CN108157173A - A kind of method that Moschus dendrobe tissue culture expands breeding seedling - Google Patents
A kind of method that Moschus dendrobe tissue culture expands breeding seedling Download PDFInfo
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- CN108157173A CN108157173A CN201711447158.3A CN201711447158A CN108157173A CN 108157173 A CN108157173 A CN 108157173A CN 201711447158 A CN201711447158 A CN 201711447158A CN 108157173 A CN108157173 A CN 108157173A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The step of the step of the step of the step of the step of the step of expanding the method for breeding seedling the invention discloses a kind of Moschus dendrobe tissue culture, being selected including explant, explant disinfection, sowing, squamous subculture, culture of rootage, hardening, rooting culture.The tissue cultures expanding propagation method of the application can effectively improve the speed of the breeding of Moschus stem of noble dendrobium seedling and breeding quantity, from the stability of maternal source control germplasm, ensure seedling quality, realize the factorial praluction of Moschus stem of noble dendrobium high quality seed seedling, meet production requirement, alleviate the present situation of scarcity of resources.
Description
Technical field
The present invention relates to the sides that a kind of tissue cultivating and seedling technology more particularly to a kind of Moschus dendrobe tissue culture expand breeding seedling
Method.
Background technology
The Moschus stem of noble dendrobium (Dendrobium parishii) is orchid family, and the Dendrobium original seed stem of noble dendrobium is mainly distributed on Vietnam, always
The countries such as Laos, Burma are distributed less within Chinese territory；The Moschus stem of noble dendrobium has denseer fragrance, spends purplish red, and petal quality is thicker, stem
Short and sturdy, the villus sense on lip is stronger, and bloom than the spring stem of noble dendrobium more a little later, has higher ornamental value.
The Moschus stem of noble dendrobium to circulate on domestic market there is no artificial mainly by from the national import such as Vietnam, Laos, Burma
Cultivation, maternal less, introduces a collection is rare；It is generally bred by way of division propagation and cuttage, breeding seedling is less, breeding
Rate is low, and growth cycle is longer, and the probability under normal habitat without artificial pollination result is smaller；Since fruit folder is few, fruit powder is thin
It is small, it is not easy the restriction of the factors such as germination and growth in a natural environment so that there is the Moschus stem of noble dendrobium seedling of high ornamental value
Breeding is obstructed.
Invention content
For overcome the deficiencies in the prior art, the purpose of the present invention is to provide a kind of Moschus dendrobe tissue cultures to expand breeding seedling
Method.The tissue culture expanding propagation method for culturing seedlings can effectively improve the speed of the breeding of Moschus stem of noble dendrobium seedling and breeding quantity, from mother
This source controls the stability of germplasm, ensures seedling quality, realizes the factorial praluction of Moschus stem of noble dendrobium high quality seed seedling, meets
Production requirement alleviates the present situation of scarcity of resources.
