CN103190343B - Key technology of organic additive for roxburgh anoectochilus terminal bud industrialization intermediate propagation - Google Patents

Key technology of organic additive for roxburgh anoectochilus terminal bud industrialization intermediate propagation Download PDF

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CN103190343B
CN103190343B CN201310083379.2A CN201310083379A CN103190343B CN 103190343 B CN103190343 B CN 103190343B CN 201310083379 A CN201310083379 A CN 201310083379A CN 103190343 B CN103190343 B CN 103190343B
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roxburgh anoectochilus
terminal bud
anoectochilus terminal
propagation
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CN103190343A (en
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何碧珠
林蔚
何官榕
肖春梅
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He Bizhu
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Fujian Agriculture and Forestry University
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Abstract

The invention provides a key technology of an organic additive for roxburgh anoectochilus terminal bud industrialization intermediate propagation, belonging to a massive propagation and cultivation technology for the roxburgh anoectochilus terminal bud. According to the key technology, a terminal bud and a stem of the roxburgh anoectochilus terminal bud are taken as the material, in industrialization, the organic additive is adjusted and added timely, and the entire plant grows up after induction cultivation, enrichment cultivation and root induction, and the rooting percentage can reach 99%; and after hardening-seedling, the plant can be transplanted into humus, and the rate of survival can reach above 99%. According to the technology, the time for enrichment cultivation is short, the expanding propagation coefficient is high, the seedling can grow up once and is healthy, the whole roxburgh anoectochilus terminal bud plant is thick, the stem is thick and the roots are flourishing, the color of leaves is dark, the leaves have obvious fine hair, the leaf vein gold threads are clear, the rate of survival is high, the weight of each plant is 4.5-5.5g, and the economic efficiency is obvious.

Description

A kind of organic additive is to roxburgh anoectochilus terminal bud industrialization Fast-propagation key technology
Technical field
The invention belongs to the industrialization Fast-propagation culture technique of roxburgh anoectochilus terminal bud.
Background technology
Roxburgh anoectochilus terminal bud ( shorthairyAntenoron) belong to the orchid family open lip Cymbidium [ anoectochilus roxburghii (wall.) Lindl] perennial Valuable Herbal Medicine plant, Chinese formal name used at school is Anoectochilus Roxburghii, rope has the laudatory titles such as " king of medicine ", " gold grass ", " refreshing medicine ", " black ginseng ".Another name Herba Anoectochili roxburghii, gold ear ring, black ginseng, gold thread taiwan anetochilus herb, gold thread disappear to the marrow, desmodium, gold thread lycopod, metal and stone silkworm, juvenile red, leaflet gold ear ring, numb leaf vegetables.Mainly be distributed in the states such as China, Japan, this single blue card, India and Nepal, be grown under remote, thickly forested mountains or thick grass that mountain stream is other moist in or angle a sylvan life, the particularly sough under broad leaved forest, generally be sparse, sporadicly distribute, seldom close life in blocks, roxburgh anoectochilus terminal bud is the traditional valuable ingredient of traditional Chinese medicine of China, because its vein is flavous beautiful vein, ornamental value is high, also have and be developed as the little potted plant bright prospects of indoor sight leaf, roxburgh anoectochilus terminal bud contains number of chemical composition, and pharmacological action is remarkable, and clinical practice is very extensive.Analyze its composition and show, its contain that cardiac glycosides, organic acid, amino acid and polysaccharose, taurine, sterol, alkaloid, Sorghumol, flavonoid glycoside and derivative thereof, parahydroxyben-zaldehyde, forulic acid, Quercetin, crude fat, carrot have and trace element as selenium, molybdenum etc.There is the effect of cool blood purging intense heat, swelling and pain relieving, the internal secretion of energy control agent, suppress the oil of hypersecretion, promote the metabolism of Skin Cell, take rear very good to whelk, acne, various blackspot, freckle, age mark effect, there is hypoglycemic, anti-ephritis, protect liver, diuresis, calmness, hypotensive, anti-oxidant, improve the effects such as osteoporosis, wherein polysaccharide component has good lethal effect to cancer cell, is very promising antineoplastic or auxiliary antineoplastic.
