CN104351056A - Method for promoting rapid propagation of dendrobium superbum - Google Patents
Method for promoting rapid propagation of dendrobium superbum Download PDFInfo
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- CN104351056A CN104351056A CN201410676585.9A CN201410676585A CN104351056A CN 104351056 A CN104351056 A CN 104351056A CN 201410676585 A CN201410676585 A CN 201410676585A CN 104351056 A CN104351056 A CN 104351056A
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Abstract
The invention establishes a tissue culture and rapid propagation system of dendrobium superbum seeds. The dendrobium superbum seeds are taken as external materials, and complete plants can be obtained sequentially by inducted germination, subculture of cluster buds and rooting culture. The method is easy to operate, low in production cost and free from environmental pollution, and is capable of realizing large-scale production. The dendrobium superbum seedlings cultured by the invention are stable in genetic characters, and have the advantages of being constant, less in input, high in yield and short in period.
Description
Technical field
The present invention relates to medicinal plant field of planting, particularly relate to a kind of method promoting santal Rapid Propagation of Dendrobium.
Background technology
The santal stem of noble dendrobium (Den parishii X Den.superbum), being hybridized by the Moschus stem of noble dendrobium and Vietnam 18 rod to form, is one of spring stem of noble dendrobium that popularity is very high, stem branch uprightly or slightly bending, blade be oblong, winter fallen leaves property.Petal is extracted out by compared with the eustipes part of top without leaf, and often joint blossoms 1 ~ 5 usually; Pattern is purplish red, petal quality is thick, have dense sandalwood fragrance, and the florescence is between 3 ~ May.Have good sight, economic worth is higher.
Along with the development of society, the people are to the pursuit of U.S. and the pursuit to high-end flowers, and the spring stem of noble dendrobium market demand potential is large, and had now external import to make the transition domestic production, restrict by output, price remains high always.The spring stem of noble dendrobium, as the cymbidium variety of up-and-coming youngster, is regarded as following most promising " star of cattleya " by potted flower manufacturing enterprise.
The tissue culture method of the santal stem of noble dendrobium of existing market sale is directly cultivated from the tender shoots of the santal stem of noble dendrobium, turns out the santal stem of noble dendrobium by subculture mode.But the training of tender shoots group need through repeatedly subculture, normally form large-scale production and need subculture more than 15-20 time, deficiency is: subculture repeatedly after just there will be variation, the situations such as vitrifying, the decline of kind matter, have a strong impact on follow-up development; Growth cycle overlong time, a Subculture Time is 40-60 days, through the production scale that repeatedly subculture can be formed, extends the growth time of the santal stem of noble dendrobium, adds cost virtually.
The present invention adopts modern biotechnology, takes seed culture pattern, large-scale production seedling, obtains the neat santal stem of noble dendrobium seedling of high-quality, solves the seedling problem of santal stem of noble dendrobium artificial planting, for the development of the santal stem of noble dendrobium contributes.
Summary of the invention
Slow in order to solve the santal stem of noble dendrobium growth existed in background technology, the problems such as subculture repeatedly can make a variation, vitrifying, the decline of kind matter, the invention provides a kind of method promoting santal Rapid Propagation of Dendrobium, its technical scheme is as follows:
1) sprout-induction: before the jaundice of santal stem of noble dendrobium seed is not ftractureed, get outstanding achievement, clean up under flowing water, be placed in the alcohol solution dipping 30-40 second that concentration is 75-80%, then being placed in concentration is that the mercuric chloride solution of 1-1.2% is sterilized 15-20 minute, sterile water wash 5-6 time, in sterilization tray, utilize scalpel to cut outstanding achievement, take out seed, be seeded in inducing culture, wherein inducing culture is that to be prepared from 1/2MS according to following material rate be that minimal medium adds methyl α-naphthyl acetate (NAA) 0.2-2.0mg/L, sugar 25-30g/L, agar 3-6g/L, Tea Polyphenols 5-30.0mg/L, banana 2%-20%, coconut milk 5%-30%, pH value is 5.6, cultivation temperature is 29-32 DEG C, intensity of illumination is 2100-2500lux, light application time is 14-16 hour/day, cultivate and obtain santal stem of noble dendrobium budlet after 16-20 days,