The purpose of the present invention adopts the following technical scheme that realization：A kind of method that Moschus dendrobe tissue culture expands breeding seedling, including,
The step of explant selects：Select the maternal plant of the Moschus stem of noble dendrobium 2-4 of health；It blooms in the spring Moschus stem of noble dendrobium
Period carries out artificial pollination to the stem of noble dendrobium, it is allowed to grow fruit pod, took pains to foster by 3-4 months, when fruit pod maturation, is picked
It puts to refrigerator and is preserved in the environment of 3-7 DEG C 2 weeks；
The step of explant sterilizes：Fruit pod is taken out, fruit pod liquid detergent or washing powder water are impregnated into 15-20min, use hairbrush
Outwash kind shell surface, and rinse 10-15min with tap water；Clean beaker is put it to, moves to transfer room, in an aseptic environment
It is impregnated with medicinal alcohol, being stirred continuously during immersion with tweezers makes its disinfection thorough, aseptic water washing 3-4 times；Hypochlorous acid is used again
Sodium sterilizes shell, aseptic water washing 3-4 times；It is finally sterilized with mercuric chloride, aseptic water washing 4-5 times is put on superclean bench
It is spare in sterile disk；
The step of sowing：The fruit folder scalpel disinfected and tweezers solution are splitted and expose yellow fruit powder, fruit powder is spread to
On 1/2MS culture mediums, lid is sealed after sterilizing bottle cap, goes in culturing room and is cultivated, 30-45 days seeds of culture start to sprout
Hair；
The step of squamous subculture：The amber seed of sprouting is 1500-2000lx, light application time 10-12 in intensity of illumination
It is grown 60 days under conditions of hour/day, Moschus seedling grows to 2-3cm high, level-one mother's bottle is formed, at this time by bottle seedling in gnotobasis
Under go in 1/2MS culture mediums and cultivate 60 days, it is long to referred to as two level mother bottle during 5-6cm；
The step of culture of rootage：The two level mother bottle for growing 60 days is gone into culture of rootage in MS culture mediums in an aseptic environment
90 days；
The step of hardening, rooting culture：Bottle seedling of taking root is cultivated 90 days in culturing room, is taken root good, seedling is sturdy, is moved into
It is placed 15-30 days in outdoor environment, it is allowed to adapt to external environment；It opens bottle cap to place 3-5 days, seedling is poured out into clean culture medium,
Moschus stem of noble dendrobium seedling root 15-20min is impregnated with fungicide, the moisture that dries in the shade is cleaned, is transplanted in matrix and is tamed.
Further, in the step of explant selects, the Moschus stem of noble dendrobium for selecting triennial is best for maternal plant；It is described
Kind shell yellow green other than during fruit pod maturation, subject to intact nothing is split.
Further, in the step of explant sterilizes, the volumetric concentration of the medicinal alcohol is 75%, and soaking time is
30-60s；The volumetric concentration of the sodium hypochlorite is 2%, disinfecting time 1-2min；The mass concentration of the mercuric chloride is
0.1%, disinfecting time 6-8min.
Further, in the step of sowing, the scalpel and tweezers are needed through 286 DEG C high disinfection and are cooled down.
Further, the methyl α-naphthyl acetate of 0.2mg/L, 20g/L are added in the step of sowing, in the 1/2MS culture mediums
The agar powder of sucrose, 4.8g/L, the pH of culture medium are adjusted to 6.0；The condition of culture of the culturing room is as follows：Temperature is 24 DEG C -26
DEG C, intensity of illumination 1500-2000lx, light application time is 10-12 hours/day.
Further, in the step of squamous subculture, in the 1/2MS culture mediums add 0.5mg/L methyl α-naphthyl acetate,
The sucrose of 6 benzyladenine 6-BA, 20g/L of 1.0mg/L, the agar powder of 4.8g/L, the murphy juice of 35g/L, 3g/L activity
Carbon dust, the pH of culture medium are adjusted to 6.0.
Further, in the step of culture of rootage, methyl α-naphthyl acetate, the 2.0mg/L of 0.5mg/L are added in the MS culture mediums
The sucrose of 6 benzyladenine 6-BA, 20g/L, the agar powder of 4.8g/L, the murphy juice of 35g/L, 25g/L bananas juice, 3g/L
Activated carbon powder, the pH of culture medium is adjusted to 6.0.
Further, in the step of hardening, rooting culture, the temperature of the outdoor environment is 22-28 DEG C, humidity is
45%-75%, and need to avoid strong light direct beam.
Further, in the step of hardening, rooting culture, the fungicide is carbendazim solution；By more bacterium during use
Spirit is diluted to 800-1000 times.
Compared with prior art, the beneficial effects of the present invention are：
(1) the tissue cultures expanding propagation method of the application can effectively improve the speed of the breeding of Moschus stem of noble dendrobium seedling and numerous
Quantity is educated, from the stability of maternal source control germplasm, ensures seedling quality, realizes the factory of Moschus stem of noble dendrobium high quality seed seedling
Metaplasia is produced, and is met production requirement, is alleviated the present situation of scarcity of resources.
