CN105104209B - The method of Dendrobidium huoshanness tissue cultures forming seedling through one step culture and the formula of the nutrient solution used - Google Patents
The method of Dendrobidium huoshanness tissue cultures forming seedling through one step culture and the formula of the nutrient solution used Download PDFInfo
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Abstract
A kind of method of Dendrobidium huoshanness tissue cultures forming seedling through one step culture, comprises the steps：The preparation of solid medium, the formula of wherein Solid nutritional base includes：A great number of elements, trace element, molysite, organic principle, hormone and additive；The preparation of liquid nutrient solution：The formula of liquid nutrient solution according to solid medium formula, without hormone, agar, cycocel and sweet apple extract solution；The selection and sterilization of Dendrobidium huoshanness Fruit pod；The preparation of Dendrobidium huoshanness seed suspension liquid；Aseptic seeding；Seedling culture；Emerge.The advantage that the cultivation of Dendrobidium huoshanness is carried out using the above method is：The cultivation cycle of Dendrobidium huoshanness is shortened, can be emerged within 6 months, i.e., sows, just can emerge the April of Second Year in the October of seed maturity, just catches up with and is planting seedling stage；The production cost of 50% Dendrobidium huoshanness tissue culture is reduced, plantation input is reduced；Increase the survival rate and germination percentage of cultivation.
Description
Technical field
The present invention relates to the technical field of plant tissue culture nursery, and in particular to a kind of side of Dendrobidium huoshanness tissue cultures
Method.
Background technology
Dendrobidium huoshanness (scientific name:Dendrobium huoshanense C.Z.Tang et S.J.Cheng) a meter dry measure used in former times is commonly called as,
It is the herbaceous plant of orchid family Dendrobium.Because the compositions such as the alkaloid contained by it, Thick many candies, amino acid, trace element have strong kidney
Voiceless sound improving eyesight, liver protection shield throat, nourishing Yin and clearing heat, nourishing the stomach to improve the production of body fluid, suppress growth of tumour cell, strengthen immunity the effects such as, always by
It is considered as first of nine big " celestial grass ".
Under wild state, Dendrobidium huoshanness growth is very slow, wherein extremely tiny, only one simple embryo and individual layer are thin
The aliform kind skin of born of the same parents is constituted, no endosperm, in its natural state, few Germination And Seedling, because excessive felling is utilized, growth is delayed
Slowly, the deterioration of ecological environment and the influence of the low reason of breeding potential, Dendrobidium huoshanness wild resource are endangered.
The method for obtaining Dendrobidium huoshanness regeneration plant by the method for Plant Tissue Breeding has two：(1) Dendrobidium huoshanness is used
Aseptic seeding is carried out after ripe seed disinfection, sprouts and produces protocorm, bud induction, stem induction and root induction is then carried out, most
Afterwards seedling is produced after strong seedling culture；(2) protocorm or Multiple Buds are intended by suitable explant induced synthesis, constantly increased
It is worth (generation certain amount), then carries out bud induction, stem induction and root induction, seedling is produced after finally carrying out strong seedling culture.
The existing tissue culture technical costs of Dendrobidium huoshanness high (needing substantial amounts of labor cost), time length are (from seed
The time of more than 12 months is at least needed to seedling), aberration rate it is high (due to carrying out to this increment and hormone induction, variation compared with
It is many), low (constantly increment makes the resistance step-down of seedling to cultivation survival rate, it is necessary to could be cultivated after hardening, and survival rate
Only 80% or so).
With continuing to develop for Dendrobidium huoshanness industrialized scale, the demand to Dendrobidium huoshanness seedling also increases constantly
Plus, the Dendrobidium huoshanness seedling of low-cost and high-quality is development trend.
The content of the invention
The technical problem to be solved of the present invention is to provide that a kind of survival rate is high, technical costs is low, the training time compared with
The method and the formula of the nutrient solution used of short Dendrobidium huoshanness tissue cultures forming seedling through one step culture.
The present invention solves above-mentioned technical problem using following technical scheme：A kind of Dendrobidium huoshanness tissue cultures forming seedling through one step culture
Method, comprise the steps：
Step one：The preparation of solid medium, the formula of wherein Solid nutritional base includes：
The a great number of elements of consisting of and proportioning：
Potassium nitrate KNO3：300~500mg/L；
Ammonium sulfate (NH4) 2SO4：400~600mg/L；
Potassium dihydrogen phosphate KH2PO4：300~500mg/L；
Magnesium sulfate MgSO4.7H2O：200~300mg/L；
Calcium nitrate Ca (NO3) 2：400~500mg/L；
The trace element of consisting of and proportioning：
KI KI：0.65~0.95mg/L；
Boric acid H3BO3：5.3~8.9mg/L；
Manganese sulfate MnSO4.4H2O：19~25mg/L；
Zinc sulfate ZnSO4.7H2O：6.5~9.8mg/L；
Sodium molybdate Na2MoO4.2H2O：0.10~0.45mg/L；
Copper sulphate CuSO4.5H2O：0.010~0.035mg/L；
Cobalt chloride CoCl2：0.010~0.035mg/L；
The molysite of consisting of and proportioning：
Disodium ethylene diamine tetraacetate Na2.EDTA：25.5~40.5mg/L；
Ferrous sulfate FeSO44.7H2O：22.5~30.5mg/L；
The organic principle of consisting of and proportioning：
Inositol C6H12O6：90~110mg/L；
Glycine C2H5NO2：1~3mg/L；
Thiamine hydrochloride VB1：0.05~0.15mg/L；
Puridoxine hydrochloride VB6：0.4~0.6mg/L；
Nicotinic acid VB5 or VPP：0.4~0.6mg/L；
The hormone of consisting of and proportioning：
Methyl α-naphthyl acetate NAA：0.6~0.9mg/L；
6-benzyl aminopurine 6-BA：0.4~0.6mg/L；
The additive of consisting of and proportioning：
Sucrose：25~35g/L；
Agar：3.5~5.5mg/L；
Spend precious No. 1：1.5~2.5mg/L；
Sweet apple mixed extract (2~4:1~2)：120~200g/L；
The production procedure of the solid medium includes：After water ebuillition of heated, the agar of above-mentioned formula is added, agar is formed
Just mixed liquid, the additive of above-mentioned composition and proportioning is added water to heat and crushes and leaches to form additive leachate, additive leaching
Mix liquid at the beginning of going out liquid and above-mentioned agar and be mixed to form agar liquid, in the agar liquid by said components and formula add a great number of elements,
Trace element, molysite, organic principle, hormone, then stir, formed culture medium just liquid, regulation culture medium just liquid ph values to
5.6-6.0, temperature is dispensed between maintaining 50 DEG C to 70 DEG C, and 100 DEG C~150 DEG C sterilizing 10min~30min are standby after packing
With；
Step 2：The preparation of liquid nutrient solution：The formula of liquid nutrient solution according to solid medium formula, without swash
