CN103947549A - Rapid propagation method of dendrobium officinale - Google Patents
Rapid propagation method of dendrobium officinale Download PDFInfo
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- CN103947549A CN103947549A CN201410153939.1A CN201410153939A CN103947549A CN 103947549 A CN103947549 A CN 103947549A CN 201410153939 A CN201410153939 A CN 201410153939A CN 103947549 A CN103947549 A CN 103947549A
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Abstract
The invention discloses a rapid propagation method of dendrobium officinale. The rapid propagation mehod comprises the following steps: washing and sterilizing an explant; inoculating the explant in an adventitious bud induction medium which comprises an MS basic culture medium, 2.0mg/L-3.0mg/L of 6-BA (6-Benayl Aminopurine) and 0.2mg/L of NAA (N-Acetyl Aspartate); inoculating an adventitious bud stem in a protocorm induction medium which comprises a B5 basic culture medium, 2.0mg/L of the NAA, 0.2mg/L of the 6-BA and 0.5mg/L-2.0mg/L of KT; inoculating a protocorm in a protocorm propagation culture medium which comprises 1/2 of the MS basic culture medium in the step 1, 50g/L of coconut juice; transferring the protocorm in a differential medium which comprises 1/2 of the MS basic culture medium in the step 1, 0.2mg/L of the NAA, 80g/L-100g/L of a potato extract; inoculating differentiated seedlings in a seedling and root growing culture medium which comprises 1/2 of the MS basic culture medium in the step 1 and 100g/L-150g/L of a banana extract. The rapid propagation method disclosed by the invention has the effects of increasing the survival rate of tissue cultured seedlings of the dendrobium officinale and by using the method, plants of the dendrobium officinale grow healthily and strongly.
Description
Technical field
The present invention relates to biological technical field, refer in particular to a kind of dendrobium candidum method for quickly breeding.
Background technology
Dendrobium candidum is the orchid family herbaceous plant that grows nonparasitically upon another plant for many years, grows in height above sea level and reaches on the rock that the mountain region half of 1600 meters is dark and damp.The effective component of dendrobium candidum is mainly dendrobium polysaccharide, dendrobine and total amino acid etc., and contains the various trace elements such as calcium, iron, zinc, selenium, sodium, has unique medical value.The traditional Chinese medical science is used as medicine with its stem, and by name " stem of noble dendrobium " belongs to the yin tonics in tonic, and " legendary god of farming's book on Chinese herbal medicine " row dendrobium candidum is top grade, is loaded with " in main wound, except numbness, lower gas, tonifying five zang organs consumptive disease, win thin, reinforcing yin essence, take thick stomach for a long time ".In Compendium of Material Medica, evaluate dendrobium candidum " reinforcing yin essence benefit essence.Thick stomach, never sufficient in benefit, flat stomach Qi, longue meat, intelligence development, except frightened, is made light of one's life by commiting suicide and is prolonged life ".The effects such as " Taoist Scriptures " records, and has enriching yin qi-restoratives, promoting production of body fluid and nourishing the lung.
Plant Tissue Breeding (Tissue culture) refers to the totipotency of utilizing plant cell, in plant corpus, take out part organ, tissue or cell, physiological environment in simulating plant body, under aseptic, proper temperature and certain condition of culture, make it existence, growth, maintain the method for its normal configuration and function.
But in prior art, most candidum tissue culturing seedling canes are partially thin, mostly even are " toothpick seedling ", root system is flourishing not, and blade does not launch, and causes later stage domestication shoot survival percent on the low side, and seedling raise period is partially long, and fresh output is lower, and production cost is higher.
Summary of the invention
The object of the present invention is to provide a kind of dendrobium candidum method for quickly breeding, to improve candidum tissue culturing seedling survival rate, and robust plant.