(2) a step of most important link is seed disinfection during the application Moschus stem of noble dendrobium seed tissue culture propagation, disinfection
It thoroughly just can guarantee that seed sowing wild Oryza species do not pollute, in the case where the specified conditions such as temperature, illumination are suitble to, just can guarantee
The germination percentage of seed seedling-raising is more than 90%, ensures that kind is pure.
(3) it is added in culture medium certain all using 1/2MS culture mediums in the sowing of tissue cultures and squamous subculture link
The auxin of concentration, by being compared with MS culture mediums, on the germination and growth of seedling without influence, using 1/2MS culture mediums
Raw material can be saved, every bottle of culture medium can save the cost of 1.0-1.5 members in production.
(4) the hardening stage, bottle seedling is grown in culturing room can move it to that temperature is 22-28 DEG C, humidity is for 90 days or so
Hardening is carried out in the greenhouse of 45%-75% 30 days, the power consumption of culturing room's fluorescent lamp and air-conditioning can be saved in this way, can monthly be saved
The cost of about 1200-1500 members or so.
Specific embodiment
In the following, with reference to specific embodiment, the present invention is described further, it should be noted that is do not collided
Under the premise of, new embodiment can be formed between various embodiments described below or between each technical characteristic in any combination.
In the present invention, if not refering in particular to, all parts, percentage are unit of weight, used equipment and raw material etc.
It is commercially available or commonly used in the art.Method in following embodiments is the normal of this field unless otherwise instructed
Rule method.
A kind of method that Moschus dendrobe tissue culture expands breeding seedling, including,
The step of explant selects：Select the maternal plant of the Moschus stem of noble dendrobium 2-4 of health；It blooms in the spring Moschus stem of noble dendrobium
Period carries out artificial pollination to the stem of noble dendrobium, it is allowed to grow fruit pod, took pains to foster by 3-4 months, when fruit pod maturation, is picked
It puts to refrigerator and is preserved in the environment of 3-7 DEG C 2 weeks.Preferably, the Moschus stem of noble dendrobium for selecting triennial is best for maternal plant；Fruit pod
Kind shell yellow green other than when ripe, subject to intact nothing is split.
The step of explant sterilizes：Fruit pod is taken out, fruit pod liquid detergent or washing powder water are impregnated into 15-20min, use hairbrush
Outwash kind shell surface, and rinse 10-15min with tap water；Clean beaker is put it to, moves to transfer room, in an aseptic environment
It is impregnated with medicinal alcohol, being stirred continuously during immersion with tweezers makes its disinfection thorough, aseptic water washing 3-4 times；Hypochlorous acid is used again
Sodium sterilizes shell, aseptic water washing 3-4 times；It is finally sterilized with mercuric chloride, aseptic water washing 4-5 times is put on superclean bench
It is spare in sterile disk；Preferably, the volumetric concentration of medicinal alcohol is 75%, soaking time 30-60s；The volume of sodium hypochlorite
A concentration of 2%, disinfecting time 1-2min；The mass concentration of mercuric chloride is 0.1%, disinfecting time 6-8min.
The step of sowing：The fruit disinfected folder is splitted into dew with through the 286 DEG C high scalpel and tweezers solution for sterilizing and cooling down
Go out yellow fruit powder, fruit powder is spread on 1/2MS culture mediums, seal lid after sterilizing bottle cap, go in culturing room and cultivated, train
30-45 days seeds are supported to start to sprout；Preferably, added in 1/2MS culture mediums the methyl α-naphthyl acetate of 0.2mg/L, 20g/L sucrose,
The agar powder of 4.8g/L, the pH of culture medium are adjusted to 6.0；The condition of culture of culturing room is as follows：Temperature is 24 DEG C -26 DEG C, and illumination is strong
It spends for 1500-2000lx, light application time is 10-12 hours/day.
