CN106472308B - A kind of preservation of cymbidium seed and method for culturing seedlings - Google Patents
A kind of preservation of cymbidium seed and method for culturing seedlings Download PDFInfo
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- CN106472308B CN106472308B CN201610876623.4A CN201610876623A CN106472308B CN 106472308 B CN106472308 B CN 106472308B CN 201610876623 A CN201610876623 A CN 201610876623A CN 106472308 B CN106472308 B CN 106472308B
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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
- 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
Abstract
The invention discloses a kind of preservation of cymbidium seed and method for culturing seedlings.The present invention develops the uncracked orchid capsule that is mature on the whole by harvesting, disinfection, and the seed in capsule is transferred in the centrifuge tube of sterilizing under aseptic condition, covers the sealing of centrifuge tube Gai Bingyong ventilated membrane, saves under the conditions of 4-6 DEG C；When needing nursery, the seed of preservation is taken out, is placed to after reaching room temperature in centrifuge tube at room temperature, seed is fabricated to seed suspension liquid with sterile water, then takes seed suspension liquid to be uniformly inoculated on once-seedling forming culture medium, once cultivates seedling.It after the present invention stores cymbidium seed 5 years under the conditions of 4-6 DEG C, still maintains higher germination rate (75.6%), and desired facility condition is simple, cheap, pollution-free and preservation capacity is huge；The seed stored through this method is not required to handle through surface sterilization again, carries out aseptic seeding using non-symbiosis germination technology, can once cultivate seedling.
Description
Technical field
The invention belongs to plant germplasm resource preservation technology fields, and in particular to a kind of preservation of cymbidium seed and nursery side
Method.
Background technique
Orchid is commonly called as orchid, has nearly cultivation history in 2000 in China, is that the important cultivation in horticulture and flower is planted
Object.Orchid is mostly rare or endangered species, and all Wild Orchids in the whole world are by column people's " wild animals and plants endangered species
International trade pact " protection scope, 90% of plant or more should be protected by accounting for the pact, be " flagship " class in plant protection
Group.The Ornamental Plants Germplasm Resources in China are lost quite serious in history, and are currently still being lost, if do not taken effective
Measure protect China's orchid germ plasm resource, it will to China's flower industry bring massive losses.
Summary of the invention
The first purpose of the invention is to provide a kind of store methods of cymbidium seed.
A kind of store method of cymbidium seed of the invention, comprising the following steps:
Harvesting development is mature on the whole and uncracked orchid capsule, sterilizes, and shifts the seed in capsule under aseptic condition
To the centrifuge tube of sterilizing, the sealing of centrifuge tube Gai Bingyong ventilated membrane is covered, is saved under the conditions of 4-6 DEG C.
It is preferred that the store method of the cymbidium seed, comprising the following steps:
Harvesting development is mature on the whole and uncracked orchid capsule, sterilizes, by the seed in single capsule under aseptic condition
It all drops on aseptic filter paper, transfers in the 1mL centrifuge tube of a sterilizing, it is close to cover centrifuge tube Gai Bingyong ventilated membrane
Envelope, numbers and is recorded to centrifuge tube the corresponding informance of wherein seed, save under the conditions of 4-6 DEG C.
It is preferred that the orchid is Doritis pulcherrima (Doritis pulcherrima), little Hua all ages orchid (Vanda
) or dendrobium candidum (Dendrobium officinale Kimura et Migo) coerulescens.
It is preferred that the development is mature on the whole and uncracked orchid capsule is 120 days or more uncracked after pollination
150 days or more uncracked little Hua all ages orchid (Vanda after Doritis pulcherrima (Doritis pulcherrima) capsule, pollination
Coerulescens) 150 days or more uncracked dendrobium candidum (Dendrobium officinale after capsule or pollination
Kimura et Migo) capsule.
It is preferred that the disinfection is orchid capsule to be impregnated to 15~30s in 70% ethanol solution of volume fraction, then use matter
It measures 0.1% mercuric chloride solution of score and sterilizes 15~20min, aseptic water washing 4~5 times, the water on capsule surface is blotted with aseptic filter paper
Point.
A second object of the present invention is to provide a kind of preservation of cymbidium seed and method for culturing seedlings.
