zinc 0.5-1.5 part
Trace element manganese 0.3-0.5 part boric acid 0.5-0.8 part shale ashes 300-500 part.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106804426A (en) * | 2016-12-26 | 2017-06-09 | 中国科学院华南植物园 | Promote the box set and method of Companumoea root vitro proliferation |
| CN107295829A (en) * | 2017-08-17 | 2017-10-27 | 河西学院 | A kind of method of yellow ginseng seed germination |
| CN107801634A (en) * | 2017-11-13 | 2018-03-16 | 陈培党 | A kind of establishing techniques of Radix Codonopsis vitro Regeneration System |
| CN109169276A (en) * | 2018-09-13 | 2019-01-11 | 甘肃源宜生物科技有限公司 | A kind of Radix Codonopsis tissue-culturing rapid propagation culture medium and method |
| CN113068611A (en) * | 2021-04-09 | 2021-07-06 | 中国热带农业科学院热带作物品种资源研究所 | Method for rapidly propagating seedlings by utilizing leaves of tetrapanax papyrifera |
-
2015
- 2015-01-08 CN CN201510008394.XA patent/CN105815212A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106804426A (en) * | 2016-12-26 | 2017-06-09 | 中国科学院华南植物园 | Promote the box set and method of Companumoea root vitro proliferation |
| CN106804426B (en) * | 2016-12-26 | 2019-07-30 | 中国科学院华南植物园 | Promote the box set and method of Companumoea root vitro proliferation |
| CN107295829A (en) * | 2017-08-17 | 2017-10-27 | 河西学院 | A kind of method of yellow ginseng seed germination |
| CN107801634A (en) * | 2017-11-13 | 2018-03-16 | 陈培党 | A kind of establishing techniques of Radix Codonopsis vitro Regeneration System |
| CN109169276A (en) * | 2018-09-13 | 2019-01-11 | 甘肃源宜生物科技有限公司 | A kind of Radix Codonopsis tissue-culturing rapid propagation culture medium and method |
| CN113068611A (en) * | 2021-04-09 | 2021-07-06 | 中国热带农业科学院热带作物品种资源研究所 | Method for rapidly propagating seedlings by utilizing leaves of tetrapanax papyrifera |
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Application publication date: 20160803 |