For reaching above-mentioned purpose, solution of the present invention is:
A kind of dendrobium candidum method for quickly breeding, comprises the following steps:
One, choose explant, explant is cleaned, sterilized;
Two, explant is inoculated in to adventitious bud induction culture base and carries out evoking adventive bud cultivation, inducing culture proportioning is: MS minimal medium, 6-BA (6-benzyl purine) 2.0mg/L-3.0mg/L, NAA(methyl α-naphthyl acetate) and 0.2mg/L, pH value 5.4;
Three, indefinite gained in step 2 leaf stem section is inoculated in protocorm inducing culture, this Medium Proportion is: B5 minimal medium, NAA2.0mg/L, 6-BA0.2mg/L, KT(kinetin) and 0.5mg/L-2.0mg/L, pH value 5.4;
Four, gained protocorm in step 3 to be transferred in Protocorm Multiplication medium, this Medium Proportion is: 1/2MS minimal medium, Coconut Juice 50g/L, pH value 5.4; Condition of culture is: light intensity 2000lx, light application time 12h/d, and shaking speed is 120r/min, switching in every 21 days is once;
Five, gained protocorm in step 4 to be transferred in differential medium, this Medium Proportion is: 1/2MS minimal medium, NAA0.2mg/L, potato extract 80-100g/L, pH value 5.4;
Six, gained differentiation seedling in step 5 is inoculated in strengthening seedling and rooting medium, this Medium Proportion is: 1/2MS minimal medium, banana extract 100-150g/L, pH value 5.4; Be placed under fluorescent lamp and cultivate after 30 days, be placed in culturing rack top layer, intensity of illumination 8000Lux-10000Lux, light application time is 12h/d, and light source is daylight, and temperature is 26 ± 2 DEG C, cultivates and emerges after 60 days;
Step 2, three and five condition of culture are: intensity of illumination 2000-3000Lux, light application time 12h/d(hour/day), light source is fluorescent lamp, temperature is 26 ± 2 DEG C.
In described step 1, cleaning, sterilisation step are: first use non-phosphide detergent aqueous solution soaking 15 minutes, then rinse 30 minutes with running water; With alcohol-pickled 15 seconds of 75%, aseptic water washing 1 time, then use 0.1%HgCl
2solution disinfection 8 minutes, aseptic water washing 5 times.
In described step 1, choose epidermis rust, stipes is obvious, entire body is transparent, fiber is few individuality as explant.
In described step 1, after explant cleans, sterilizes, suck dry moisture is cut to the stem section of 1.0-1.5cm, and access inducing culture is induced cultivation.
Adopt after such scheme, the invention solves candidum tissue culturing seedling cane partially thin, root system is flourishing not, the technical problems such as blade does not launch, and survival rate is on the low side, realize dendrobium candidum bottle seedling leaf thickening, fully launch, leaf look dark green, and cane is sturdy, well developed root system, survival rate reaches more than 98%, in whole breeding flow process, do not add hormone, greatly in limit, reduced the use of hormone, further ensured the safety as the dendrobium candidum of medicinal plant.
Embodiment
Embodiment
The present invention discloses a kind of dendrobium candidum method for quickly breeding, comprises the following steps:
One, the choosing and the acquisition of sterilizable material of explant material
Choose epidermis rust, stipes obviously, entire body is comparatively transparent, fiber is less individuality is as explant.First explant is cleaned, sterilized, concrete steps are as follows: first use non-phosphide detergent aqueous solution soaking 15 minutes, then rinse 30 minutes with running water flowing water; With alcohol-pickled 15 seconds of 75%, aseptic water washing 1 time, then use 0.1%HgCl
2solution disinfection 8 minutes, aseptic water washing 5 times.
After above-mentioned explant cleans, sterilizes, suck dry moisture is cut to the stem section of 1.0～1.5cm, then accesses inducing culture and induces cultivation.
Two, the induction of indefinite bud
Gained explant in step 1 is inoculated in to adventitious bud induction culture base and induces cultivation, inducing culture proportioning is: MS minimal medium, 6-BA (6-benzyl purine) 2.0mg/L-3.0mg/L, NAA(methyl α-naphthyl acetate) 0.2mg/L, pH value 5.4, condition of culture is: intensity of illumination is 2000-3000Lux, light application time is 12h/d, light source is fluorescent lamp, and temperature is 26 ± 2 DEG C.Explant approximately induces indefinite bud for 3 months in this medium.
Three, the induction of protocorm
Indefinite gained in step 2 leaf stem section is inoculated in protocorm inducing culture, this Medium Proportion is: B5 minimal medium, NAA2.0mg/L, 6-BA0.2mg/L, KT(kinetin) 0.5mg/L-2.0mg/L, pH value 5.4, condition of culture is: intensity of illumination is 2000-3000Lux, light application time is 12h/d, light source is fluorescent lamp, and temperature is 26 ± 2 DEG C.Stem section was through the cultivation of 3 months, and two ends induce the protocorm of peak green, full grains.