2) squamous subculture: by step 1) a large amount of santal stem of noble dendrobium budlet of obtaining is placed in subculture medium, wherein subculture medium is prepared from according to following material rate, 1/2MS minimal medium adds (6-benzyl aminopurine (6-BA) 0.5-4.0mg/L, heteroauxin (IAA) 0.5-2.0mg/L, sugar 25-30g/L, agar 3-6g/L, Tea Polyphenols 5-30.0mg/L, banana 2%-20%, coconut milk 5%-30%, pH value is 5.6, cultivation temperature is 29-32 DEG C, incubation time is 14-16 hour/day, intensity of illumination is 2100-25001ux, the santal dendrobe tissue culture differentiation seedling of about 2cm height within about 62-70 days, can be obtained,
3) culture of rootage of santal dendrobe tissue culture differentiation seedling: by step 2) in a large amount of santal stem of noble dendrobium differentiation transplantation of seedlings of obtaining to root media, wherein root media is prepared from according to following material rate, 1/2MS is that minimal medium adds banana 5%-20%, IBA 0.1-2.0, sugar 2-10%, Tea Polyphenols 5-30.0mg/L, banana 2%-20%, coconut milk 5%-30%, active carbon 0.2-0.4%, pH value 5.6 cultivation temperature is 28 DEG C, intensity of illumination 2100-25001ux, light application time is after cultivating 62-70 days under the condition of 14-16 hour/day, cultivate under plantlet in vitro is moved on to natural daylight, the santal stem of noble dendrobium that can obtain having 3-4 bar root after 25-28 days is taken root plantlet in vitro,
4) hardening and transplanting: wash step 3) in the santal stem of noble dendrobium that obtains to take root the medium of plantlet in vitro root, dry steam, the whole plant of plantlet in vitro band root of being taken root by the santal stem of noble dendrobium is transplanted on the seedbed of carrying substrates, after 1-2 month, the growth of santal stem of noble dendrobium seedling is stable, grow new root and bud just can be transplanted in planting greenhouse, will suitably shelter from heat or light during greenhouse cultivation, intensity of illumination is 800-1000lux, humid control is at 50-60%, and temperature controls at 20-25 DEG C.
Preferably, the method of described promotion santal Rapid Propagation of Dendrobium, it is characterized in that, step 1) in inducing culture be: 1/2MS is that minimal medium adds methyl α-naphthyl acetate (NAA) 1-2.0mg/L, sugar 30g/L, agar 4g/L, Tea Polyphenols 25mg/L, banana 2%, coconut milk 5%.
Preferably, the method of described promotion santal Rapid Propagation of Dendrobium, it is characterized in that, step 2) in subculture medium be: 1/2MS minimal medium adds (6-benzyl aminopurine (6-BA) 1.5-4.0mg/L, heteroauxin (IAA) 1.5-2.0mg/L, sugared 30g/L, agar 3-6g/L, Tea Polyphenols 15-30.0mg/L, banana 10-20%, coconut milk 25-30%.
Preferably, the method of described promotion santal Rapid Propagation of Dendrobium, it is characterized in that, step 3) in root media be: 1/2MS is that minimal medium adds banana 15%-20%, IBA 1.0-2.0, sugared 2-5%, Tea Polyphenols 15mg/L, banana 4%, coconut milk 15%, active carbon 0.2-0.4%.
Beneficial effect of the present invention:
1, the starting material of group training, tender shoots and seed can, santal stem of noble dendrobium tender shoots is the portion of material that plant is from the outside got, because a strain stem of noble dendrobium only has a bud, also several bud can only be obtained by stem section, each bud adopt after sterilization, the sterilizable material finally obtained probably only has about 40%, then sterilizable material is utilized could to continue propagation, induction protocorm, expand numerous, will through the very long time, generally need could obtain abundant material through 10-20 generation, but group training process relaying counts longer from generation to generation, cost raises, vitrifying variation strengthens, therefore material now has not had very large value, along with the increase of subculture.Material degeneration is also very serious problem.
2, stem of noble dendrobium seed advantage is, amount is large, the stem of noble dendrobium seed containing about 10-100 about ten thousand quantity in each stem of noble dendrobium outstanding achievement, and the medium percentage of seedgermination through screening can reach about more than 95%, and therefore seed just can accomplish the seedling of hundreds of thousands.And tender shoots needs just can accomplish that this is measured through 15-20 for the time of left and right, and quality can't ensure.
3, seed sprouting is significantly shorter than tender shoots subculture to required time of finally emerging the time, it is almost the odd of tender shoots, this has saved fund to a great extent, save the energy, improve competitiveness, utilize needed for seed and only within more than 200 day, can emerge, and utilize tender shoots required time of accomplishing to emerge will be more than 1000 day, therefore consider also to be far superior to tender shoots induction seedling from market angle.