The step of squamous subculture：The amber seed of sprouting is 1500-2000lx, light application time 10-12 in intensity of illumination
It is grown 60 days under conditions of hour/day, Moschus seedling grows to 2-3cm high, level-one mother's bottle is formed, at this time by bottle seedling in gnotobasis
Under go in 1/2MS culture mediums and cultivate 60 days, it is long to referred to as two level mother bottle during 5-6cm；Preferably, it is added in 1/2MS culture mediums
The methyl α-naphthyl acetate of 0.5mg/L, the sucrose of 6 benzyladenine 6-BA, 20g/L of 1.0mg/L, the agar powder of 4.8g/L, 35g/L soil
The activated carbon powder of fermented bean drink, 3g/L, the pH of culture medium are adjusted to 6.0.
The step of culture of rootage：The two level mother bottle for growing 60 days is gone into culture of rootage in MS culture mediums in an aseptic environment
90 days；Preferably, the sugarcane of the methyl α-naphthyl acetate of 0.5mg/L, 6 benzyladenine 6-BA, 20g/L of 2.0mg/L are added in MS culture mediums
The activated carbon powder of sugar, the agar powder of 4.8g/L, the murphy juice of 35g/L, the bananas juice of 25g/L, 3g/L, the pH of culture medium are adjusted to
6.0。
The step of hardening, rooting culture：Bottle seedling of taking root is cultivated 90 days in culturing room, is taken root good, seedling is sturdy, is moved into
It is placed 15-30 days in outdoor environment, the temperature of outdoor environment is 22-28 DEG C, humidity 45%-75%, and needs to avoid strong light
Direct projection allows it to adapt to external environment；It opens bottle cap to place 3-5 days, seedling is poured out into clean culture medium, impregnate Moschus stone with fungicide
Dry measure used in former times seedling root 15-20min cleans the moisture that dries in the shade, is transplanted in matrix and is tamed.Fungicide is carbendazim solution；Make
Carbendazim is diluted to 800-1000 times by the used time.
Embodiment 1：
A kind of method that Moschus dendrobe tissue culture expands breeding seedling, including,
The step of explant selects：Select the maternal plant of the Moschus stem of noble dendrobium triennial of health；It blooms in the spring Moschus stem of noble dendrobium
Period carries out artificial pollination to the stem of noble dendrobium, it is allowed to grow fruit pod, was taken pains to foster by 3-4 months, when fruit pod maturation, plants shell in addition
Yellow green subject to intact nothing is split, is picked to put to refrigerator and is preserved in the environment of 3-7 DEG C 2 weeks.
The step of explant sterilizes：Fruit pod is taken out, fruit pod liquid detergent or washing powder water are impregnated into 15-20min, use hairbrush
Outwash kind shell surface, and rinse 10-15min with tap water；Clean beaker is put it to, moves to transfer room, in an aseptic environment
50s is impregnated for 75% medicinal alcohol with volumetric concentration, being stirred continuously during immersion with tweezers makes its disinfection thorough, aseptic water washing
3-4 times；Volumetric concentration is used again as 2% hypochlorite disinfectant shell 1.5min, aseptic water washing 3-4 times；Finally use mass concentration
7min is sterilized for 0.1% mercuric chloride, aseptic water washing 4-5 times is put into spare in the sterile disk on superclean bench.
The step of sowing：The fruit disinfected folder is splitted into dew with through the 286 DEG C high scalpel and tweezers solution for sterilizing and cooling down
Go out yellow fruit powder, fruit powder be spread on 1/2MS culture mediums, sterilize bottle cap after seal lid, go in culturing room temperature be 24
DEG C -26 DEG C, intensity of illumination 1500-2000lx, light application time is to be cultivated under conditions of 10-12 hour/day, culture 30-
Seed starts to sprout within 45 days；The agar powder of the methyl α-naphthyl acetate of 0.2mg/L, the sucrose of 20g/L, 4.8g/L are added in 1/2MS culture mediums,
The pH of culture medium is adjusted to 6.0.