Four, the propagation of protocorm
Gained protocorm in step 3 is transferred in Protocorm Multiplication medium, this Medium Proportion is: 1/2MS minimal medium (is got 1/2 of macroelement, other trace element remains unchanged, and this stage medium is liquid nutrient medium), Coconut Juice 50g/L, pH value 5.4.This stage condition of culture is: intensity of illumination 2000Lux, light application time 12h/d, and shaking speed is 120r/min, switching in every 21 days is once.
Five, the differentiation of protocorm
Gained protocorm in step 4 is transferred in differential medium, and this Medium Proportion is: 1/2MS minimal medium (get 1/2 of macroelement, other trace element remains unchanged), NAA0.2mg/L, potato extract 80-100g/L, pH value 5.4.Condition of culture is: intensity of illumination is 2000-3000Lux, and light application time is 12h/d, and light source is fluorescent lamp, and temperature is 26 ± 2 DEG C.Approximately, after 2.5 months, about 75% protocorm differentiation, obtains dendrobium candidum differentiation seedling.
Six, strengthening seedling and rooting and hardening
Gained differentiation seedling in step 5 is inoculated in strengthening seedling and rooting medium, and this Medium Proportion is: 1/2MS minimal medium (get 1/2 of macroelement, other trace element remains unchanged), banana extract 100-150g/L, pH value 5.4.Dendrobium candidum differentiation seedling is in this medium, and under fluorescent lamp condition, intensity of illumination is 2000-3000Lux, light application time is 12h/d, and light source is fluorescent lamp, and temperature is 26 ± 2 DEG C, cultivate after 30 days, can be placed in culturing rack top layer, accept solar radiation, intensity of illumination 8000Lux-10000Lux, light application time is 12h/d, and light source is daylight, and temperature is 26 ± 2 DEG C, cultivate after 60 days, can emerge.
The composition of MS medium is routinely:
The composition of B5 medium is routinely:
Dendrobium candidum method for quickly breeding of the present invention can utilize natural daylight to greatest extent, has not only saved electric cost, and can make dendrobium candidum bottle seedling leaf thickening, fully launches, and leaf look dark green, and cane is sturdy, well developed root system, and survival rate reaches more than 98%.
Claims (4)
1. a dendrobium candidum method for quickly breeding, is characterized in that, comprises the following steps:
One, choose explant, explant is cleaned, sterilized;
Two, explant is inoculated in to adventitious bud induction culture base and carries out evoking adventive bud cultivation, inducing culture proportioning is: MS minimal medium, 6-BA 2.0mg/L-3.0mg/L, NAA0.2mg/L, pH value 5.4;
Three, indefinite gained in step 2 leaf stem section is inoculated in protocorm inducing culture, this Medium Proportion is: B5 minimal medium, NAA2.0mg/L, 6-BA0.2mg/L, KT0.5mg/L-2.0mg/L, pH value 5.4;
Four, gained protocorm in step 3 to be transferred in Protocorm Multiplication medium, this Medium Proportion is: 1/2MS minimal medium, Coconut Juice 50g/L, pH value 5.4; Condition of culture is: intensity of illumination 2000Lux, light application time 12h/d, and shaking speed is 120r/min, switching in every 21 days is once;
Five, gained protocorm in step 4 to be transferred in differential medium, this Medium Proportion is: 1/2 MS minimal medium, NAA0.2mg/L, potato extract 80-100g/L, pH value 5.4;
Six, gained differentiation seedling in step 5 is inoculated in strengthening seedling and rooting medium, this Medium Proportion is: 1/2MS minimal medium, banana extract 100-150g/L, pH value 5.4; Be placed under fluorescent lamp and cultivate after 30 days, be placed in culturing rack top layer, intensity of illumination 8000 Lux-10000 Lux, light application time is 12h/d, and light source is daylight, and temperature is 26 ± 2 DEG C, cultivates and emerges after 60 days;
Step 2, three and five condition of culture are all: intensity of illumination 2000-3000Lux, and light application time 12h/d, light source is fluorescent lamp, temperature is 26 ± 2 DEG C.
2. a kind of dendrobium candidum method for quickly breeding as claimed in claim 1, is characterized in that, in described step 1, cleaning, sterilisation step are: first use non-phosphide detergent aqueous solution soaking 15 minutes, then rinse 30 minutes with running water; With alcohol-pickled 15 seconds of 75%, aseptic water washing 1 time, then use 0.1%HgCl
2solution disinfection 8 minutes, aseptic water washing 5 times.