Tender shoots is as group training material: the material collected needed for enough materials is also very large, and the stem of noble dendrobium cost of institute's loss is also very high, and seed almost has no effect to plant.Therefore
Adopt seed group culture method first cost low, second promotes easily, and the 3rd, good economy performance, advanced technology, the 4th: market value is high, largely can reduce costs, interest concessions plantation, be conducive to expanding and promote, new thinking is provided to the protection of Dendrobium Plants.
4, medium is preferred, coordinate variable concentrations hormone to add different organic matters and Tea Polyphenols again by minimal medium form and showing the medium used, the Tea Polyphenols wherein added acts on somatic cells, the integrality of its cell wall of energy progressive failure, alkaline phosphatase is oozed out, then membrane passage is made to strengthen, cause metal ion, the seepage of protein makes cell generation get muddled, destroy cell structure gradually, thus play antibacterial effect, quote the adding of banana, coconut milk and naturally meet thing, short callus, promotes that seedling is grown up strong and sturdy.On the basis of repeatedly attempting, what screen according to certain principle has fine induction to the santal stem of noble dendrobium, can in the good characteristic situation keeping former plant, and in not variation situation, appreciation rate is high, grows fast excellent culture medium.
Embodiment
Embodiment 1
1) Fiber differentiation: before the jaundice of santal stem of noble dendrobium seed is not ftractureed, get outstanding achievement, clean up under flowing water, utilize 75% alcohol immersion 30 seconds, concentration be 1% mercuric chloride sterilize 15 minutes, sterile water wash 5 times, in sterilization tray, utilize scalpel to cut outstanding achievement, take out seed, be seeded in suitable medium, cultivation temperature is 29 DEG C, intensity of illumination is 2100lux, light application time is 14 hours/day, cultivate 16 days afterwards visible santal stem of noble dendrobium budlet send, wherein medium is: 1/2MS is that minimal medium adds methyl α-naphthyl acetate (NAA) 2.0mg/L, sugar 30g/L, agar 4.0g/L, banana 25g, coconut milk 25g, Tea Polyphenols 15g, pH value is 5.6,
2) squamous subculture: by step 1) to be placed in 1/2MS be that minimal medium adds 6-benzyl aminopurine (6-BA) 0.5mg/L for a large amount of santal stem of noble dendrobium budlet of obtaining, heteroauxin (IAA) 2.0mg/L, sugar 30g/L, agar 4.0g/L, banana 25g, coconut milk 25g, Tea Polyphenols 15g, pH value is cultivate in the medium of 5.6, and cultivation temperature is 29 DEG C, and incubation time is 14 hours/day, intensity of illumination is 2100lux, within about 44 days, can obtain the neat santal dendrobe tissue culture differentiation seedling of about 2cm height;
3) santal stem of noble dendrobium differentiation seedling rooting is cultivated, by step 2) a large amount of santal stem of noble dendrobium differentiation transplantation of seedlings of obtaining are to root media, cultivation temperature is 29 DEG C, intensity of illumination 2500lux, light application time is cultivate after 40 days under the condition of 14 hours/day, cultivates under plantlet in vitro is moved on to natural daylight, the santal stem of noble dendrobium that can obtain having 4 roots after 50 days is taken root plantlet in vitro, wherein root media is: 1/2MS is minimal medium 4%, Tea Polyphenols 3.5%, banana 5%, coconut milk 5%, growth hormone IBA 0.1%, sugar 2%, active carbon 0.2%,, pH value is 5.6;
4) hardening and transplanting: wash step 3) in the santal stem of noble dendrobium that obtains to take root the medium of plantlet in vitro root, dry steam, the whole plant of plantlet in vitro band root of being taken root by the santal stem of noble dendrobium is transplanted on the seedbed of carrying substrates, after 2 months, the growth of santal stem of noble dendrobium seedling is stable, grow new root and bud just can be transplanted in planting greenhouse, will suitably shelter from heat or light during greenhouse cultivation, intensity of illumination is 800lux, humid control is 60%, and temperature controls at 20 DEG C.