Roxburgh anoectochilus terminal bud seed is small, embryonic development is incomplete, and the difficulty ` that sprouts extremely under field conditions (factors), if with root division or cottage propagation, length consuming time and reproduction coefficient are low, the natural environment that roxburgh anoectochilus terminal bud is depended on for existence changes, and wild resource falls sharply, now on the verge of extinction, and the roxburgh anoectochilus terminal bud of natural environment growth because of artificially excavate in a large number and the cattle grazing such as bird, mouse oneself greatly reduce nature storage, germ plasm resource is rare, endangered, can not meet clinical needs.The work of artificial propagation roxburgh anoectochilus terminal bud is in recent years corresponding extensive expansion also; though tissue-culturing rapid propagation roxburgh anoectochilus terminal bud has been reported; the research of the group culturation rapid propagating technology of roxburgh anoectochilus terminal bud on links also obtains greater advance; but in producing at present, still lack complete tissue culture technique system; in the key technology of producing in scale, industrialization, need further study and break through; the present invention relates to a kind of roxburgh anoectochilus terminal bud industrialization Fast-propagation culture technique; by application the present invention, can provide the good purifying group training seedling of establishing in large scale, to providing technical support for roxburgh anoectochilus terminal bud industrialization.
Summary of the invention
The object of this invention is to provide a kind of method for tissue culture of roxburgh anoectochilus terminal bud industrialization Fast-propagation, the good purifying group training seedling of establishing in large scale is provided, in solution prior art, seed is small, embryonic development is incomplete, difficulty problems such as reproduction coefficient is low of sprouting extremely under field conditions (factors), select the medium of suitable interpolation organic additive to breed anoectochilus blume seedling product quality, finished product seedling product look like to have a direct impact, it is short that application the method is cultivated generation time, expand numerous coefficient high, disposable seedling, seedling health, stem is thick, well developed root system, leaf color is deep obvious flannelette shape, vein gold thread is distinct, survival rate is high, every strain weight reaches 4.5-5.5 gram, there is significant economic benefit, apply the present invention at implement scale simultaneously, in the key technology that industrialization is produced, obtained further breakthrough, for popularization and the development of roxburgh anoectochilus terminal bud kind resource and be applied to plant genetic engineering and the foundation of genetic conversion system and improve significant.
A kind of organic additive is to roxburgh anoectochilus terminal bud industrialization Fast-propagation key technology:
Described method concrete steps comprise:
1) draw materials: explant material is wild roxburgh anoectochilus terminal bud; In season in spring and autumn, pluck, after harvesting, use the cloth that wets to wrap that to take back laboratory standby;
2) preparation of medium:
Bud inducing culture: MS+BA 0.5-2.0mgL -1+ KT 0.5-2mgL -1+ NAA 0.5-2mgL -1+ sucrose 30gL -1+ active carbon 1-2gL -1+ agar 6.5gL -1;
Clump bud inducing culture: MS+BA 0.5-3mgL -1+ KT 0.5-2mgL -1+ NAA 0.5-2mgL -1+ sucrose 30gL -1+ bananas juice 50-150 gL -1+ peptone 0.5-2.5gL -1+ active carbon 1-2gL -1+ agar 6.5gL -1;
Bud proliferated culture medium: 1/2MS +bA 0.5-3mgL -1+ KT 0.5-2mgL -1 +nAA 0.5-2mgL -1+ bananas juice 50-150 gL -1+ peptone 0.5-2.5gL -1+ spend precious No. 1 0.5-1.5gL -1+ sucrose 30gL -1+ active carbon 0.5-1.5gL -1+ agar 6.5gL -1;
Root media: 1/3MS+ IBA 0.5-1mgL -1+ NAA 0.5-1mgL -1+ potato pulp 50-150 gL -1+ sucrose 30gL -1+ active carbon 0.5-2gL -1+ agar 6.5gL -1;
The preparation method of described bananas juice is: will after banana peeling, be ground into mud shape;
The preparation method of described potato pulp is: will after peeling potatoes, be ground into pulpous state;
3) materials disinfection processing: the wild roxburgh anoectochilus terminal bud gathering is rinsed well with running water, apart from bud point 1-2 centimeters clip stem section, remove unnecessary blade, put in saturated bleaching powder supernatant rinsing 15min and scrub gently with banister brush, after rinsing well, be placed in flowing water punching and drip a 0.5-1 hour, then rinse 2-3 time with distilled water, in superclean bench, with 75% alcohol disinfecting 30s, add 0.1wt%HgCl 2process 5-10min; With sterile water, rush 4-6 more standby all over rearmounted workbench.