A kind of preservation of cymbidium seed and method for culturing seedlings, comprising the following steps:
A. save: harvesting development is mature on the whole and uncracked orchid capsule, sterilizes, by the kind in capsule under aseptic condition
Son is transferred in the centrifuge tube of sterilizing, is covered the sealing of centrifuge tube Gai Bingyong ventilated membrane, is saved under the conditions of 4-6 DEG C；
B. nursery: the seed of preservation is taken out, and is placed to after reaching room temperature in centrifuge tube at room temperature, with sterile water by seed
It is fabricated to seed suspension liquid, then takes seed suspension liquid to be uniformly inoculated on once-seedling forming culture medium, once cultivates seedling；It is described
Once-seedling forming culture medium: every liter containing spend No. 1 1~2g of treasured, No. 2 1~2g of Hua Bao, 0.5~2g of peptone, 40~80g of mashed potatoes,
50~100g of banana puree, 10~20g of apple butter, 500~1000mg of activated carbon, 80~120mg of inositol, glycine 1.5~
2.5mg, thiamine hydrochloride (VB1) 0.05~0.2mg, puridoxine hydrochloride (VB6) 0.4~0.8mg, 0.4~0.8mg of niacin, 6-
0.05~0.1mg of benzyl purine, methyl α-naphthyl acetate (NAA) 0.2~2mg, 15~30g of sucrose and agar 6~7g, pH 5.4-5.6.
It is preferred that the orchid is Doritis pulcherrima (Doritis pulcherrima), little Hua all ages orchid (Vanda
) or dendrobium candidum (Dendrobium officinale Kimura et Migo) coerulescens.
It is preferred that the development is mature on the whole and uncracked orchid capsule is 120 days or more uncracked after pollination
150 days or more uncracked little Hua all ages orchid (Vanda after Doritis pulcherrima (Doritis pulcherrima) capsule, pollination
Coerulescens) 150 days or more uncracked dendrobium candidum (Dendrobium officinale after capsule or pollination
Kimura et Migo) capsule.
It is preferred that the disinfection is orchid capsule to be impregnated to 15~30s in 70% ethanol solution of volume fraction, then use matter
It measures 0.1% mercuric chloride solution of score and sterilizes 15~20min, aseptic water washing 4~5 times, the water on capsule surface is blotted with aseptic filter paper
Point.
It is preferred that the concentration of the seed suspension liquid is 10-15 seeds/mL sterile water, the seed suspension liquid
Inoculum concentration is 20-30 seeds/150mL once-seedling forming culture medium.Through this method store seed be not required to again through surface sterilization at
Reason carries out aseptic seeding using non-symbiosis germination technology, can obtain seedling rapidly.
After the present invention stores cymbidium seed 5 years under the conditions of 4-6 DEG C, although percentage of seedgermination has decline, but still protect
It holds higher germination rate (75.2%), which can be used as a kind of selection scheme of orchid Germ-plasma resources protection, excellent
Point is that required storage space is small, and facility condition is simple, cheap, pollution-free, saves that capacity is huge, 25cm*10cm*5cm's
Plastic casing can store the cymbidium seed up to counted in terms of hundred million, calculate that storage life can be up to 10 years from the result obtained at present
More than, it is a kind of efficient orchid germplasm resource preservation method.The present invention has storage space small, facility condition is simple, it is cheap,
Pollution-free, preservation capacity is huge, saves the features such as low in cost, the meaning especially for countries and regions underdeveloped
It is very great, it inexpensive, of less demanding can be stored in a large amount of orchid germ plasm resource, some economy are solved and owe
Though the country germ plasm resource that reaches is abundant, but powerless build Freezing room saving germ plasm resource and cause seriously to lose
Contradiction.
Influence during Seed storage its shelf-life factor soil will there are two, one is seed moisture content, another
It is reserve temperature.When seed moisture content is higher than 14%, microorganism is infected on the surface of seed and internal breeding, significantly and promptly
Shorten the service life of seed.The plastic centrifuge tube low temperature storage cymbidium seed sterile with certain air permeability that the present invention uses,
Seed slow dehydration in storing process, respiratory metabolism level is relatively low, and Conservation environment relative humidity is lower, and to seed into
Capable is Preservation in sterile condition, reduces destruction of the microorganism to seed vitality, and slowly new old generation is carried out under conditions of seed asepsis
It thanks, remains higher vitality, therefore substantially prolong the storage life of seed.Program is established in cymbidium seed library of the invention is
Artificial pollination is carried out to orchid, at fruit development 120~180 days, takes fruit after surface sterilization, by orchid kind under aseptic condition
Son is collected in sterilized plastic centrifuge tube, is not required to handle by dehydration and drying, is sealed with ventilated membrane and is placed in 4-6 DEG C and keeps away
Light saves, i.e., breathes freely and save under aseptic conditions, can be used as long-term store method in orchid.