The step of squamous subculture：The amber seed of sprouting is 1500-2000lx, light application time 10-12 in intensity of illumination
It is grown 60 days under conditions of hour/day, Moschus seedling grows to 2-3cm high, level-one mother's bottle is formed, at this time by bottle seedling in gnotobasis
Under go in 1/2MS culture mediums and cultivate 60 days, it is long to referred to as two level mother bottle during 5-6cm；0.5mg/L is added in 1/2MS culture mediums
Methyl α-naphthyl acetate, the sucrose of 6 benzyladenine 6-BA, 20g/L of 1.0mg/L, the agar powder of 4.8g/L, 35g/L murphy juice,
The activated carbon powder of 3g/L, the pH of culture medium are adjusted to 6.0.
The step of culture of rootage：The two level mother bottle for growing 60 days is gone into culture of rootage in MS culture mediums in an aseptic environment
90 days；Sucrose, the 4.8g/ of the methyl α-naphthyl acetate of 0.5mg/L, 6 benzyladenine 6-BA, 20g/L of 2.0mg/L are added in MS culture mediums
The agar powder of L, the murphy juice of 35g/L, the bananas juice of 25g/L, 3g/L activated carbon powder, the pH of culture medium is adjusted to 6.0.
The step of hardening, rooting culture：Bottle seedling of taking root is cultivated 90 days in culturing room, is taken root good, seedling is sturdy, is moved into
It is placed 15-30 days in outdoor environment, the temperature of outdoor environment is 22-28 DEG C, humidity 45%-75%, and needs to avoid strong light
Direct projection allows it to adapt to external environment；It opens bottle cap to place 3-5 days, seedling is poured out into clean culture medium, impregnate Moschus stone with fungicide
Dry measure used in former times seedling root 18min cleans the moisture that dries in the shade, is transplanted in matrix and is tamed.Fungicide is carbendazim solution；During use
Carbendazim is diluted to 900 times.
Embodiment 2：
A kind of method that Moschus dendrobe tissue culture expands breeding seedling, including,
The step of explant selects：Select 2 years raw maternal plants of the Moschus stem of noble dendrobium of health；It blooms in the spring Moschus stem of noble dendrobium
Period carries out artificial pollination to the stem of noble dendrobium, it is allowed to grow fruit pod, was taken pains to foster by 3-4 months, when fruit pod maturation, plants shell in addition
Yellow green subject to intact nothing is split, is picked to put to refrigerator and is preserved in the environment of 3-7 DEG C 2 weeks.
The step of explant sterilizes：Fruit pod is taken out, fruit pod liquid detergent or washing powder water are impregnated into 15-20min, use hairbrush
Outwash kind shell surface, and rinse 10-15min with tap water；Clean beaker is put it to, moves to transfer room, in an aseptic environment
30s is impregnated for 75% medicinal alcohol with volumetric concentration, being stirred continuously during immersion with tweezers makes its disinfection thorough, aseptic water washing
3-4 times；Volumetric concentration is used again as 2% hypochlorite disinfectant shell 1min, aseptic water washing 3-4 times；It is with mass concentration finally
0.1% mercuric chloride sterilizes 6min, and aseptic water washing 4-5 times is put into spare in the sterile disk on superclean bench.
The step of sowing：The fruit disinfected folder is splitted into dew with through the 286 DEG C high scalpel and tweezers solution for sterilizing and cooling down
Go out yellow fruit powder, fruit powder be spread on 1/2MS culture mediums, sterilize bottle cap after seal lid, go in culturing room temperature be 24
DEG C -26 DEG C, intensity of illumination 1500-2000lx, light application time is to be cultivated under conditions of 10-12 hour/day, culture 30-
Seed starts to sprout within 45 days；The agar powder of the methyl α-naphthyl acetate of 0.2mg/L, the sucrose of 20g/L, 4.8g/L are added in 1/2MS culture mediums,
The pH of culture medium is adjusted to 6.0.