4) bud induction is cultivated: the material after sterilization is placed on sterilized culture dish, with scalpel, cut away the cut out portion 0.2-0.4cm contacting with mercuric chloride, then material is cut into the long stem section of 1-2cm and is inoculated in respectively in high temperature, autoclaved inducing culture; Condition of culture: culturing room's temperature is 23 ± 2 ℃, light application time 12h/d, intensity of illumination 500-1000lx, bud induction is cultivated 30 days; Obtain Multiple Buds.
5) propagation is cultivated: it is 1.5-2cm that the Multiple Buds obtaining through step 4) induction cultivation is cut and be cut into length, is seeded in respectively in proliferated culture medium; During inoculation, stem section is lain on medium, through 50-70d, breed and cultivate, it is the expansion propagation cultivation that the individual stem section of 5-10 schedules to last 50-70d that the Multiple Buds after propagation is cut into bud number; Culturing room's temperature of whole process is 23 ± 2 ℃, light application time 12h/d, intensity of illumination 1500-2000lx;
6) root induction: step 5) is bred to the seedling individual plant of cultivating out and cut, with 2-3 sheet leaf, plant height 1-2cm, transfers in root media; The culture of rootage time is 30 days, and culturing room's temperature is 23 ± 2 ℃, light application time 12h/d, intensity of illumination 1500-2000lx;
7) bottle transplantation of seedlings: through step 6) after root induction, when plant grows to 10cm, test-tube plantlet was placed on to natural daylight lower refining seedling after 3-5 days, then opens bottle cap hardening 2-3 days, then from blake bottle, take out, wash away the medium that is attached to root system, in the mixed-matrix that to move into quality be 1:2 than vermiculite and humus soil mass ratio, temperature is controlled at 15-30 ℃, and humidity should remain between 70%-85%, avoid direct sunlight, light transmittance 50%-60%.
Described roxburgh anoectochilus terminal bud is the wild roxburgh anoectochilus terminal bud gathering from Nanjing County, ZhangZhou, Fujian Province sea of trees forest.
Remarkable advantage of the present invention is: adopt the inventive method to carry out, stem section is cultivated and within 15 ~ 20 days, can be sprouted the strong sprouting of band white villus growth key through induction, the propagation strong seedling culture of selecting suitable medium to pass through again 50-70 days, expands numerous coefficient high, by height approximately 3-4cm stalwartness without offspring, be divided into individual plant, be seeded in root induction on root media, after 1 month, can grow into whole plant, there is the root of 2-5cm to grow, aerial root is obvious, rooting rate can reach 99%, in industrialization production process, adjusts interpolation organic additive in good time, organic additive consumption 50-150 gL -1, add the medium of organic additive to anoectochilus blume seedling product quality, finished product seedling product look like to have a direct impact, it is short that the method is cultivated generation time, expand numerous coefficient high, disposable seedling, seedling health, roxburgh anoectochilus terminal bud stem plant is thick, well developed root system, survival rate is high, leaf color is deep obvious flannelette shape, vein gold thread is distinct, every strain weight reaches 4.5-5.5 gram, there is significant economic benefit, the good purifying group training seedling of establishing in large scale can be provided, in solution prior art, seed is small, embryonic development is incomplete, difficulty problems such as reproduction coefficient is low of sprouting extremely under field conditions (factors), the present invention is at implement scale in application, in the key technology that industrialization is produced, obtained further breakthrough, for popularization and the development of roxburgh anoectochilus terminal bud kind resource and be applied to plant genetic engineering and the foundation of genetic conversion system and improve significant.