Embodiment 2
1) Fiber differentiation: before the jaundice of santal stem of noble dendrobium seed is not ftractureed, get outstanding achievement, clean up under flowing water, utilize 80% alcohol immersion 40 seconds, concentration be 1.2% mercuric chloride sterilize 15 minutes, sterile water wash 6 times, in sterilization tray, utilize scalpel to cut outstanding achievement, take out seed, be seeded in suitable medium, cultivation temperature is 30 DEG C, intensity of illumination is 2200lux, light application time is 16 hours/day, cultivate 20 days afterwards visible santal stem of noble dendrobium budlet send, wherein medium is: 1/2MS is that minimal medium adds methyl α-naphthyl acetate (NAA) 1.Smg/L, sugar 28g/L, agar 3.8g/L, coconut milk 25g, Tea Polyphenols 15g, pH value is 5.6,
2) squamous subculture: by step 1) to be placed in 1/2MS be that minimal medium adds 6-benzyl aminopurine (6-BA) 4.0mg/L for a large amount of santal stem of noble dendrobium budlet of obtaining, heteroauxin (IAA) 2.0mg/L, sugar 30g/L, agar 4.0g/L, coconut milk 25g, Tea Polyphenols 15g, pH value is cultivate in the medium of 5.6, cultivation temperature is 30 DEG C, incubation time is 16 hours/day, intensity of illumination is 2200lux, within about 70 days, can obtain the neat santal dendrobe tissue culture differentiation seedling of about 2cm height;
3) santal stem of noble dendrobium differentiation seedling rooting is cultivated, by step 2) a large amount of santal stem of noble dendrobium differentiation transplantation of seedlings of obtaining are to root media, cultivation temperature is 30 DEG C, intensity of illumination 2500lux, light application time is cultivate after 35 days under the condition of 16 hours/day, cultivate under plantlet in vitro is moved on to natural daylight, the santal stem of noble dendrobium that can obtain having 4 roots after 65 days is taken root plantlet in vitro, and wherein root media is: 1/2MS is that minimal medium adds coconut milk 20%, growth hormone IBA 2.0%, sugar 2%, active carbon 0.2%, Tea Polyphenols 15g, pH value is 5.6;
4) hardening and transplanting: wash step 3) in the santal stem of noble dendrobium that obtains to take root the medium of plantlet in vitro root, dry steam, the whole plant of plantlet in vitro band root of being taken root by the santal stem of noble dendrobium is transplanted on the seedbed of carrying substrates, after 2 months, the growth of santal stem of noble dendrobium seedling is stable, grow new root and bud just can be transplanted in planting greenhouse, will suitably shelter from heat or light during greenhouse cultivation, intensity of illumination is 800lux, humid control is 60%, and temperature controls at 20 DEG C.
Embodiment 3
1) Fiber differentiation: before the jaundice of santal stem of noble dendrobium seed is not ftractureed, get outstanding achievement, clean up under flowing water, utilize 75% alcohol immersion 35 seconds, concentration be 1.2% mercuric chloride sterilize 15 minutes, sterile water wash 6 times, in sterilization tray, utilize scalpel to cut outstanding achievement, take out seed, be seeded in suitable medium, cultivation temperature is 32 DEG C, intensity of illumination is 2300lux, light application time is 16 hours/day, cultivate 20 days afterwards visible santal stem of noble dendrobium budlet send, wherein medium is: 1/2MS is that minimal medium adds methyl α-naphthyl acetate (NAA) 1.5mg/L, , sugar 28g/L, agar 3.8g/L, banana 25g, Tea Polyphenols 15g, pH value is 5.6,
2) squamous subculture: by step 1) to be placed in 1/2MS be that minimal medium adds 6-benzyl aminopurine (6-BA) 4.0mg/L for a large amount of santal stem of noble dendrobium budlet of obtaining, heteroauxin (IAA) 2.0mg/L, sugar 30g/L, agar 4.0g/L, banana 25g, Tea Polyphenols 15g, pH value is cultivate in the medium of 5.6, cultivation temperature is 30 DEG C, incubation time is 16 hours/day, and intensity of illumination is 2200lux, within about 55 days, can obtain the neat santal dendrobe tissue culture differentiation seedling of about 2cm height;
3) santal stem of noble dendrobium differentiation seedling rooting is cultivated, by step 2) a large amount of santal stem of noble dendrobium differentiation transplantation of seedlings of obtaining are to root media, cultivation temperature is 30 DEG C, intensity of illumination 2500lux, light application time is cultivate after 35 days under the condition of 16 hours/day, cultivates under plantlet in vitro is moved on to natural daylight, and the santal stem of noble dendrobium that can obtain having 4 roots after 60 days is taken root plantlet in vitro, wherein root media is: 1/2MS is minimal medium 4%, banana 20%, Tea Polyphenols 12%, growth hormone IBA2.0%, sugar 2%, active carbon 0.2%, pH value is 5.6;
4) hardening and transplanting: wash step 3) in the santal stem of noble dendrobium that obtains to take root the medium of plantlet in vitro root, dry steam, the whole plant of plantlet in vitro band root of being taken root by the santal stem of noble dendrobium is transplanted on the seedbed of carrying substrates, after 2 months, the growth of santal stem of noble dendrobium seedling is stable, grow new root and bud just can be transplanted in planting greenhouse, will suitably shelter from heat or light during greenhouse cultivation, intensity of illumination is 800lux, humid control is 60%, and temperature controls at 20 DEG C.