3. a kind of dendrobium candidum method for quickly breeding as claimed in claim 1, is characterized in that, in described step 1, chooses epidermis rust, stipes is obvious, entire body is transparent, fiber is few individuality as explant.
4. a kind of dendrobium candidum method for quickly breeding as claimed in claim 1, is characterized in that, in described step 1, after explant cleans, sterilizes, suck dry moisture is cut to the stem section of 1.0-1.5cm, and access inducing culture is induced cultivation.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104303765A (en) * | 2014-10-10 | 2015-01-28 | 江苏神草生物科技有限公司 | High-yield planting method for noble dendrobium |
| CN104322374A (en) * | 2014-11-12 | 2015-02-04 | 柳州市天姿园艺有限公司 | Culture medium solution for tissue culture seedling of dendrobium officinale for propagating by using pedicel |
| CN104472350A (en) * | 2014-11-12 | 2015-04-01 | 柳州市天姿园艺有限公司 | Dendrobium officinale kimura et migo tissue culture seedling growing composite culture medium fluid for breeding by using leaf tip as explant |
| CN104542277A (en) * | 2014-12-19 | 2015-04-29 | 广西壮族自治区农业科学院生物技术研究所 | Rapid propagation method for sagittaria sagittifolia tissue culture seedling and culture medium |
| CN105145351A (en) * | 2015-07-30 | 2015-12-16 | 长沙绿天生物技术有限公司 | Dendrobium officinale Kimura et Migo tissue culture batch production method through one-step seedling formation |
| CN105613291A (en) * | 2015-12-31 | 2016-06-01 | 厦门涌泉科技有限公司 | Fast breeding method for dendrobium candidum protocorm tissue culture |
| CN105886452A (en) * | 2014-12-25 | 2016-08-24 | 廉美兰 | Method for improving active substances in protocorm of dendrobium officinale kimura et migo by virtue of abiotic elicitors |
| CN106342687A (en) * | 2016-08-30 | 2017-01-25 | 柳州市泓吉农业科技有限公司 | A rapid propagation method of Dendrobium candidum |
| CN112088760A (en) * | 2020-07-28 | 2020-12-18 | 灵山县山霖生物技术有限公司 | Seedling growing method for dendrobium officinale |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104303765A (en) * | 2014-10-10 | 2015-01-28 | 江苏神草生物科技有限公司 | High-yield planting method for noble dendrobium |
| CN104303765B (en) * | 2014-10-10 | 2017-05-31 | 泉州惠安长圣生物科技有限公司 | The high-yield planting method of the stem of noble dendrobium |
| CN104322374A (en) * | 2014-11-12 | 2015-02-04 | 柳州市天姿园艺有限公司 | Culture medium solution for tissue culture seedling of dendrobium officinale for propagating by using pedicel |
| CN104472350A (en) * | 2014-11-12 | 2015-04-01 | 柳州市天姿园艺有限公司 | Dendrobium officinale kimura et migo tissue culture seedling growing composite culture medium fluid for breeding by using leaf tip as explant |
| CN104542277A (en) * | 2014-12-19 | 2015-04-29 | 广西壮族自治区农业科学院生物技术研究所 | Rapid propagation method for sagittaria sagittifolia tissue culture seedling and culture medium |
| CN105886452A (en) * | 2014-12-25 | 2016-08-24 | 廉美兰 | Method for improving active substances in protocorm of dendrobium officinale kimura et migo by virtue of abiotic elicitors |
| CN105145351A (en) * | 2015-07-30 | 2015-12-16 | 长沙绿天生物技术有限公司 | Dendrobium officinale Kimura et Migo tissue culture batch production method through one-step seedling formation |
| CN105145351B (en) * | 2015-07-30 | 2017-06-13 | 长沙绿天生物技术有限公司 | The method of candidum tissue culturing forming seedling through one step culture mass production |
| CN105613291A (en) * | 2015-12-31 | 2016-06-01 | 厦门涌泉科技有限公司 | Fast breeding method for dendrobium candidum protocorm tissue culture |
| CN106342687A (en) * | 2016-08-30 | 2017-01-25 | 柳州市泓吉农业科技有限公司 | A rapid propagation method of Dendrobium candidum |
| CN112088760A (en) * | 2020-07-28 | 2020-12-18 | 灵山县山霖生物技术有限公司 | Seedling growing method for dendrobium officinale |
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Application publication date: 20140730 |