Accompanying drawing explanation
Fig. 1: bud induction cultivation-stem section inoculation
Fig. 2, the growth of Multiple Buds
Fig. 3, propagation is cultivated
Fig. 4, root induction
Fig. 5, transplants roxburgh anoectochilus terminal bud
Embodiment
embodiment 1
1) draw materials: explant material collection is from the wild roxburgh anoectochilus terminal bud of Nanjing County, ZhangZhou, Fujian Province sea of trees forest,
2) preparation of medium:
Bud inducing culture: MS+BA 2.0mgL -1+ KT 0.5.mgL -1+ NAA 0.5.mgL -1+ sucrose 30gL -1+ active carbon 1gL -1+ agar 6.5gL -1;
Clump bud inducing culture: MS+BA 0.5mgL -1+ KT 0.5mgL -1+ NAA 0.5mgL -1+ sucrose 30gL -1+ bananas juice 100 gL -1+ peptone 2.5gL -1+ active carbon 1gL -1+ agar 6.5gL -1;
Bud proliferated culture medium: 1/2MS +bA 0.5mgL -1+ KT 0.5mgL -1 +nAA 0.5mgL -1+ bananas juice 100 gL -1+ peptone 2.5gL -1+ spend precious No. 1 1.5gL -1+ sucrose 30gL -1+ active carbon 1.5gL -1+ agar 6.5gL -1;
Root media: 1/3MS+ IBA 1mgL -1+ NAA 0.5mgL -1+ potato pulp 100 gL -1+ sucrose 30gL -1+ active carbon 2gL -1+ agar 6.5gL -1;
The preparation method of bananas juice is: will after banana peeling, be ground into mud shape;
The preparation method of potato pulp is: will after peeling potatoes, be ground into pulpous state;
3) materials disinfection processing: the wild roxburgh anoectochilus terminal bud gathering is rinsed well with running water and carried out suitable pruning, apart from bud point 1-2 centimeters clip stem section, remove unnecessary blade, put in saturated bleaching powder supernatant rinsing 15min and scrub gently with banister brush, after rinsing well, be placed in flowing water punching and drip 1 hour; Distilled water rinses 3 times, in superclean bench, with removing alcohol after 75% alcohol disinfecting 30s, adds 0.1% mercuric chloride (HgCl 2) process 10min, pour mercury solution into useless mercury bottle; With after sterile water punching 4 times, put workbench standby.
4) bud induction is cultivated: the material of inoculation is placed on sterilized culture dish, with scalpel, cut away the cut out portion 0.2-0.4cm contacting with mercuric chloride, the stem section that then material is cut into 1-2cm length is seeded to respectively and is inoculated in respectively in high temperature, autoclaved inducing culture; In the blake bottle that is 10 at diameter, add the medium of 80-90ML, 5-10 of inoculation stem section; 10 days rear section materials of access medium are sprouted and are expanded in the resting bud of rhizome junction, cultivate 15d observable milky main bud and form; The condition of culture that this one-phase is used: culturing room's temperature is 23 ± 2 ℃, light application time 12h/d, intensity of illumination 500-1000lx; After cultivating 30d, cultivation is bred in taking-up;
6) propagation is cultivated: will cultivate through above-mentioned induction, the well-grown Multiple Buds obtaining cuts and is cut into long 1.5-2 cm and is seeded in respectively in proliferated culture medium; During inoculation, stem section to be lain on medium and cultivate through 50d propagation, can induce a large amount of Multiple Buds, every Multiple Buds approximately has 5-10 bud, be cut into that bud number equates or expansion propagation that close bud clump carries out 70d is cultivated, culturing room's temperature of whole process is 23 ± 2 ℃, light application time 12h/d, intensity of illumination 1500-2000lx;
7) root induction: the seedling individual plant that propagation is cultivated out cuts, every strain is with 2-3 sheet leaf, and plant height 1-2cm, transfers in root media; Cultivate after 30d, near on each sections of medium, grow 2 of 1-, the aerial root that is meat shape penetrates medium, on it, there is fine and closely woven, a milky hair, shorter and the branch not of root, have the root of 1-2cm to grow, rooting rate can reach 99 %, completes the plant regeneration of roxburgh anoectochilus terminal bud and the cultivation of Fast-propagation test-tube plantlet raw when aerial root is more obvious; The condition of culture that this one-phase is used: culturing room's temperature is 23 ± 2 ℃, light application time 12h/d, intensity of illumination 1500-2000lx;
8) bottle transplantation of seedlings: when bottle seedling grows to 3cm, 2-3 bar root, 5 leaves of 4-, leaf is about 2-3cm, from the each sections near medium, grow 2 of 1-, the aerial root that is meat shape penetrates medium, on it, have fine and closely woven, milky hair, shorter and the branch not of root, cultivate 30d left and right, when growing to 10cm, plant can carry out outdoor transplanting, test-tube plantlet is placed on to natural daylight lower refining seedling 3 days, open bottle cap hardening 2 days, then from blake bottle, take out, wash away the medium that is attached to root system, move in the matrix of vermiculite and humus soil (1:2) mixing, moisturizing is sheltered from heat or light, temperature is controlled at 15-30 ℃, humidity should remain between 70%-85%, avoid direct sunlight light transmittance 50%-60%.Survival rate can reach more than 99%.