The step of squamous subculture：The amber seed of sprouting is 1500-2000lx, light application time 10-12 in intensity of illumination
It is grown 60 days under conditions of hour/day, Moschus seedling grows to 2-3cm high, level-one mother's bottle is formed, at this time by bottle seedling in gnotobasis
Under go in 1/2MS culture mediums and cultivate 60 days, it is long to referred to as two level mother bottle during 5-6cm；0.5mg/L is added in 1/2MS culture mediums
Methyl α-naphthyl acetate, the sucrose of 6 benzyladenine 6-BA, 20g/L of 1.0mg/L, the agar powder of 4.8g/L, 35g/L murphy juice,
The activated carbon powder of 3g/L, the pH of culture medium are adjusted to 6.0.
The step of culture of rootage：The two level mother bottle for growing 60 days is gone into culture of rootage in MS culture mediums in an aseptic environment
90 days；Sucrose, the 4.8g/ of the methyl α-naphthyl acetate of 0.5mg/L, 6 benzyladenine 6-BA, 20g/L of 2.0mg/L are added in MS culture mediums
The agar powder of L, the murphy juice of 35g/L, the bananas juice of 25g/L, 3g/L activated carbon powder, the pH of culture medium is adjusted to 6.0.
The step of hardening, rooting culture：Bottle seedling of taking root is cultivated 90 days in culturing room, is taken root good, seedling is sturdy, is moved into
It is placed 15-30 days in outdoor environment, the temperature of outdoor environment is 22-28 DEG C, humidity 45%-75%, and needs to avoid strong light
Direct projection allows it to adapt to external environment；It opens bottle cap to place 3-5 days, seedling is poured out into clean culture medium, impregnate Moschus stone with fungicide
Dry measure used in former times seedling root 15min cleans the moisture that dries in the shade, is transplanted in matrix and is tamed.Fungicide is carbendazim solution；During use
Carbendazim is diluted to 800 times.
Embodiment 3：
A kind of method that Moschus dendrobe tissue culture expands breeding seedling, including,
The step of explant selects：Select 4 years raw maternal plants of the Moschus stem of noble dendrobium of health；It blooms in the spring Moschus stem of noble dendrobium
Period carries out artificial pollination to the stem of noble dendrobium, it is allowed to grow fruit pod, was taken pains to foster by 3-4 months, when fruit pod maturation, plants shell in addition
Yellow green subject to intact nothing is split, is picked to put to refrigerator and is preserved in the environment of 3-7 DEG C 2 weeks.
The step of explant sterilizes：Fruit pod is taken out, fruit pod liquid detergent or washing powder water are impregnated into 15-20min, use hairbrush
Outwash kind shell surface, and rinse 10-15min with tap water；Clean beaker is put it to, moves to transfer room, in an aseptic environment
60s is impregnated for 75% medicinal alcohol with volumetric concentration, being stirred continuously during immersion with tweezers makes its disinfection thorough, aseptic water washing
3-4 times；Volumetric concentration is used again as 2% hypochlorite disinfectant shell 2min, aseptic water washing 3-4 times；It is with mass concentration finally
0.1% mercuric chloride sterilizes 8min, and aseptic water washing 4-5 times is put into spare in the sterile disk on superclean bench.
The step of sowing：The fruit disinfected folder is splitted into dew with through the 286 DEG C high scalpel and tweezers solution for sterilizing and cooling down
Go out yellow fruit powder, fruit powder be spread on 1/2MS culture mediums, sterilize bottle cap after seal lid, go in culturing room temperature be 24
DEG C -26 DEG C, intensity of illumination 1500-2000lx, light application time is to be cultivated under conditions of 10-12 hour/day, culture 30-
Seed starts to sprout within 45 days；The agar powder of the methyl α-naphthyl acetate of 0.2mg/L, the sucrose of 20g/L, 4.8g/L are added in 1/2MS culture mediums,
The pH of culture medium is adjusted to 6.0.