Embodiment 4
1) Fiber differentiation: before the jaundice of santal stem of noble dendrobium seed is not ftractureed, get outstanding achievement, clean up under flowing water, utilize 75% alcohol immersion 35 seconds, concentration be 1.2% mercuric chloride sterilize 15 minutes, sterile water wash 6 times, in sterilization tray, utilize scalpel to cut outstanding achievement, take out seed, be seeded in suitable medium, cultivation temperature is 32 DEG C, intensity of illumination is 2300lux, light application time is 16 hours/day, cultivate 20 days afterwards visible santal stem of noble dendrobium budlet send, wherein medium is: 1/2MS is that minimal medium adds methyl α-naphthyl acetate (NAA) 1.5mg/L, , sugar 28g/L, agar 3.8g/L, banana 25g, coconut milk 25g, pH value is 5.6,
2) squamous subculture: by step 1) to be placed in 1/2MS be that minimal medium adds 6-benzyl aminopurine (6-BA) 4.0mg/L for a large amount of santal stem of noble dendrobium budlet of obtaining, heteroauxin (IAA) 2.0mg/L, sugar 30g/L, agar 4.0g/L, banana 25g, coconut milk 25g, pH value is cultivate in the medium of 5.6, cultivation temperature is 30 DEG C, incubation time is 16 hours/day, and intensity of illumination is 2200lux, within about 60 days, can obtain the neat santal dendrobe tissue culture differentiation seedling of about 2cm height;
3) santal stem of noble dendrobium differentiation seedling rooting is cultivated, by step 2) a large amount of santal stem of noble dendrobium differentiation transplantation of seedlings of obtaining are to root media, cultivation temperature is 30 DEG C, intensity of illumination 2500lux, light application time is cultivate after 35 days under the condition of 16 hours/day, cultivates under plantlet in vitro is moved on to natural daylight, the santal stem of noble dendrobium that can obtain having 4 roots after 45 days is taken root plantlet in vitro, wherein root media is: 1/2MS is minimal medium 4%, banana 20%, coconut milk 20%, growth hormone IBA2.0%, sugar 2%, active carbon 0.2%,, pH value is 5.6;
4) hardening and transplanting: wash step 3) in the santal stem of noble dendrobium that obtains to take root the medium of plantlet in vitro root, dry steam, the whole plant of plantlet in vitro band root of being taken root by the santal stem of noble dendrobium is transplanted on the seedbed of carrying substrates, after 2 months, the growth of santal stem of noble dendrobium seedling is stable, grow new root and bud just can be transplanted in planting greenhouse, will suitably shelter from heat or light during greenhouse cultivation, intensity of illumination is 800lux, humid control is 60%, and temperature controls at 20 DEG C.
Embodiment 5
1) Fiber differentiation: before the jaundice of santal stem of noble dendrobium seed is not ftractureed, get outstanding achievement, clean up under flowing water, utilize 75% alcohol immersion 35 seconds, concentration be 1.2% mercuric chloride sterilize 15 minutes, sterile water wash 6 times, in sterilization tray, utilize scalpel to cut outstanding achievement, take out seed, be seeded in suitable medium, cultivation temperature is 32 DEG C, intensity of illumination is 2300lux, light application time is 16 hours/day, cultivate 30 days afterwards visible santal stem of noble dendrobium budlet send, wherein medium is: 1/2MS is that minimal medium adds methyl α-naphthyl acetate (NAA) 1.5mg/L, , sugar 28g/L, agar 3.8g/L, pH value is 5.6,
2) squamous subculture: by step 1) to be placed in 1/2MS be that minimal medium adds 6-benzyl aminopurine (6-BA) 4.0mg/L for a large amount of santal stem of noble dendrobium budlet of obtaining, heteroauxin (IAA) 2.0mg/L, sugar 30g/L, agar 4.0g/L, pH value is cultivate in the medium of 5.6, and cultivation temperature is 30 DEG C, and incubation time is 16 hours/day, intensity of illumination is 2200lux, within about 70 days, can obtain the neat santal dendrobe tissue culture differentiation seedling of about 2cm height;
3) santal stem of noble dendrobium differentiation seedling rooting is cultivated, by step 2) a large amount of santal stem of noble dendrobium differentiation transplantation of seedlings of obtaining are to root media, cultivation temperature is 30 DEG C, intensity of illumination 2500lux, light application time is cultivate after 45 days under the condition of 16 hours/day, cultivate under plantlet in vitro is moved on to natural daylight, the santal stem of noble dendrobium that can obtain having 4 roots after 65 days is taken root plantlet in vitro, and wherein root media is: 1/2MS is minimal medium 4%, growth hormone IBA 2.0%, sugar 2%, active carbon 0.2%, pH value is 5.6;
4) hardening and transplanting: wash step 3) in the santal stem of noble dendrobium that obtains to take root the medium of plantlet in vitro root, dry steam, the whole plant of plantlet in vitro band root of being taken root by the santal stem of noble dendrobium is transplanted on the seedbed of carrying substrates, after 2 months, the growth of santal stem of noble dendrobium seedling is stable, grow new root and bud just can be transplanted in planting greenhouse, will suitably shelter from heat or light during greenhouse cultivation, intensity of illumination is 800lux, humid control is 60%, and temperature controls at 20 DEG C.