Seed amount is huge in orchid fruit, its seed bank is established and long-term effectively in carrying out in the space that can use very little
Germ-plasma resources protection, this store method has many advantages, such as that equipment is simple, easy to operate, at low cost, can be by depositing in seed bank
The sprouting of the vibrant seed of the orchid of storage and reach restore group, culture new varieties the purpose of, the present invention have important scientific research
Value and application value.The grain weight that the present invention saves is big, and seed is light, takes up space small, and 1 cubic space can store 80,000 fruits.
If a fruit is one kind, that can store 80,000 kinds, if a fruit has 100,000 seeds, that can store 8,000,000,000
Cymbidium seed.Cymbidium seed of the invention is that single fruit single tube saves, and guarantees the specificity and pure of seed.Utilize present invention side
Method, it is expected to find a simple, easy to operate, low-cost approach in orchid germ plasm resource storage method, can be effective
It saves orchid germ plasm resource, rearing new variety, the seeling industry of endangered breed and ecological recovery etc. and establishes its material base, thus
It can lay the foundation for the sustainable development of China orchid rearing new variety and its industry, enhancing China is in the world flower industry
Competitiveness, and obtain preferable economic benefit and social benefit, ecological benefits.
Specific embodiment
The following examples are further illustrations of the invention, rather than limiting the invention.
It is used in the following embodiment that spend precious No. 1, Hua Bao 2 be the production of U.S. HYPONeX chemical company, Taiwan and garden
Skill enterprise stock Co., Ltd dispenses product.The ventilated membrane used isM sealed membrane (PM-996), the sealed membrane produce
The thickness of product is 0.005, and " (127 μm) (4*1.25), the place of production U.S. are bought from domestic relevant device sales company.
Embodiment 1: the preservation of Doritis pulcherrima (Doritis pulcherrima) seed and method for culturing seedlings
1. artificial pollination: Doritis pulcherrima selects robust growth plant when current year August is just bloomed, when the Yu Baitian morning ten, with
The toothpick of bacterium of having gone out is tool, and the vibrant massula on open two days or so flowers of picking is invested other one and opened
On the column cap for putting two days or so flowers, 20 flowers of pollinating altogether.Maternal back lip bagging is extractd after pollination, is gone after about 1 week
Bag will hang up label, have the information such as parent name, hybridization date and pollination people on label.
2. cultivating capsule: after successfully carrying out artificial pollination, reinforcing the fertilizer and water management of maternal plant, guarantee artificial pollination capsule
Normal development until mature.
3. disinfection capsule and make seed bank: choose fruit age be 120 days capsule (at this time capsule development be mature on the whole and not
Cracking), in 70% ethanol solution of volume fraction impregnate 15~30s, then with 0.1% mercuric chloride solution of mass fraction sterilize 15~
20min aseptic water washing 4~5 times, blots the moisture on capsule surface under aseptic condition on superclean bench with aseptic filter paper.
Then capsule is cut with aseptic operation knife, the seed in capsule is all dropped on aseptic filter paper, then from same
The seed of capsule is put together in the plastic centrifuge tube of the 1mL of high-temperature sterilization, is closed the lid, then with ventilated membrane seal from
Heart pipe.An early fixed number is indicated on centrifuge tube, the corresponding phase for understanding the Doritis pulcherrima seed of storage number on computer file
Information is closed, then centrifuge tube is placed in and adheres to separately or divides in the plastic casing (25cm*10cm*5cm) of kind, stores the kind of 10 capsules
Son, what this plastic casing loaded is the seed bank of Doritis pulcherrima.
4. cryo-conservation: the plastics box for housing centrifuge tube being placed and is stored under 4 DEG C of refrigerated conditions.