The step of squamous subculture：The amber seed of sprouting is 1500-2000lux, light application time 10-12 in intensity of illumination
It is grown 60 days under conditions of hour/day, Moschus seedling grows to 2-3cm high, level-one mother's bottle is formed, at this time by bottle seedling in gnotobasis
Under go in 1/2MS culture mediums and cultivate 60 days, it is long to referred to as two level mother bottle during 5-6cm；0.5mg/L is added in 1/2MS culture mediums
Methyl α-naphthyl acetate, the sucrose of 6 benzyladenine 6-BA, 20g/L of 1.0mg/L, the agar powder of 4.8g/L, 35g/L murphy juice,
The activated carbon powder of 3g/L, the pH of culture medium are adjusted to 6.0.
The step of culture of rootage：The two level mother bottle for growing 60 days is gone into culture of rootage in MS culture mediums in an aseptic environment
90 days；Sucrose, the 4.8g/ of the methyl α-naphthyl acetate of 0.5mg/L, 6 benzyladenine 6-BA, 20g/L of 2.0mg/L are added in MS culture mediums
The agar powder of L, the murphy juice of 35g/L, the bananas juice of 25g/L, 3g/L activated carbon powder, the pH of culture medium is adjusted to 6.0.
The step of hardening, rooting culture：Bottle seedling of taking root is cultivated 90 days in culturing room, is taken root good, seedling is sturdy, is moved into
It is placed 15-30 days in outdoor environment, the temperature of outdoor environment is 22-28 DEG C, humidity 45%-75%, and needs to avoid strong light
Direct projection allows it to adapt to external environment；It opens bottle cap to place 3-5 days, seedling is poured out into clean culture medium, impregnate Moschus stone with fungicide
Dry measure used in former times seedling root 20min cleans the moisture that dries in the shade, is transplanted in matrix and is tamed.Fungicide is carbendazim solution；During use
Carbendazim is diluted to 1000 times.
The above embodiment is only the preferred embodiment of the present invention, it is impossible to the scope of protection of the invention is limited with this,
The variation and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed range.
Claims (9)
1. a kind of method that Moschus dendrobe tissue culture expands breeding seedling, it is characterised in that including,
The step of explant selects：Select the maternal plant of the Moschus stem of noble dendrobium 2-4 of health；In spring Moschus stem of noble dendrobium duration of flowering
Artificial pollination is carried out to the stem of noble dendrobium, it is allowed to grow fruit pod, was taken pains to foster by 3-4 months, when fruit pod maturation, picked put to
Refrigerator preserves 2 weeks in the environment of 3-7 DEG C；
The step of explant sterilizes：Fruit pod is taken out, fruit pod liquid detergent or washing powder water are impregnated into 15-20min, with hairbrush outwash
Kind shell surface, and rinse 10-15min with tap water；Clean beaker is put it to, moves to transfer room, in an aseptic environment with doctor
It is impregnated with alcohol, being stirred continuously during immersion with tweezers makes its disinfection thorough, aseptic water washing 3-4 times；Disappeared again with sodium hypochlorite
Malicious shell, aseptic water washing 3-4 times；It is finally sterilized, aseptic water washing 4-5 times, is put into sterile on superclean bench with mercuric chloride
It is spare in disk；
The step of sowing：The fruit folder scalpel disinfected and tweezers solution are splitted and expose yellow fruit powder, fruit powder is spread to 1/2MS
On culture medium, lid is sealed after sterilizing bottle cap, goes in culturing room and is cultivated, 30-45 days seeds of culture start to sprout；
The step of squamous subculture：The amber seed of sprouting intensity of illumination be 1500-2000lx, light application time for 10-12 hours/
It is grown 60 days under conditions of it, Moschus seedling grows to 2-3cm high, forms level-one mother's bottle, at this time turns bottle seedling in an aseptic environment
It is cultivated 60 days into 1/2MS culture mediums, it is long to referred to as two level mother bottle during 5-6cm；
The step of culture of rootage：The two level mother bottle for growing 60 days is gone into culture of rootage 90 in MS culture mediums in an aseptic environment
My god；
The step of hardening, rooting culture：Bottle seedling of taking root is cultivated 90 days in culturing room, is taken root good, seedling is sturdy, is moved into outdoor
It is placed 15-30 days in environment, it is allowed to adapt to external environment；It opens bottle cap to place 3-5 days, seedling is poured out into clean culture medium, with killing
Microbial inoculum impregnates Moschus stem of noble dendrobium seedling root 15-20min, cleans the moisture that dries in the shade, is transplanted in matrix and is tamed.