Element, agar, precious No. 1 and sweet apple extract solution are spent, the production procedure of the liquid nutrient solution includes：After water ebuillition of heated, press
Added according to the formula of liquid nutrient solution after each component, mixing constant volume, just liquid ph values are to 5.6-6.0 for regulation culture medium, and temperature is maintained
Dispensed between 50 DEG C to 70 DEG C, 100 DEG C~150 DEG C sterilizing 10min~30min are standby after packing；
Step 3：The selection and sterilization of Dendrobidium huoshanness Fruit pod：The Dendrobidium huoshanness outstanding achievement of selection mature and plump then, surface without
Scab, damage by worms and ftracture, the Dendrobidium huoshanness outstanding achievement of harvesting is cleared up, remove the withered residual flower in outstanding achievement surface, and use clear water
Rinse, be then placed in 75% alcohol untill flooding repeatedly, soak 20min~50min, be then placed on sterile inoculation disk
In dried on aseptic operating platform, it is standby；
Step 4：The preparation of Dendrobidium huoshanness seed suspension liquid：On desinfection chamber superclean bench face, with scalpel in outstanding achievement
A small openning is cut in one end, clamps carpopodium with tweezers, by cut alignment bottleneck, is gently beaten with another tweezers and clamp carpopodium
Tweezers, seed is uniformly scattered in nutrient solution, and suspension is stirred with magnetic stirring apparatus under sterile environment,
And detect density of the seed in suspension with tally under the microscope with suspension counting method, it is desirable to reach 80~120/ml's
Density；
Step 5：Aseptic seeding：On sterile behaviour's platform, 1ml is extracted from seed suspension liquid with the liquid-transfering gun after sterilizing, is put
Enter in solid medium, cover bottle cap and slowly rock, until the uniform suspension in bottle be laid in media surface untill, put
Enter to cultivate in frame, deliver to culturing room's culture；
Step 6：Seedling culture, has just started to be within 10 days that seed sprouts the stage, using light culture, no light, temperature control exists
24 DEG C~27 DEG C, -70 days 10 days, be the development stage, in this period, it is ensured that light application time 7~9 hours, intensity of illumination
1500lux-2000lux, temperature control, to occur in root stage, this period, is protected at 25 DEG C~28 DEG C, 70 days~120 days
Demonstrate,prove light application time 9~12 hours, intensity of illumination 1800lux-2100lux, temperature control is in 24 DEG C~27 DEG C, 120 days~180
My god, it is in strong sprout stage, this period, it is ensured that light application time 9~12 hours, intensity of illumination 2500lux-3000lux, temperature
Control is at 24 DEG C~27 DEG C.During training, continue to give birth to it is impossible to meet seedling if finding that solid medium has been absorbed by seedling
When length needs, the liquid nutrient solution after sterilizing is added in blake bottle on aseptic operating platform with sterile liquid-transfering gun, seedling is supplied
Continued growth；
Step 7：Emerge.
Present invention also offers the liquid nutrient solution used in a kind of method of Dendrobidium huoshanness tissue cultures forming seedling through one step culture, institute
Stating the formula of liquid nutrient solution includes：
The a great number of elements of consisting of and proportioning：
Potassium nitrate KNO3：300~500mg/L；
Ammonium sulfate (NH4) 2SO4：400~600mg/L；
Potassium dihydrogen phosphate KH2PO4：300~500mg/L；
Magnesium sulfate MgSO4.7H2O：200~300mg/L；
Calcium nitrate Ca (NO3) 2：400~500mg/L；
The trace element of consisting of and proportioning：
KI KI：0.65~0.95mg/L；
Boric acid H3BO3：5.3~8.9mg/L；
Manganese sulfate MnSO4.4H2O：19~25mg/L；
Zinc sulfate ZnSO4.7H2O：6.5~9.8mg/L；
Sodium molybdate Na2MoO4.2H2O：0.10~0.45mg/L；
Copper sulphate CuSO4.5H2O：0.010~0.035mg/L；
Cobalt chloride CoCl2：0.010~0.035mg/L；
The molysite of consisting of and proportioning：
Disodium ethylene diamine tetraacetate Na2.EDTA：25.5~40.5mg/L；
Ferrous sulfate FeSO44.7H2O：22.5~30.5mg/L；
The organic principle of consisting of and proportioning：
Inositol C6H12O6：90~110mg/L；
Glycine C2H5NO2：1~3mg/L；
Thiamine hydrochloride VB1：0.05~0.15mg/L；
Puridoxine hydrochloride VB6：0.4~0.6mg/L；
Nicotinic acid VB5 or VPP：0.4~0.6mg/L；
The additive of consisting of and proportioning：
Sucrose：25~35g/L.
The advantage that the cultivation of the lightning stem of noble dendrobium is carried out using the above method is：
1st, the cultivation cycle of Dendrobidium huoshanness is shortened, can be emerged within 6 months, both seed maturity sowing in October, Second Year
April can seedling, just catch up with plant seedling stage；
2nd, reduce 50% Dendrobidium huoshanness tissue culture into production cost, reduce Cotton Varieties by Small Farming Households input；
3rd, in seedling raising process, do not change its nutritional ingredient substantially, the appropriate liquid that has additional nutrients need to be only needed according to its growth
, greatly reduce the variation caused by excessive turn point and the change of growing environment；
4th, in seedling raising process, nutritional ingredient needed for active absorption transformation increases the survival rate and germination percentage of cultivation.
Brief description of the drawings
Fig. 1 for the present invention in solid medium flow sheet.
Fig. 2 for the present invention in liquid nutrient solution flow sheet.
Fig. 3 for the present invention in seedling culture flow sheet.
Embodiment
The present invention is described in detail below in conjunction with accompanying drawing.
Embodiment one
A kind of method of Dendrobidium huoshanness tissue cultures forming seedling through one step culture, comprises the following steps：
Step one：The preparation of solid medium, the formula of wherein Solid nutritional base includes：
The a great number of elements of consisting of and proportioning：
Potassium nitrate KNO3：400mg/L；
Ammonium sulfate (NH4) 2SO4：500mg/L；
Potassium dihydrogen phosphate (KH2PO4)：400mg/L；
Magnesium sulfate MgSO4.7H2O：250mg/L；
Calcium nitrate Ca (NO3) 2：450mg/L；
The trace element of consisting of and proportioning：
KI KI：0.83mg/L；
Boric acid H3BO3：6.2mg/L；
Manganese sulfate MnSO4.4H2O：22.3mg/L；
Zinc sulfate ZnSO4.7H2O：8.6mg/L；
Sodium molybdate Na2MoO4.2H2O：0.25mg/L；
Copper sulphate CuSO4.5H2O：0.025mg/L；
Cobalt chloride CoCl2：0.025mg/L；
The molysite of consisting of and proportioning：
Disodium ethylene diamine tetraacetate Na2.EDTA：37.3mg/L；
Ferrous sulfate FeSO44.7H2O：27.8mg/L；
The organic principle of consisting of and proportioning：
Inositol C6H12O6：100mg/L；
Glycine C2H5NO2：2mg/L；
Thiamine hydrochloride VB1：0.1mg/L；
Puridoxine hydrochloride VB6：0.5mg/L；
Nicotinic acid VB5 or VPP：0.5mg/L；
The hormone of consisting of and proportioning：
Methyl α-naphthyl acetate NAA：0.8mg/L；
6-benzyl aminopurine 6-BA：0.5mg/L；
The additive of consisting of and proportioning：
Sucrose：30g/L；
Agar：4.5mg/L；
Spend precious No. one：2.0mg/L；
Sweet apple mixed extract (3:13)：150g/L.