Claims (1)

1. a roxburgh anoectochilus terminal bud industrialization method for quickly breeding: described method concrete steps comprise:
1) draw materials: explant material is wild roxburgh anoectochilus terminal bud; In season in spring and autumn, pluck, after harvesting, use the cloth that wets to wrap that to take back laboratory standby;
2) preparation of medium:
Bud inducing culture: MS+BA 0.5-2.0mgL -1+ KT 0.5-2mgL -1+ NAA 0.5-2mgL -1+ sucrose 30gL -1+ active carbon 1-2gL -1+ agar 6.5gL -1;
Clump bud inducing culture: MS+BA 0.5-3mgL -1+ KT 0.5-2mgL -1+ NAA 0.5-2mgL -1+ sucrose 30gL -1+ bananas juice 50-150 gL -1+ peptone 0.5-2.5gL -1+ active carbon 1-2gL -1+ agar 6.5gL -1;
Bud proliferated culture medium: 1/2MS+BA 0.5-3mgL -1+ KT 0.5-2mgL -1+ NAA 0.5-2mgL -1+ bananas juice 50-150 gL -1+ peptone 0.5-2.5gL -1+ spend precious No. 1 0.5-1.5gL -1+ sucrose 30gL -1+ active carbon 0.5-1.5gL -1+ agar 6.5gL -1;
Root media: 1/3MS+ IBA 0.5-1mgL -1+ NAA 0.5-1mgL -1+ potato pulp 50-150 gL -1+ sucrose 30gL -1+ active carbon 0.5-2gL -1+ agar 6.5gL -1;
The preparation method of described bananas juice is: will after banana peeling, be ground into mud shape;
The preparation method of described potato pulp is: will after peeling potatoes, be ground into pulpous state;
3) materials disinfection processing: the wild roxburgh anoectochilus terminal bud gathering is rinsed well with running water, apart from bud point 1-2 centimeters clip stem section, remove unnecessary blade, put in saturated bleaching powder supernatant rinsing 15min and scrub gently with banister brush, after rinsing well, be placed in flowing water punching and drip a 0.5-1 hour, then rinse 2-3 time with distilled water, in superclean bench, with 75% alcohol disinfecting 30s, add 0.1wt%HgCl2 to process 5-10min; With sterile water, rush 4-6 more standby all over rearmounted workbench;
4) bud induction is cultivated: the material after sterilization is placed on sterilized culture dish, with scalpel, cut away the cut out portion 0.2-0.4cm contacting with mercuric chloride, then material is cut into the long stem section of 1-2cm and is inoculated in respectively in high temperature, autoclaved inducing culture; Condition of culture: culturing room's temperature is 23 ± 2 ℃, light application time 12h/d, intensity of illumination 500-1000lx, bud induction is cultivated 30 days; Obtain Multiple Buds;
5) propagation is cultivated: it is 1.5-2cm that the Multiple Buds obtaining through step 4) induction cultivation is cut and be cut into length, is seeded in respectively in proliferated culture medium; During inoculation, stem section is lain on medium, through 50-70d, breed and cultivate, it is the expansion propagation cultivation that the individual stem section of 5-10 schedules to last 50-70d that the Multiple Buds after propagation is cut into bud number; Culturing room's temperature of whole process is 23 ± 2 ℃, light application time 12h/d, intensity of illumination 1500-2000lx;
6) root induction: cut breeding through step 5) the seedling individual plant of cultivating out, with 2-3 sheet leaf, plant height 1-2cm, transfers in root media; The culture of rootage time is 30 days, and culturing room's temperature is 23 ± 2 ℃, light application time 12h/d, intensity of illumination 1500-2000lx;
7) bottle transplantation of seedlings: through step 6) after root induction, when plant grows to 10cm, test-tube plantlet was placed on to natural daylight lower refining seedling after 3-5 days, then opens bottle cap hardening 2-3 days, then from blake bottle, take out, wash away the medium that is attached to root system, in the mixed-matrix that to move into quality be 1: 2 than vermiculite and humus soil mass ratio, temperature is controlled at 15-28 ℃, and humidity remains between 70%-85%, avoid direct sunlight, light transmittance 50%-60%;
Described roxburgh anoectochilus terminal bud is the wild roxburgh anoectochilus terminal bud gathering from Nanjing County, ZhangZhou, Fujian Province sea of trees forest.
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CN103461118A (en) * 2013-07-31 2013-12-25 湖州市农业科学研究院 Industrialized production method for anoectochilus roxburghii seedlings
CN103416310B (en) * 2013-08-28 2015-05-20 叶宝鑫 Culture medium used for roxburgh anoectochilus terminal bud organic tissue culture
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CN105165630B (en) * 2015-10-27 2017-12-22 河池乐康生态农业科技有限公司 The breeding method of roxburgh anoectochilus terminal bud tissue-cultured seedling
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