2. the method that Moschus dendrobe tissue culture as described in claim 1 expands breeding seedling, which is characterized in that in the step of explant selection
In rapid, the Moschus stem of noble dendrobium for selecting triennial is best for maternal plant；Kind shell yellow green, intact nothing are split other than during the fruit pod maturation
Subject to.
3. the method that Moschus dendrobe tissue culture as described in claim 1 expands breeding seedling, which is characterized in that in the step of explant disinfection
In rapid, the volumetric concentration of the medicinal alcohol is 75%, soaking time 30-60s；The volumetric concentration of the sodium hypochlorite is
2%, disinfecting time 1-2min；The mass concentration of the mercuric chloride is 0.1%, disinfecting time 6-8min.
4. the method that Moschus dendrobe tissue culture as described in claim 1 expands breeding seedling, which is characterized in that in the step of sowing,
The scalpel and tweezers are needed through 286 DEG C high disinfection and are cooled down.
5. the method that Moschus dendrobe tissue culture as described in claim 1 expands breeding seedling, which is characterized in that in the step of sowing,
The agar powder of the methyl α-naphthyl acetate of 0.2mg/L, the sucrose of 20g/L, 4.8g/L, the pH tune of culture medium are added in the 1/2MS culture mediums
To 6.0；The condition of culture of the culturing room is as follows：Temperature is 24 DEG C -26 DEG C, intensity of illumination 1500-2000lx, light application time
For 10-12 hours/day.
6. the method that Moschus dendrobe tissue culture as described in claim 1 expands breeding seedling, which is characterized in that the squamous subculture the step of
In, added in the 1/2MS culture mediums methyl α-naphthyl acetate of 0.5mg/L, 1.0mg/L 6 benzyladenine 6-BA, 20g/L sucrose,
The agar powder of 4.8g/L, the murphy juice of 35g/L, 3g/L activated carbon powder, the pH of culture medium is adjusted to 6.0.
7. the method that Moschus dendrobe tissue culture as described in claim 1 expands breeding seedling, which is characterized in that the culture of rootage the step of
In, added in the MS culture mediums methyl α-naphthyl acetate of 0.5mg/L, 2.0mg/L 6 benzyladenine 6-BA, 20g/L sucrose,
The agar powder of 4.8g/L, the murphy juice of 35g/L, the bananas juice of 25g/L, 3g/L activated carbon powder, the pH of culture medium is adjusted to 6.0.
8. the method that Moschus dendrobe tissue culture as described in claim 1 expands breeding seedling, which is characterized in that in hardening, rooting culture
The step of in, the temperature of the outdoor environment is 22-28 DEG C, humidity 45%-75%, and needs to avoid strong light direct beam.
9. the method that Moschus dendrobe tissue culture as described in claim 1 expands breeding seedling, which is characterized in that in hardening, rooting culture
The step of in, the fungicide be carbendazim solution；Carbendazim is diluted to 800-1000 times during use.
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Effective date of registration: 20190401 Address after: 650000 6th Floor, Block A, Sunshine Building, 999 Kegao Road, Wuhua District, Kunming City, Yunnan Province Applicant after: Kangmei Pharmaceutical (Kunming) Germplasm Resources Co., Ltd. Address before: Wenshan Chinese Traditional Medicine Biovalley 4-21, South Kaihua Road, Wenshan, Wenshan Zhuang and Miao Autonomous Prefecture, Yunnan Province Applicant before: Kangmei Pharmaceutical (Wenshan) medicinal material planting Management Co., Ltd. |
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