5. aseptic seeding: the Doritis pulcherrima seed for storing refrigerator certain time (5~10 years) being taken out, is placed under room temperature
Reach room temperature in two hours to centrifuge tube, it is then dense in Doritis pulcherrima seed is fabricated to seed with sterile water on superclean bench
Degree is the seed suspension liquid of the inoculation of 10-15/mL.The seed suspension liquid of inoculation is drawn under aseptic condition with liquid-transfering gun
On 2mL to once-seedling forming culture medium and it is uniformly distributed in seed on the culture medium, it should equipped with 150mL in each orchid culture bottle
Culture medium, seed amount 20-30 of inoculation.It is counted, the Doritis pulcherrima seed germination rate after saving 5 years is 74.8%.It is used
Once-seedling forming culture medium: every liter containing spending No. 2 1g of No. 1 1g, Hua Bao of treasured, peptone 0.5g, mashed potatoes 40g, banana puree 50g, apple
Puree 10g, activated carbon 500mg, inositol 80mg, glycine 1.5mg, thiamine hydrochloride (VB1) 0.05mg, puridoxine hydrochloride
(VB6) 0.4mg, niacin 0.4mg, 6- benzyl purine 0.05mg, methyl α-naphthyl acetate (NAA) 0.2mg, sucrose 15g and agar 6g, pH
5.4；Its preparation method is: after mixing by above-mentioned each component, in 1.06kg/cm2Sterilize 20min under the conditions of (121 DEG C), to
It is carried out before culture medium is cooling aseptic subpackaged.
6. once-seedling forming culture: inoculated Doritis pulcherrima seed is placed in natral light cahure room (26 ± 2) DEG C culture,
Culture was once trained seedling after 6~8 months.
Embodiment 2: the little Hua all ages preservation of blue (Vanda coerulescens) seed and method for culturing seedlings
1. artificial pollination: little Hua is all ages blue when current year September is just bloomed, and selects robust growth plant, the Yu Baitian morning ten
When, using the toothpick for bacterium of having gone out as tool, picking opens the vibrant massula on two days or so flowers, is invested other one
On the column cap of pieces open two days or so flowers, artificial pollination 20 altogether.Maternal back lip bagging, about 1 star are extractd after pollination
Bag is removed after phase, to hang up label, there are the information such as parent name, hybridization date and pollination people on label.
2. cultivating capsule: after successfully carrying out artificial pollination, reinforcing the fertilizer and water management of maternal plant, guarantee artificial pollination capsule
Normal development until mature.
3. disinfection capsule and make seed bank: choose fruit age be 150 days capsule (at this time capsule development be mature on the whole and not
Cracking), in 70% ethanol solution of volume fraction impregnate 15~30s, then with 0.1% mercuric chloride solution of mass fraction sterilize 15~
20min aseptic water washing 4~5 times, blots the moisture on capsule surface under aseptic condition on superclean bench with aseptic filter paper.
Then capsule is cut with aseptic operation knife, the seed in capsule is all dropped on aseptic filter paper, then from same
The seed of capsule is put together in the plastic centrifuge tube of the 1mL of high-temperature sterilization, is closed the lid, then with ventilated membrane seal from
Heart pipe.An early fixed number is indicated on centrifuge tube, storage number is corresponding on computer file understands the little Hua all ages orchid species
Relevant information, then centrifuge tube is placed in adhere to separately or point kind plastic casing (25cm*10cm*5cm) in, store 10 capsules
Seed, what this plastic casing loaded is little Hua all ages blue seed bank.
4. cryo-conservation: the plastics box for housing centrifuge tube being placed and is stored under 5 DEG C of refrigerated conditions.