The production procedure of the solid medium includes：After water (preferably pure water) ebuillition of heated, above-mentioned formula is added
Agar, formed agar at the beginning of mix liquid, by the additive of above-mentioned composition and proportioning add water heating crush and leach to be formed additive leaching
Go out liquid, liquid is mixed at the beginning of the additive leachate and above-mentioned agar and is mixed to form agar liquid, by said components and match somebody with somebody in the agar liquid
Side adds a great number of elements, trace element, molysite, organic principle, hormone, then stirs, and forms culture medium just liquid, regulation training
Supporting base, just liquid ph values are to 5.8, and practical operation needs to be dispensed between temperature maintains 60 degree, 121 DEG C of sterilizing 20min after packing
It is standby.
Step 2：The preparation of liquid nutrient solution：The formula of liquid nutrient solution according to solid medium formula, without swash
Element, agar, precious No. one and sweet apple extract solution are spent, the production procedure of the liquid nutrient solution includes：Water is (preferably pure
Water) after ebuillition of heated, added according to the formula of liquid nutrient solution after each component, mixing constant volume, regulation culture medium just liquid ph values to
5.8, temperature maintains 50~70 DEG C to be needed to be dispensed according to practical operation, and 121 DEG C of sterilizing 20min are standby after packing；
Step 3：The selection and sterilization of Dendrobidium huoshanness Fruit pod：The ripe then warming Dendrobidium huoshanness fruit of selection, surface without
Scab, pityriasis simplex, manual cleanup is carried out by the Dendrobidium huoshanness Fruit pod of harvesting, removes the withered residual flower in outstanding achievement surface, and anti-with clear water
It is multiple to rinse, it is then placed in 75% alcohol untill flooding, soaks 30min, be then placed in sterile inoculation disk sterile
Dried on operating desk, it is standby；
Step 4：The preparation of Dendrobidium huoshanness seed suspension liquid：On desinfection chamber superclean bench face, with scalpel in Fruit pod
Afterbody cuts a small openning, clamps carpopodium with tweezers, by cut alignment bottleneck, is gently beaten with another tweezers and clamp carpopodium
Tweezers, seed is uniformly scattered in nutrient solution, and stirred under sterile environment with magnetic agitation machine suspension, and
Detect that seed, in the density of suspension, is advisable with 100/ml density with tally under the microscope with suspension counting method；
Step 5：Aseptic seeding：On sterile behaviour's platform, 1ml is extracted from seed suspension liquid with the liquid-transfering gun after sterilizing, is put
Enter in solid medium, cover bottle cap and slowly rock, until the uniform suspension in bottle be laid in media surface untill, put
Enter to cultivate in frame, deliver to culturing room's culture；
Step 6：Seedling culture, has just started to be within 10 days that seed sprouts the stage, using light culture, no light, temperature control exists
25 DEG C ± 2 DEG C, -70 days 10 days, be the development stage, in this period, it is ensured that light application time 8 hours, intensity of illumination
1500lux-2000lux, temperature control is at 27 DEG C ± 2 DEG C, -120 days 70 days, to send out in lateral bud stage, this period, it is ensured that
Light application time 10 hours, intensity of illumination 2000lux, temperature control is at 25 DEG C ± 2 DEG C, and -180 days 120 days, be the strong sprout stage, this
In one period, it is ensured that light application time 10 hours, intensity of illumination 2500lux-3000lux, temperature control is at 25 DEG C ± 2 DEG C.
During training, if finding, solid medium has been absorbed by seedling when being needed it is impossible to meet seedling continued growth, can be with sterile
Liquid-transfering gun adds to the liquid nutrient solution after sterilizing in blake bottle on aseptic operating platform, supplies seedling continued growth；
Step 7：Emerge, for cultivation Dendrobidium huoshanness commercial seedling should for uncontamination, without rotten stem, rotten；2, blade
More than, normal expansion, leaf color is light green or emerald green；Qualified seedling in root more than 1 and unit bottle accounts for more than the 95% of total seedling amount.
Using above step, emerge within 0 26 days 5 months.