Embodiment 6
1) Fiber differentiation: get santal stem of noble dendrobium tender shoots in 75% alcohol immersion 30 seconds, concentration be 1% mercuric chloride sterilize 15 minutes, sterile water wash 5 times, in sterilization tray, utilize scalpel to cut outstanding achievement, take out seed, be seeded in suitable medium, cultivation temperature is 29 DEG C, intensity of illumination is 2100lux, light application time is 14 hours/day, cultivate 40 days afterwards visible santal stem of noble dendrobium budlet send, wherein medium is: 1/2MS is that minimal medium adds methyl α-naphthyl acetate (NAA) 2.0mg/L, , sugar 30g/L, agar 4.0g/L, banana 25g, coconut milk 25g, Tea Polyphenols 15g, pH value is 5.6,
2) squamous subculture: by step 1) to be placed in 1/2MS be that minimal medium adds 6-benzyl aminopurine (6-BA) 0.5mg/L for a large amount of santal stem of noble dendrobium budlet of obtaining, heteroauxin (IAA) 2.0mg/L, sugar 30g/L, agar 4.0g/L, banana 25g, coconut milk 25g, Tea Polyphenols 15g, pH value is cultivate in the medium of 5.6, cultivation temperature is 29 DEG C, incubation time is 14 hours/day, and intensity of illumination is 2100lux, within about 70 days, can obtain santal dendrobe tissue culture differentiation seedling;
3) santal stem of noble dendrobium differentiation seedling rooting is cultivated, by step 2) a large amount of santal stem of noble dendrobium differentiation transplantation of seedlings of obtaining are to root media, cultivation temperature is 29 DEG C, intensity of illumination 2500lux, light application time is cultivate after 60 days under the condition of 14 hours/day, cultivate under plantlet in vitro is moved on to natural daylight, the santal stem of noble dendrobium that can obtain having 4 roots after 80 days is taken root plantlet in vitro, wherein root media is: 1/2MS is minimal medium 4%, wood chip 20%, Tea Polyphenols 3.5%, banana 5%, coconut milk 10%, growth hormone IBA 0.1%, sugar 2%, active carbon 0.2%, , pH value is 5.6,
4) hardening and transplanting: wash step 3) in the santal stem of noble dendrobium that obtains to take root the medium of plantlet in vitro root, dry steam, the whole plant of plantlet in vitro band root of being taken root by the santal stem of noble dendrobium is transplanted on the seedbed of carrying substrates, after 3 months, the growth of santal stem of noble dendrobium seedling is stable, grow new root and bud just can be transplanted in planting greenhouse, will suitably shelter from heat or light during greenhouse cultivation, intensity of illumination is 800lux, humid control is 60%, and temperature controls at 20 DEG C.