5. aseptic seeding: by the little Hua for storing refrigerator certain time (5~10 years), all ages orchid species is taken out, under room temperature
It places in two hours to centrifuge tube and reaches room temperature, then in all ages orchid species makes by little Hua with sterile water on superclean bench
At the seed suspension liquid for the inoculation that seed concentration is 10-15/mL.The seed of inoculation is drawn under aseptic condition with liquid-transfering gun
Suspension 2mL is to once-seedling forming culture medium and is uniformly distributed in seed on the culture medium, is equipped in each orchid culture bottle
The 150mL culture medium, seed amount 20-30 of inoculation.It is counted, all ages blue seed germination rate is the little Hua after saving 5 years
75.2%.Once-seedling forming culture medium used: every liter containing spending No. 2 2g of No. 1 2g, Hua Bao of treasured, peptone 2g, mashed potatoes 80g, banana
Mud 100g, apple butter 20g, activated carbon 1000mg, inositol 120mg, glycine 2.5mg, thiamine hydrochloride (VB1) 0.2mg, hydrochloric acid
Pyridoxol (VB6) 0.8mg, niacin 0.8mg, 6- benzyl purine 0.1mg, methyl α-naphthyl acetate (NAA) 2mg, sucrose 30g and agar 7g, pH
5.6；Its preparation method is: after mixing by above-mentioned each component, in 1.06kg/cm2Sterilize 20min under the conditions of (121 DEG C), to
It is carried out before culture medium is cooling aseptic subpackaged.
6. once-seedling forming culture: by inoculated little Hua, all ages orchid species is placed in natral light cahure room (26 ± 2) DEG C
Culture, culture were once trained seedling after 6~8 months.
Embodiment 3: it the preservation of dendrobium candidum (Dendrobium officinale Kimura et Migo) seed and educates
Seedling-growing method
1. artificial pollination: at the beginning of 3 months current years of dendrobium candidum when blooming, select robust growth plant, when the Yu Baitian morning ten, with
The toothpick of bacterium of having gone out is tool, and the vibrant massula on open two days or so flowers of picking is invested other one and opened
On the column cap for putting two days or so flowers, pollinate 20 altogether.Maternal back lip bagging is extractd after pollination, removes bag after about 1 week,
Label is hung up, there are the information such as parent name, hybridization date and pollination people on label.
2. cultivating capsule: after successfully carrying out artificial pollination, reinforcing the fertilizer and water management of maternal plant, guarantee artificial pollination capsule
Normal development until mature.
3. disinfection capsule and make seed bank: choose fruit age be 150 days capsule (at this time capsule development be mature on the whole and not
Cracking), in 70% ethanol solution of volume fraction impregnate 15~30s, then with 0.1% mercuric chloride solution of mass fraction sterilize 15~
20min aseptic water washing 4~5 times, blots the moisture on capsule surface under aseptic condition on superclean bench with aseptic filter paper.
Then capsule is cut with aseptic operation knife, the seed in capsule is all dropped on aseptic filter paper, then from same
The seed of capsule is put together in the plastic centrifuge tube of the 1mL of high-temperature sterilization, is closed the lid, then with ventilated membrane seal from
Heart pipe.An early fixed number is indicated on centrifuge tube, storage number correspondence understands the Seeds of Dendrobium Candidum on computer file
Relevant information, then centrifuge tube is placed in and adheres to separately or divides in the plastic casing (25cm*10cm*5cm) of kind, store the kind of 10 capsules
Son, what this plastic casing loaded is the seed bank of dendrobium candidum.
4. cryo-conservation: the plastics box for housing centrifuge tube being placed and is stored under 6 DEG C of refrigerated conditions.
5. aseptic seeding: the Seeds of Dendrobium Candidum for storing refrigerator certain time (5~10 years) being taken out, room temperature condition decentralization
It sets in two hours to centrifuge tube and reaches room temperature, then in Seeds of Dendrobium Candidum is fabricated to kind with sterile water on superclean bench
Sub- concentration is the seed suspension liquid of the inoculation of 10-15/mL.The seed suspension of inoculation is drawn under aseptic condition with liquid-transfering gun
Liquid 2mL is to once-seedling forming culture medium and is uniformly distributed in seed on the culture medium, should equipped with 150mL in each orchid culture bottle
Culture medium, seed amount 20-30 of inoculation.It is counted, the Seeds of Dendrobium Candidum germination rate after saving 5 years is 74.3%.Institute
Once-seedling forming culture medium: every liter containing spend No. 2 2g of No. 1 2g, Hua Bao of treasured, peptone 1.5g, mashed potatoes 60g, banana puree 80g,
Apple butter 20g, activated carbon 800mg, inositol 100mg, glycine 2mg, thiamine hydrochloride (VB1) 0.2mg, puridoxine hydrochloride
(VB6) 0.6mg, niacin 0.8mg, 6- benzyl purine 0.1mg, methyl α-naphthyl acetate (NAA) 2mg, sucrose 25g and agar 7g, pH 5.6；Its
Preparation method is: after mixing by above-mentioned each component, in 1.06kg/cm2Sterilize 20min under the conditions of (121 DEG C), to culture medium
It is carried out before cooling aseptic subpackaged.