Embodiment two
A kind of method of Dendrobidium huoshanness tissue cultures forming seedling through one step culture, including the preparation of solid medium, liquid nutrient solution
Prepare, the selection of Dendrobidium huoshanness Fruit pod and sterilization, the preparation of Dendrobidium huoshanness seed suspension liquid, aseptic seeding, seedling culture and
Emerge standard, specific step is as follows：
Step one：The preparation of solid medium, the formula of wherein Solid nutritional base includes：
The a great number of elements of consisting of and proportioning：
Potassium nitrate KNO3：300mg/L；
Ammonium sulfate (NH4) 2SO4：400mg/L；
Potassium dihydrogen phosphate (KH2PO4)：300mg/L；
Magnesium sulfate MgSO4.7H2O：200mg/L；
Calcium nitrate Ca (NO3) 2：400mg/L；
The trace element of consisting of and proportioning：
KI KI：0.65mg/L；
Boric acid H3BO3：5.3mg/L；
Manganese sulfate MnSO4.4H2O：19mg/L；
Zinc sulfate ZnSO4.7H2O：9.8mg/L；
Sodium molybdate Na2MoO4.2H2O：0.45mg/L；
Copper sulphate CuSO4.5H2O：0.035mg/L；
Cobalt chloride CoCl2：0.010mg/L；
The molysite of consisting of and proportioning：
Disodium ethylene diamine tetraacetate Na2.EDTA：25.5mg/L；
Ferrous sulfate FeSO44.7H2O：30.5mg/L；
The organic principle of consisting of and proportioning：
Inositol C6H12O6：90mg/L；
Glycine C2H5NO2：3mg/L；
Thiamine hydrochloride VB1：0.05mg/L；
Puridoxine hydrochloride VB6：0.4mg/L；
Nicotinic acid VB5 or VPP：0.4mg/L；
The hormone of consisting of and proportioning：
Methyl α-naphthyl acetate NAA：0.6mg/L；
6-benzyl aminopurine 6-BA：0.4mg/L；
The additive of consisting of and proportioning：
Sucrose：25g/L；
Agar：5.5mg/L；
Spend precious No. 1：1.5mg/L；
Sweet apple mixed extract (2:1)：200g/L；
The production procedure of the solid medium includes：After water (preferably pure water) ebuillition of heated, above-mentioned formula is added
Agar, formed agar at the beginning of mix liquid, by the additive of above-mentioned composition and proportioning add water heating crush and leach to be formed additive leaching
Go out liquid, liquid is mixed at the beginning of the additive leachate and above-mentioned agar and is mixed to form agar liquid, by said components and match somebody with somebody in the agar liquid
Side adds a great number of elements, trace element, molysite, organic principle, hormone, then stirs, and forms culture medium just liquid, regulation training
Supporting base, just liquid ph values are to 5.6, and temperature maintains 50 DEG C to be needed to be dispensed according to practical operation, 100 DEG C of sterilizing 30min after packing
It is standby；
Step 2：The preparation of liquid nutrient solution：The formula of liquid nutrient solution according to solid medium formula, without swash
Element, agar, precious No. 1 and sweet apple extract solution are spent, the production procedure of the liquid nutrient solution includes：After water ebuillition of heated, press
Added according to the formula of liquid nutrient solution after each component, mixing constant volume, just liquid ph values are to 5.4 for regulation culture medium, and temperature maintains 50
DEG C need to be dispensed according to practical operation, 100 DEG C of sterilizing 30min are standby after packing；
Step 3：The selection and sterilization of Dendrobidium huoshanness Fruit pod：The ripe then warming Dendrobidium huoshanness fruit of selection, surface without
Scab, pityriasis simplex, manual cleanup is carried out by the Dendrobidium huoshanness Fruit pod of harvesting, removes the withered residual flower in outstanding achievement surface, and anti-with clear water
It is multiple to rinse, it is then placed in 75% alcohol untill flooding, soaks 50min, be then placed in sterile inoculation disk sterile
Dried on operating desk, it is standby；
Step 4：The preparation of Dendrobidium huoshanness seed suspension liquid：On desinfection chamber superclean bench face, with scalpel in Fruit pod
Afterbody cuts a small openning, clamps carpopodium with tweezers, by cut alignment bottleneck, is gently beaten with another tweezers and clamp carpopodium
Tweezers, seed is uniformly scattered in nutrient solution, and stirred under sterile environment with magnetic agitation machine suspension, and
Detect that seed, in the density of suspension, reaches 80/ml density with tally under the microscope with suspension counting method；
Step 5：Aseptic seeding：On sterile behaviour's platform, 1ml is extracted from seed suspension liquid with the liquid-transfering gun after sterilizing, is put
Enter in solid medium, cover bottle cap and slowly rock, until the uniform suspension in bottle be laid in media surface untill, put
Enter to cultivate in frame, deliver to culturing room's culture；
Step 6：Seedling culture, has just started to be within 10 days that seed sprouts the stage, using light culture, no light, temperature control exists
24 DEG C, -70 days 10 days, be the development stage, in this period, it is ensured that light application time 7 hours, intensity of illumination 2000lux, temperature
Control is at 28 DEG C, -120 days 70 days, to send out in lateral bud stage, this period, it is ensured that light application time 12 hours, intensity of illumination
1800lux, temperature control, at 24 DEG C, -180 days 120 days, is in strong sprout stage, this period, it is ensured that light application time 9 hours,
Intensity of illumination 3000lux, temperature control, at 24 DEG C, -200 days 180 days, is in hardening stage, this period, using nature
Light, intensity of illumination is below 8000lux, and temperature control is below 32 DEG C, during training, if finding solid medium by seedling
Absorb when being needed it is impossible to meet seedling continued growth, sought the liquid after sterilizing on aseptic operating platform with sterile liquid-transfering gun
Nutrient solution is added in blake bottle, supplies seedling continued growth.
Step 7：Emerge, for cultivation Dendrobidium huoshanness commercial seedling should for uncontamination, without rotten stem, rotten；2, blade
More than, normal expansion, leaf color is light green or emerald green；Qualified seedling in root more than 1 and unit bottle accounts for more than the 95% of total seedling amount.
Using above step, emerge within 6 months.
Embodiment three
A kind of method of Dendrobidium huoshanness tissue cultures forming seedling through one step culture, including the preparation of solid medium, liquid nutrient solution
Prepare, the selection of Dendrobidium huoshanness Fruit pod and sterilization, the preparation of Dendrobidium huoshanness seed suspension liquid, aseptic seeding, seedling culture and
Emerge standard, specific step is as follows：
Step one：The preparation of solid medium, the formula of wherein Solid nutritional base includes：
The a great number of elements of consisting of and proportioning：
Potassium nitrate KNO3：500mg/L；
Ammonium sulfate (NH4) 2SO4：600mg/L；
Potassium dihydrogen phosphate (KH2PO4)：500mg/L；
Magnesium sulfate MgSO4.7H2O：300mg/L；
Calcium nitrate Ca (NO3) 2：500mg/L；
The trace element of consisting of and proportioning：
KI KI：0.95mg/L；
Boric acid H3BO3：8.9mg/L；
Manganese sulfate MnSO4.4H2O：25mg/L；
Zinc sulfate ZnSO4.7H2O：6.5mg/L；
Sodium molybdate Na2MoO4.2H2O：0.10mg/L；
Copper sulphate CuSO4.5H2O：0.010mg/L；
Cobalt chloride CoCl2：0.035mg/L；
The molysite of consisting of and proportioning：
Disodium ethylene diamine tetraacetate Na2.EDTA：40.5mg/L；
Ferrous sulfate FeSO44.7H2O：22.5mg/L；
The organic principle of consisting of and proportioning：
Inositol C6H12O6：110mg/L；