Embodiment 7
1) Fiber differentiation: get santal stem of noble dendrobium tender shoots in 75% alcohol immersion 30 seconds, concentration be 1% mercuric chloride sterilize 15 minutes, sterile water wash 5 times, in sterilization tray, utilize scalpel to cut outstanding achievement, take out seed, be seeded in suitable medium, cultivation temperature is 29 DEG C, intensity of illumination is 2100lux, light application time is 14 hours/day, cultivate 40 days afterwards visible santal stem of noble dendrobium budlet send, wherein medium is: 1/2MS is that minimal medium adds methyl α-naphthyl acetate (NAA) 2.0mg/L, sugar 30g/L, agar 4.0g/L, banana 25g, coconut milk 25g, Tea Polyphenols 15g, pH value is 5.6,
2) squamous subculture: by step 1) to be placed in 1/2MS be that minimal medium adds 6-benzyl aminopurine (6-BA) 0.5mg/L for a large amount of santal stem of noble dendrobium budlet of obtaining, heteroauxin (IAA) 2.0mg/L, sugar 30g/L, agar 4.0g/L, banana 25g, coconut milk 25g, Tea Polyphenols 15g, , pH value is cultivate in the medium of 5.6, cultivation temperature is 29 DEG C, incubation time is 14 hours/day, intensity of illumination is 2100lux, within 60 days, complete a squamous subculture, repeat subculture step 14 times, squamous subculture is to after 15 times, the santal stem of noble dendrobium obtained differentiation seedling is carried out culture of rootage,
3) santal stem of noble dendrobium differentiation seedling rooting is cultivated, by step 2) a large amount of santal stem of noble dendrobium differentiation transplantation of seedlings of obtaining are to root media, cultivation temperature is 29 DEG C, intensity of illumination 2500lux, light application time is cultivate after 40 days under the condition of 14 hours/day, cultivate under plantlet in vitro is moved on to natural daylight, the santal stem of noble dendrobium that can obtain having 4 roots after 40 days is taken root plantlet in vitro, wherein root media is: 1/2MS is minimal medium 4%, coconut milk 10%, Tea Polyphenols 3.5%, banana 5% growth hormone IBA 0.1%, sugar 2%, active carbon 0.2%, pH value is 5.6;
4) hardening and transplanting: wash step 3) in the santal stem of noble dendrobium that obtains to take root the medium of plantlet in vitro root, dry steam, the whole plant of plantlet in vitro band root of being taken root by the santal stem of noble dendrobium is transplanted on the seedbed of carrying substrates, after 2 months, the growth of santal stem of noble dendrobium seedling is stable, grow new root and bud just can be transplanted in planting greenhouse, will suitably shelter from heat or light during greenhouse cultivation, intensity of illumination is 800lux, humid control is 60%, and temperature controls at 20 DEG C.
The tissue culture method test data contrast of embodiment 1-embodiment 7
| Sequence number | Seedling-growing time/sky | Germination rate | Subculture planting percent | Rooting rate | Hardening survival rate |
| Embodiment 1 | 210 | 96％ | 95％ | 98％ | 99％ |
| Embodiment 2 | 250 | 86％ | 83％ | 82％ | 83％ |
| Embodiment 3 | 230 | 88％ | 86％ | 88％ | 86％ |
| Embodiment 4 | 220 | 90％ | 88％ | 90％ | 90％ |
| Embodiment 5 | 270 | 84％ | 82％ | 78％ | 82％ |
| Embodiment 6 | 340 | 42％ | 41％ | 40％ | 50％ |
| Embodiment 7 | 1080 | 80％ | 80％ | 78％ | 76％ |
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the embodiment described.
Claims (4)
1. promote a method for santal Rapid Propagation of Dendrobium, it is characterized in that, comprise the following steps:
1) sprout-induction: before the jaundice of santal stem of noble dendrobium seed is not ftractureed, get outstanding achievement, clean up under flowing water, be placed in the alcohol solution dipping 30-40 second that concentration is 75-80%, then being placed in concentration is that the mercuric chloride solution of 1-1.2% is sterilized 15-20 minute, sterile water wash 5-6 time, in sterilization tray, utilize scalpel to cut outstanding achievement, take out seed, be seeded in inducing culture, wherein inducing culture is that to be prepared from 1/2MS according to following material rate be that minimal medium adds methyl α-naphthyl acetate (NAA) 0.2-2.0mg/L, sugar 25-30g/L, agar 3-6g/L, Tea Polyphenols 5-30.0mg/L, banana 2%-20%, coconut milk 5%-30%, pH value is 5.6, cultivation temperature is 29-32 DEG C, intensity of illumination is 2100-2500lux, light application time is 14-16 hour/day, cultivate and obtain santal stem of noble dendrobium budlet after 16-20 days,