6. once-seedling forming culture: inoculated Seeds of Dendrobium Candidum is placed in natral light cahure room (26 ± 2) DEG C training
It supports, culture was once trained seedling after 6~8 months.
Claims (4)
1. preservation and the method for culturing seedlings of a kind of cymbidium seed, which comprises the following steps:
A. save: harvesting development is mature on the whole and uncracked orchid capsule, sterilizes, and turns the seed in capsule under aseptic condition
It moves in the centrifuge tube of sterilizing, covers the sealing of centrifuge tube Gai Bingyong ventilated membrane, saved under the conditions of 4-6 DEG C；The orchid is
Doritis pulcherrima (Doritis pulcherrima), little Hua all ages blue (Vanda coerulescens) or dendrobium candidum
(Dendrobium officinale Kimura et Migo)；
B. nursery: the seed of preservation is taken out, and is placed to after reaching room temperature in centrifuge tube, is made seed at room temperature of sterile water
At seed suspension liquid, then takes seed suspension liquid to be uniformly inoculated on once-seedling forming culture medium, once cultivate seedling；Described one
Secondary seedling culture medium: every liter containing spending No. 1 1~2g of treasured, No. 2 1~2g of Hua Bao, 0.5~2g of peptone, 40~80g of mashed potatoes, banana
50~100g of mud, 10~20g of apple butter, 500~1000mg of activated carbon, 80~120mg of inositol, 1.5~2.5mg of glycine, salt
0.05~0.2mg of allithiamine element, 0.4~0.8mg of puridoxine hydrochloride, 0.4~0.8mg of niacin, 6- benzyl purine 0.05~
0.1mg, 0.2~2mg of methyl α-naphthyl acetate, 15~30g of sucrose and agar 6~7g, pH 5.4-5.6.
2. preservation and the method for culturing seedlings of cymbidium seed according to claim 1, which is characterized in that the development substantially at
Uncracked Doritis pulcherrima (Doritis pulcherrima) capsule that ripe and uncracked orchid capsule is 120 days or more after pollination
After fruit, pollination 150 days or more uncracked little Hua all ages after blue (Vanda coerulescens) capsule or pollination 150 days with
On uncracked dendrobium candidum (Dendrobium officinale Kimura et Migo) capsule.
3. preservation and the method for culturing seedlings of cymbidium seed according to claim 1, which is characterized in that the disinfection is will be blue
Flower capsule impregnates 15~30s in 70% ethanol solution of volume fraction, then 15 are sterilized with 0.1% mercuric chloride solution of mass fraction~
Aseptic water washing 4~5 times, the moisture on capsule surface is blotted with aseptic filter paper by 20min.
4. preservation and the method for culturing seedlings of cymbidium seed according to claim 1, which is characterized in that the seed suspension liquid
Concentration be 10-15 seeds/mL sterile water, the inoculum concentration of the seed suspension liquid is primary for 20-30 seed/150mL
Seedling culture medium.
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| CN107484461A (en) * | 2017-09-12 | 2017-12-19 | 中国科学院华南植物园 | A kind of method that the pocket orchid seed germination rate for improving cryopreservation is handled using sodium hypochlorite |
| CN107466861A (en) * | 2017-09-28 | 2017-12-15 | 南京大花滩生物科技有限公司 | A kind of little Hua all ages orchid species seedling aseptic seeding rapid propagation methods |
| CN107494269A (en) * | 2017-09-29 | 2017-12-22 | 南京仙草堂生物科技有限公司 | A kind of elimination little Hua all ages in blue tissue culture procedures endophyte method |
| CN107581067A (en) * | 2017-09-29 | 2018-01-16 | 南京仙草堂生物科技有限公司 | A kind of little Hua all ages orchid species seedling aseptic seeding propagation methods |
| CN110651559A (en) * | 2019-11-07 | 2020-01-07 | 安徽祥枫生物科技有限公司 | Preservation method of dendrobium huoshanense capsules for tissue culture seedling |
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