Glycine C2H5NO2：1mg/L；
Thiamine hydrochloride VB1：0.15mg/L；
Puridoxine hydrochloride VB6：0.6mg/L；
Nicotinic acid VB5 or VPP：0.6mg/L；
The hormone of consisting of and proportioning：
Methyl α-naphthyl acetate NAA：0.9mg/L；
6-benzyl aminopurine 6-BA：0.6mg/L；
The additive of consisting of and proportioning：
Sucrose：35g/L；
Agar：3.5mg/L；
Spend precious No. 1：2.5mg/L；
Sweet apple mixed extract (2~4:1~2)：120g/L；
The production procedure of the solid medium includes：After water ebuillition of heated, the agar of above-mentioned formula is added, agar is formed
Just mixed liquid, the additive of above-mentioned composition and proportioning is added water to heat and crushes and leaches to form additive leachate, additive leaching
Mix liquid at the beginning of going out liquid and above-mentioned agar and be mixed to form agar liquid, in the agar liquid by said components and formula add a great number of elements,
Trace element, molysite, organic principle, hormone, then stir, formed culture medium just liquid, regulation culture medium just liquid ph values to
6, temperature maintains 70 DEG C to be needed to be dispensed according to practical operation, and 150 DEG C of sterilizing 10min are standby after packing；
Step 2：The preparation of liquid nutrient solution：The formula of liquid nutrient solution according to solid medium formula, without swash
Element, agar, precious No. 1 and sweet apple extract solution are spent, the production procedure of the liquid nutrient solution includes：After water ebuillition of heated, press
Added according to the formula of liquid nutrient solution after each component, mixing constant volume, just liquid ph values are to 6 for regulation culture medium, and temperature maintains 70 DEG C
Need to be dispensed according to practical operation, 150 DEG C of sterilizing 10min are standby after packing；
Step 3：The selection and sterilization of Dendrobidium huoshanness Fruit pod：The ripe then warming Dendrobidium huoshanness fruit of selection, surface without
Scab, pityriasis simplex, manual cleanup is carried out by the Dendrobidium huoshanness Fruit pod of harvesting, removes the withered residual flower in outstanding achievement surface, and anti-with clear water
It is multiple to rinse, it is then placed in 75% alcohol untill flooding, soaks 20min, be then placed in sterile inoculation disk sterile
Dried on operating desk, it is standby；
Step 4：The preparation of Dendrobidium huoshanness seed suspension liquid：On desinfection chamber superclean bench face, with scalpel in Fruit pod
Afterbody cuts a small openning, clamps carpopodium with tweezers, by cut alignment bottleneck, is gently beaten with another tweezers and clamp carpopodium
Tweezers, seed is uniformly scattered in nutrient solution, and stirred under sterile environment with magnetic agitation machine suspension, and
Detect that seed, in the density of suspension, is advisable with 120/ml density with tally under the microscope with suspension counting method；
Step 5：Aseptic seeding：On sterile behaviour's platform, 1ml is extracted from seed suspension liquid with the liquid-transfering gun after sterilizing, is put
Enter in solid medium, cover bottle cap and slowly rock, until the uniform suspension in bottle be laid in media surface untill, put
Enter to cultivate in frame, deliver to culturing room's culture；
Step 6：Seedling culture, has just started to be within 10 days that seed sprouts the stage, using light culture, no light, temperature control exists
27 DEG C, -70 days 10 days, be the development stage, in this period, it is ensured that light application time 9 hours, intensity of illumination 1500lux, temperature
Control is at 25 DEG C, -120 days 70 days, to send out in lateral bud stage, this period, it is ensured that light application time 9 hours, intensity of illumination
2100lux, temperature control is in strong sprout stage, this period, it is ensured that light application time 12 is small at 27 DEG C, -180 days 120 days
When, intensity of illumination 2500lux, temperature control, at 27 DEG C, -200 days 180 days, is in hardening stage, this period, using certainly
Right light, intensity of illumination is below 8000lux, and temperature control is below 32 DEG C, during training, if finding, solid medium is planted
Seedling has been absorbed when being needed it is impossible to meet seedling continued growth, with sterile liquid-transfering gun on aseptic operating platform by the liquid after sterilizing
Nutrient solution is added in blake bottle, supplies seedling continued growth；
Step 7：Emerge, for cultivation Dendrobidium huoshanness commercial seedling should for uncontamination, without rotten stem, rotten；2, blade
More than, normal expansion, leaf color is light green or emerald green；Qualified seedling in root more than 1 and unit bottle accounts for more than the 95% of total seedling amount.
Using above step, emerge within 03 days 6 months.
The preferred embodiment of the invention is the foregoing is only, creation is not intended to limit the invention, it is all at this
Any modifications, equivalent substitutions and improvements made within the spirit and principle of innovation and creation etc., should be included in the invention
Protection domain within.
Claims (5)
1. a kind of method of Dendrobidium huoshanness tissue cultures forming seedling through one step culture, it is characterised in that：Comprise the steps：
Step one：The preparation of solid medium, the formula of wherein Solid nutritional base includes：
The a great number of elements of consisting of and proportioning：
Potassium nitrate KNO3：300~500mg/L；
Ammonium sulfate (NH4)2SO4：400~600mg/L；
Potassium dihydrogen phosphate KH2PO4：300~500mg/L；
Magnesium sulfate MgSO4.7H2O：200~300mg/L；
Calcium nitrate Ca (NO3)2：400~500mg/L；
The trace element of consisting of and proportioning：
KI KI：0.65~0.95mg/L；
Boric acid H3BO3：5.3~8.9mg/L；
Manganese sulfate MnSO4.4H2O：19~25mg/L；
Zinc sulfate ZnSO4.7H2O：6.5~9.8mg/L；
Sodium molybdate Na2MoO4.2H2O：0.10~0.45mg/L；
Copper sulphate CuSO4.5H2O：0.010~0.035mg/L；
Cobalt chloride CoCl2：0.010~0.035mg/L；
The molysite of consisting of and proportioning：
Disodium ethylene diamine tetraacetate Na2.EDTA：25.5~40.5mg/L；
Ferrous sulfate FeSO4.7H2O：22.5~30.5mg/L；
The organic principle of consisting of and proportioning：
Inositol C6H12O6：90~110mg/L；
Glycine C2H5NO2：1~3mg/L；
Thiamine hydrochloride VB1：0.05~0.15mg/L；
Puridoxine hydrochloride VB6：0.4~0.6mg/L；
Nicotinic acid VB5 or VPP：0.4~0.6mg/L；
The hormone of consisting of and proportioning：
Methyl α-naphthyl acetate NAA：0.6~0.9mg/L；
6-benzyl aminopurine 6-BA：0.4~0.6mg/L；
The additive of consisting of and proportioning：
Sucrose：25~35g/L；
Agar：3.5~5.5mg/L；
Spend precious No. 1：1.5~2.5mg/L；
Sweet apple mixed extract 2~4:1~2：120~200g/L；
The production procedure of the solid medium includes：After water ebuillition of heated, the agar of above-mentioned formula is added, is mixed at the beginning of forming agar
Liquid, the additive of above-mentioned composition and proportioning is added water to heat and crushes and leaches to form additive leachate, the additive leachate
Agar liquid is mixed to form with mixing liquid at the beginning of above-mentioned agar, a great number of elements, micro is added by said components and formula in the agar liquid
Element, molysite, organic principle, hormone, then stir, and form culture medium just liquid, regulation ph values to 5.6-6.0, temperature dimension
Hold and dispensed between 50 DEG C to 70 DEG C, 100 DEG C~150 DEG C sterilizing 10min~30min are standby after packing；