2) squamous subculture: by step 1) a large amount of santal stem of noble dendrobium budlet of obtaining is placed in subculture medium, wherein subculture medium is prepared from according to following material rate, 1/2MS minimal medium adds (6-benzyl aminopurine (6-BA) 0.5-4.0mg/L, heteroauxin (IAA) 0.5-2.0mg/L, sugar 25-30g/L, agar 3-6g/L, Tea Polyphenols 5-30.0mg/L, banana 2%-20%, coconut milk 5%-30%, pH value is 5.6, cultivation temperature is 29-32 DEG C, incubation time is 14-16 hour/day, intensity of illumination is 2100-2500lux, the santal dendrobe tissue culture differentiation seedling of about 2cm height within about 62-70 days, can be obtained,
3) culture of rootage of santal dendrobe tissue culture differentiation seedling: by step 2) in a large amount of santal stem of noble dendrobium differentiation transplantation of seedlings of obtaining to root media, wherein root media is prepared from according to following material rate, 1/2MS is that minimal medium adds banana 5%-20%, IBA 0.1-2.0, sugar 2-10%, Tea Polyphenols 5-30.0mg/L, banana 2%-20%, coconut milk 5%-30%, active carbon 0.2-0.4%, pH value 5.6 cultivation temperature is 28 DEG C, intensity of illumination 2100-2500lux, light application time is after cultivating 62-70 days under the condition of 14-16 hour/day, cultivate under plantlet in vitro is moved on to natural daylight, the santal stem of noble dendrobium that can obtain having 3-4 bar root after 25-28 days is taken root plantlet in vitro,
4) hardening and transplanting: wash step 3) in the santal stem of noble dendrobium that obtains to take root the medium of plantlet in vitro root, dry steam, the whole plant of plantlet in vitro band root of being taken root by the santal stem of noble dendrobium is transplanted on the seedbed of carrying substrates, after 1-2 month, the growth of santal stem of noble dendrobium seedling is stable, grow new root and bud just can be transplanted in planting greenhouse, will suitably shelter from heat or light during greenhouse cultivation, intensity of illumination is 800-1000lux, humid control is at 50-60%, and temperature controls at 20-25 DEG C.
2. the method for promotion santal Rapid Propagation of Dendrobium according to claim 1, it is characterized in that, step 1) in inducing culture be: 1/2MS is that minimal medium adds methyl α-naphthyl acetate (NAA) 1-2.0mg/L, sugar 30g/L, agar 4g/L, Tea Polyphenols 25mg/L, banana 2%, coconut milk 5%.
3. the method for promotion santal Rapid Propagation of Dendrobium according to claim 1, it is characterized in that, step 2) in subculture medium be: 1/2MS minimal medium adds (6-benzyl aminopurine (6-BA) 1.5-4.0mg/L, heteroauxin (IAA) 1.5-2.0mg/L, sugar 30g/L, agar 3-6g/L, Tea Polyphenols 15-30.0mg/L, banana 10-20%, coconut milk 25-30%.
4. the method for promotion santal Rapid Propagation of Dendrobium according to claim 1, it is characterized in that, step 3) in root media be: 1/2MS is that minimal medium adds banana 15%-20%, IBA1.0-2.0, sugar 2-5%, Tea Polyphenols 15mg/L, banana 4%, coconut milk 15%, active carbon 0.2-0.4%.
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| CN110050696A (en) * | 2019-04-28 | 2019-07-26 | 北京市辐射中心 | Ion beam mutagenesis obtains the methods and applications of the dendrobium candidum of high polysaccharide high yield |
| CN111972291A (en) * | 2020-09-03 | 2020-11-24 | 云南华农农业有限公司 | Artificial efficient clonal propagation method of dendrobium santalinum |
| CN116171855A (en) * | 2023-02-09 | 2023-05-30 | 云南山里红生物科技有限公司 | Dendrobium officinale tissue culture method and culture medium formula thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108934924A (en) * | 2018-05-30 | 2018-12-07 | 江苏圣福来生态农业有限公司 | A kind of dendrobium candidum axenic culture seed broadcasts seedling culture medium and its application |
| CN108934924B (en) * | 2018-05-30 | 2020-06-09 | 江苏圣福来生态农业有限公司 | Dendrobium officinale sterile culture seed seeding culture medium and application thereof |
| CN110050696A (en) * | 2019-04-28 | 2019-07-26 | 北京市辐射中心 | Ion beam mutagenesis obtains the methods and applications of the dendrobium candidum of high polysaccharide high yield |
| CN111972291A (en) * | 2020-09-03 | 2020-11-24 | 云南华农农业有限公司 | Artificial efficient clonal propagation method of dendrobium santalinum |
| CN116171855A (en) * | 2023-02-09 | 2023-05-30 | 云南山里红生物科技有限公司 | Dendrobium officinale tissue culture method and culture medium formula thereof |
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