Step 2：The preparation of liquid nutrient solution：The formula of liquid nutrient solution according to solid medium formula, without hormone,
Agar, precious No. 1 and sweet apple extract solution are spent, the production procedure of the liquid nutrient solution includes：After water ebuillition of heated, according to liquid
The formula of body nutrient solution is added after each component, mixing constant volume, regulation ph values to 5.6-6.0, temperature maintain 50 DEG C to 70 DEG C it
Between dispensed, after packing 100 DEG C~150 DEG C sterilizing 10min~30min it is standby；
Step 3：The selection and sterilization of Dendrobidium huoshanness Fruit pod：The Dendrobidium huoshanness outstanding achievement of mature and plump then is selected, surface is disease-free
Spot, damage by worms and ftracture, the Dendrobidium huoshanness outstanding achievement of harvesting is cleared up, remove the withered residual flower in outstanding achievement surface, and it is anti-with clear water
It is multiple to rinse, it is then placed in 75% alcohol untill flooding, soaks 20min~50min, be then placed in sterile inoculation disk
Dried on aseptic operating platform, it is standby；
Step 4：The preparation of Dendrobidium huoshanness seed suspension liquid：On desinfection chamber superclean bench face, with scalpel in one end of outstanding achievement
A small openning is cut, carpopodium is clamped with tweezers, by cut alignment bottleneck, the tweezer for clamping carpopodium is gently beaten with another tweezers
Son, seed is uniformly scattered in nutrient solution, and suspension is stirred with magnetic stirring apparatus under sterile environment, is used in combination
Suspension counting method detects density of the seed in suspension with tally under the microscope, it is desirable to reach that 80~120/ml's is close
Degree；
Step 5：Aseptic seeding：On aseptic operating platform, 1ml is extracted from seed suspension liquid with the liquid-transfering gun after sterilizing, is put into
In solid medium, cover bottle cap and slowly rock, until the uniform suspension in bottle be laid in media surface untill, be put into
Cultivate in frame, deliver to culturing room's culture；
Step 6：Seedling culture, has just started to be within 10 days that seed sprouts the stage, using light culture, no light, temperature control is at 24 DEG C
~27 DEG C, -70 days 10 days, be the development stage, in this period, it is ensured that light application time 7~9 hours, intensity of illumination
1500lux-2000lux, temperature control, to occur in root stage, this period, is protected at 25 DEG C~28 DEG C, 70 days~120 days
Demonstrate,prove light application time 9~12 hours, intensity of illumination 1800lux-2100lux, temperature control is in 24 DEG C~27 DEG C, 120 days~180
My god, it is in strong sprout stage, this period, it is ensured that light application time 9~12 hours, intensity of illumination 2500lux-3000lux, temperature
Control is at 24 DEG C~27 DEG C；In incubation, if discovery solid medium has been absorbed by seedling, it is impossible to meet seedling continuation
When growth needs, the liquid nutrient solution after sterilizing is added in blake bottle on aseptic operating platform with sterile liquid-transfering gun, supply kind
Seedling continued growth；
Step 7：Emerge.
2. the method for Dendrobidium huoshanness tissue cultures forming seedling through one step culture according to claim 1, it is characterised in that：Step 7 goes out
Seedling standard is：For cultivation Dendrobidium huoshanness commercial seedling for uncontamination, without rotten stem, rotten；More than 2, blade, normal expansion, leaf
Color is light green or emerald green, and the qualified seedling in root more than 1 and unit bottle accounts for more than the 95% of total seedling amount.
3. the method for Dendrobidium huoshanness tissue cultures forming seedling through one step culture according to claim 1, it is characterised in that：The step one
It is specially to step 6：
Step one：The preparation of solid medium, the formula of wherein Solid nutritional base includes：
The a great number of elements of consisting of and proportioning：
Potassium nitrate KNO3：400mg/L；
Ammonium sulfate (NH4)2SO4：500mg/L；
Potassium dihydrogen phosphate (KH2PO4)：400mg/L；
Magnesium sulfate MgSO4.7H2O：250mg/L；
Calcium nitrate Ca (NO3)2：450mg/L；
The trace element of consisting of and proportioning：
KI KI：0.83mg/L；
Boric acid H3BO3：6.2mg/L；
Manganese sulfate MnSO4.4H2O：22.3mg/L；
Zinc sulfate ZnSO4.7H2O：8.6mg/L；
Sodium molybdate Na2MoO4.2H2O：0.25mg/L；
Copper sulphate CuSO4.5H2O：0.025mg/L；
Cobalt chloride CoCl2：0.025mg/L；
The molysite of consisting of and proportioning：
Disodium ethylene diamine tetraacetate Na2.EDTA：37.3mg/L；
Ferrous sulfate FeSO4.7H2O：27.8mg/L；
The organic principle of consisting of and proportioning：
Inositol C6H12O6：100mg/L；
Glycine C2H5NO2：2mg/L；
Thiamine hydrochloride VB1：0.1mg/L；
Puridoxine hydrochloride VB6：0.5mg/L；
Nicotinic acid VB5 or VPP：0.5mg/L；
The hormone of consisting of and proportioning：
Methyl α-naphthyl acetate NAA：0.8mg/L；
6-benzyl aminopurine 6-BA：0.5mg/L；
The additive of consisting of and proportioning：
Sucrose：30g/L；
Agar：4.5mg/L；
Spend precious No. 1：2.0mg/L；
Sweet apple mixed extract 3:1：150g/L；
The production procedure of the solid medium includes：After water ebuillition of heated, the agar of above-mentioned formula is added, is mixed at the beginning of forming agar
Liquid, the additive of above-mentioned composition and proportioning is added water to heat and crushes and leaches to form additive leachate, the additive leachate
Agar liquid is mixed to form with mixing liquid at the beginning of above-mentioned agar, a great number of elements, micro is added by said components and formula in the agar liquid
Element, molysite, organic principle, hormone, then stir, and form culture medium just liquid, regulation ph values are to 5.4, and temperature is maintained
60 DEG C need to be dispensed according to practical operation, and 121 DEG C of sterilizing 20min are standby after packing；
Step 2：The preparation of liquid nutrient solution：The formula of liquid nutrient solution according to solid medium formula, without hormone,
Agar, precious No. 1 and sweet apple extract solution are spent, the production procedure of the liquid nutrient solution includes：After water ebuillition of heated, according to liquid
The formula of body nutrient solution is added after each component, mixing constant volume, and regulation ph values are to 5.4, and temperature maintains 60 DEG C according to practical operation
Need to be dispensed, 121 DEG C of sterilizing 20min are standby after packing；
Step 3：The selection and sterilization of Dendrobidium huoshanness Fruit pod：Selection warming Dendrobidium huoshanness fruit ripe then, surface is disease-free
Spot, pityriasis simplex, manual cleanup is carried out by the Dendrobidium huoshanness Fruit pod of harvesting, removes the withered residual flower in outstanding achievement surface, and with clear water repeatedly
Rinse, be then placed in 75% alcohol untill flooding, soak 30min, be then placed in sterile inoculation disk in sterile behaviour
Make to dry on platform, it is standby；
Step 4：The preparation of Dendrobidium huoshanness seed suspension liquid：On desinfection chamber superclean bench face, with scalpel Fruit pod afterbody
A small openning is cut, carpopodium is clamped with tweezers, by cut alignment bottleneck, the tweezer for clamping carpopodium is gently beaten with another tweezers
Son, seed is uniformly scattered in nutrient solution, and is stirred under sterile environment with magnetic agitation machine suspension, and with outstanding
Floating counting method detects that seed, in the density of suspension, reaches 100/ml density with tally under the microscope；
Step 5：Aseptic seeding：On aseptic operating platform, 1ml is extracted from seed suspension liquid with the liquid-transfering gun after sterilizing, is put into
In solid medium, cover bottle cap and slowly rock, until the uniform suspension in bottle be laid in media surface untill, be put into
Cultivate in frame, deliver to culturing room's culture；
Step 6：Seedling culture, has just started to be within 10 days that seed sprouts the stage, using light culture, no light, temperature control is at 25 DEG C
± 2 DEG C, -70 days 10 days, be the development stage, in this period, it is ensured that light application time 8 hours, intensity of illumination 1500lux-
2000lux, temperature control is at 27 DEG C ± 2 DEG C, -120 days 70 days, to send out in lateral bud stage, this period, it is ensured that light application time
10 hours, intensity of illumination 2000lux, temperature control, at 25 DEG C ± 2 DEG C, -180 days 120 days, was strong sprout stage, this period
It is interior, it is ensured that light application time 10 hours, intensity of illumination 2500lux-3000lux, temperature control is at 25 DEG C ± 2 DEG C, 180 days -200
My god, be that, using natural light, intensity of illumination is below 8000lux in hardening stage, this period, temperature control 32 DEG C with
Under, in incubation, if discovery solid medium has been absorbed by seedling when being needed it is impossible to meet seedling continued growth, with nothing
Bacterium liquid-transfering gun adds to the liquid nutrient solution after sterilizing in blake bottle on aseptic operating platform, supplies seedling continued growth.
4. the method for Dendrobidium huoshanness tissue cultures forming seedling through one step culture according to claim 1, it is characterised in that：The step one
It is specially to step 6：
Step one：The preparation of solid medium, the formula of wherein Solid nutritional base includes：
The a great number of elements of consisting of and proportioning：
Potassium nitrate KNO3：500mg/L；
Ammonium sulfate (NH4)2SO4：600mg/L；
Potassium dihydrogen phosphate (KH2PO4)：500mg/L；
Magnesium sulfate MgSO4.7H2O：300mg/L；
Calcium nitrate Ca (NO3)2：500mg/L；
The trace element of consisting of and proportioning：
KI KI：0.95mg/L；
Boric acid H3BO3：8.9mg/L；
Manganese sulfate MnSO4.4H2O：25mg/L；
Zinc sulfate ZnSO4.7H2O：6.5mg/L；
Sodium molybdate Na2MoO4.2H2O：0.10mg/L；
Copper sulphate CuSO4.5H2O：0.010mg/L；
Cobalt chloride CoCl2：0.035mg/L；
The molysite of consisting of and proportioning：
Disodium ethylene diamine tetraacetate Na2.EDTA：40.5mg/L；
Ferrous sulfate FeSO4.7H2O：22.5mg/L；
The organic principle of consisting of and proportioning：
Inositol C6H12O6：110mg/L；
Glycine C2H5NO2：1mg/L；
Thiamine hydrochloride VB1：0.15mg/L；
Puridoxine hydrochloride VB6：0.6mg/L；
Nicotinic acid VB5 or VPP：0.6mg/L；
The hormone of consisting of and proportioning：
Methyl α-naphthyl acetate NAA：0.9mg/L；
6-benzyl aminopurine 6-BA：0.6mg/L；
The additive of consisting of and proportioning：
Sucrose：35g/L；
Agar：3.5mg/L；
Spend precious No. 1：2.5mg/L；
Sweet apple mixed extract 2~4:1~2：120g/L；
The production procedure of the solid medium includes：After water ebuillition of heated, the agar of above-mentioned formula is added, is mixed at the beginning of forming agar
Liquid, the additive of above-mentioned composition and proportioning is added water to heat and crushes and leaches to form additive leachate, the additive leachate
Agar liquid is mixed to form with mixing liquid at the beginning of above-mentioned agar, a great number of elements, micro is added by said components and formula in the agar liquid
Element, molysite, organic principle, hormone, then stir, and form culture medium just liquid, regulation ph values are to 6, and temperature maintains 70
DEG C need to be dispensed according to practical operation, 150 DEG C of sterilizing 10min are standby after packing；
Step 2：The preparation of liquid nutrient solution：The formula of liquid nutrient solution according to solid medium formula, without hormone,
Agar, precious No. 1 and sweet apple extract solution are spent, the production procedure of the liquid nutrient solution includes：After water ebuillition of heated, according to liquid
The formula of body nutrient solution is added after each component, mixing constant volume, and regulation ph values to 6, temperature maintains 70 DEG C and needed according to practical operation
Dispensed, 150 DEG C of sterilizing 10min are standby after packing；
Step 3：The selection and sterilization of Dendrobidium huoshanness Fruit pod：Selection warming Dendrobidium huoshanness fruit ripe then, surface is disease-free
Spot, pityriasis simplex, manual cleanup is carried out by the Dendrobidium huoshanness Fruit pod of harvesting, removes the withered residual flower in outstanding achievement surface, and with clear water repeatedly
Rinse, be then placed in 75% alcohol untill flooding, soak 20min, be then placed in sterile inoculation disk in sterile behaviour
Make to dry on platform, it is standby；
Step 4：The preparation of Dendrobidium huoshanness seed suspension liquid：On desinfection chamber superclean bench face, with scalpel Fruit pod afterbody
A small openning is cut, carpopodium is clamped with tweezers, by cut alignment bottleneck, the tweezer for clamping carpopodium is gently beaten with another tweezers
Son, seed is uniformly scattered in nutrient solution, and is stirred under sterile environment with magnetic agitation machine suspension, and with outstanding
Floating counting method detects that seed, in the density of suspension, reaches 120/ml density with tally under the microscope；
Step 5：Aseptic seeding：On aseptic operating platform, 1ml is extracted from seed suspension liquid with the liquid-transfering gun after sterilizing, is put into
In solid medium, cover bottle cap and slowly rock, until the uniform suspension in bottle be laid in media surface untill, be put into
Cultivate in frame, deliver to culturing room's culture；
Step 6：Seedling culture, has just started to be within 10 days that seed sprouts the stage, using light culture, no light, temperature control is 27
DEG C, -70 days 10 days, be the development stage, in this period, it is ensured that light application time 9 hours, intensity of illumination 1500lux, temperature control
System is at 25 DEG C, -120 days 70 days, to send out in lateral bud stage, this period, it is ensured that light application time 9 hours, intensity of illumination
2100lux, temperature control is in strong sprout stage, this period, it is ensured that light application time 12 is small at 27 DEG C, -180 days 120 days
When, intensity of illumination 2500lux, temperature control, at 27 DEG C, -200 days 180 days, is in hardening stage, this period, using certainly
Right light, intensity of illumination is below 8000lux, and temperature control is below 32 DEG C, in incubation, if finding solid medium quilt
Seedling has been absorbed when being needed it is impossible to meet seedling continued growth, with sterile liquid-transfering gun on aseptic operating platform by the liquid after sterilizing
Body nutrient solution is added in blake bottle, supplies seedling continued growth.
5. the method for Dendrobidium huoshanness tissue cultures forming seedling through one step culture according to claim 1, it is characterised in that：The step one
It is specially to step 6：
Step one：The preparation of solid medium, the formula of wherein Solid nutritional base includes：
The a great number of elements of consisting of and proportioning：
Potassium nitrate KNO3：300mg/L；
Ammonium sulfate (NH4)2SO4：400mg/L；
Potassium dihydrogen phosphate (KH2PO4)：300mg/L；
Magnesium sulfate MgSO4.7H2O：200mg/L；
Calcium nitrate Ca (NO3)2：400mg/L；
The trace element of consisting of and proportioning：
KI KI：0.65mg/L；
Boric acid H3BO3：5.3mg/L；
Manganese sulfate MnSO4.4H2O：19mg/L；
Zinc sulfate ZnSO4.7H2O：9.8mg/L；
Sodium molybdate Na2MoO4.2H2O：0.45mg/L；
Copper sulphate CuSO4.5H2O：0.035mg/L；
Cobalt chloride CoCl2：0.01mg/L；
The molysite of consisting of and proportioning：
Disodium ethylene diamine tetraacetate Na2.EDTA：25.5mg/L；
Ferrous sulfate FeSO4.7H2O：30.5mg/L；
The organic principle of consisting of and proportioning：
Inositol C6H12O6：90mg/L；
Glycine C2H5NO2：3mg/L；
Thiamine hydrochloride VB1：0.05mg/L；
Puridoxine hydrochloride VB6：0.4mg/L；
Nicotinic acid VB5 or VPP：0.4mg/L；
The hormone of consisting of and proportioning：
Methyl α-naphthyl acetate NAA：0.6mg/L；
6-benzyl aminopurine 6-BA：0.4mg/L；
The additive of consisting of and proportioning：
Sucrose：25g/L；
Agar：5.5mg/L；
Spend precious No. 1：1.5mg/L；
Sweet apple mixed extract 2~4:1~2：200g/L
The production procedure of the solid medium includes：After water ebuillition of heated, the agar of above-mentioned formula is added, is mixed at the beginning of forming agar
Liquid, the additive of above-mentioned composition and proportioning is added water to heat and crushes and leaches to form additive leachate, the additive leachate
Agar liquid is mixed to form with mixing liquid at the beginning of above-mentioned agar, a great number of elements, micro is added by said components and formula in the agar liquid
Element, molysite, organic principle, hormone, then stir, and form culture medium just liquid, regulation ph values are to 5.6, and temperature is maintained
50 DEG C need to be dispensed according to practical operation, and 100 DEG C of sterilizing 30min are standby after packing；
Step 2：The preparation of liquid nutrient solution：The formula of liquid nutrient solution according to solid medium formula, without hormone,
Agar, precious No. 1 and sweet apple extract solution are spent, the production procedure of the liquid nutrient solution includes：After water ebuillition of heated, according to liquid
The formula of body nutrient solution is added after each component, mixing constant volume, and regulation ph values are to 5.4, and temperature maintains 50 DEG C according to practical operation
Need to be dispensed, 100 DEG C of sterilizing 30min are standby after packing；
Step 3：The selection and sterilization of Dendrobidium huoshanness Fruit pod：Selection warming Dendrobidium huoshanness fruit ripe then, surface is disease-free
Spot, pityriasis simplex, manual cleanup is carried out by the Dendrobidium huoshanness Fruit pod of harvesting, removes the withered residual flower in outstanding achievement surface, and with clear water repeatedly
Rinse, be then placed in 75% alcohol untill flooding, soak 50min, be then placed in sterile inoculation disk in sterile behaviour
Make to dry on platform, it is standby；
Step 4：The preparation of Dendrobidium huoshanness seed suspension liquid：On desinfection chamber superclean bench face, with scalpel Fruit pod afterbody
A small openning is cut, carpopodium is clamped with tweezers, by cut alignment bottleneck, the tweezer for clamping carpopodium is gently beaten with another tweezers
Son, seed is uniformly scattered in nutrient solution, and is stirred under sterile environment with magnetic agitation machine suspension, and with outstanding
Floating counting method detects that seed, in the density of suspension, reaches 80/ml density with tally under the microscope；
Step 5：Aseptic seeding：On aseptic operating platform, 1ml is extracted from seed suspension liquid with the liquid-transfering gun after sterilizing, is put into
In solid medium, cover bottle cap and slowly rock, until the uniform suspension in bottle be laid in media surface untill, be put into
Cultivate in frame, deliver to culturing room's culture；
Step 6：Seedling culture, has just started to be within 10 days that seed sprouts the stage, using light culture, no light, temperature control is 24
DEG C, -70 days 10 days, be the development stage, in this period, it is ensured that light application time 7 hours, intensity of illumination 2000lux, temperature control
System is at 28 DEG C, -120 days 70 days, to send out in lateral bud stage, this period, it is ensured that light application time 12 hours, intensity of illumination
1800lux, temperature control, at 24 DEG C, -180 days 120 days, is in strong sprout stage, this period, it is ensured that light application time 9 hours,
Intensity of illumination 3000lux, temperature control, at 24 DEG C, -200 days 180 days, is in hardening stage, this period, using nature
Light, intensity of illumination is below 8000lux, and temperature control is below 32 DEG C, in incubation, if finding, solid medium is planted
Seedling has been absorbed when being needed it is impossible to meet seedling continued growth, with sterile liquid-transfering gun on aseptic operating platform by the liquid after sterilizing
Nutrient solution is added in blake bottle, supplies seedling continued growth.
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| CN105613296A (en) * | 2016-01-25 | 2016-06-01 | 浙江欧银农业发展有限公司 | Tissue culture inoculating method for dendrobium officinale |
| CN106386482B (en) * | 2016-08-31 | 2018-06-08 | 南京仙草堂生物科技有限公司 | Dendrobium candidum high quality seedling nature optical culture once-seedling forming quick propagating method |
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