CN103918552B - A kind of quick tissue is cultivated the method for stem of noble dendrobium seedling - Google Patents
A kind of quick tissue is cultivated the method for stem of noble dendrobium seedling Download PDFInfo
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Abstract
The invention belongs to planting technology field, be specifically related to a kind of tissue fast and cultivate stem of noble dendrobium kind seedling-growing method, described cultural method comprises: step 1: the separation of symbiosis fungi and cultivation; Step 2: the cultivation of dendrobe tissue culture seedling; Step 3: group training seedling and fungal component co-incubation; Step 4: the hardening of Mycorrhizal group training seedling is cultivated. Method of the present invention can obtain fast stem of noble dendrobium seedling and survival rate reaches 98%.
Description
Technical field
The invention belongs to planting technology field, be specifically related to a kind of tissue fast and cultivate stem of noble dendrobium kind seedling-growing method.
Background technology
The stem of noble dendrobium is China's tradition rare traditional Chinese medicine, and it is all on the books that edition pharmacopeia is gone through by China. The former plant of the Chinese medicine stem of noble dendrobium isThe orchid family (Orchidaceae) Dendrobium (DendrobiumSw.) herbaceos perennial. The stem of noble dendrobium has tasteCloudy heat-clearing, the beneficial stomach that promotes the production of body fluid, the effect such as moisten the lung and relieve the cough, be usually used in that consumption of body fluid caused by febrile disease, dry are fidgety, abnormal heat after being illEtc. various disease conditions. Modern pharmacological research shows, that the stem of noble dendrobium also has is antitumor, anti-ageing, strengthen human immunityThe effect of power and hemangiectasis. But because long-term immoderate the excavating of people makes depletion of natural resources, addThe breeding potential of dendrobium candidum itself is extremely low, and existing dendrobium candidum becomes one of medicinal material kind of special-protection-by-the-State.Therefore artificial growth dendrobium candidum has broad prospects, and is dendrobium candidum plant husbandry but lack high quality seedlingOne of bottleneck.
Summary of the invention
The object of the invention is to develop a kind of method that quick tissue is cultivated stem of noble dendrobium seedling, survival rate reaches98％。
The present invention is achieved through the following technical solutions:
A kind of tissue is fast cultivated stem of noble dendrobium kind seedling-growing method, and its feature comprises the following steps:
Quick tissue is cultivated a method for stem of noble dendrobium seedling, comprises the steps:
Step 1: the separation of symbiosis fungi and cultivation
A. the isolation and purification of symbiosis fungi
Take the life root of wild Dendrodium, 0.1% mercuric chloride solution soaks 2-5 minute, aseptic water washing 3-5 time;Be cut to 0.05-0.3cm2Tissue segments be inoculated on symbiosis fungi culture medium and cultivate, condition of culture is:20-30 DEG C, dark culturing; In the time that notching edge grows hypha,hyphae, adopt mycelia top method of purification progressively pureChange and obtain pure bacterial strain;
Described symbiosis fungi culture medium is: glucose or sucrose 10-40g/L, oatmeal 10-100g/L, pineTrailing plants juice 1-150g/L, chloramphenicol or penicillin 0.001-1g/L, potassium dihydrogen phosphate 0.01-10g/L, magnesium sulfate0.01-10g/L, yeast extract 1-10g/L, agar 1-30g/L, pH value is 4-9, sterilising conditions is: 121 DEG C,20 minutes;
B. the fermented and cultured of symbiosis fungi
By after the slant tube actication of culture of mycorrhizal fungi, transfer in symbiosis fungi culture medium, in 20-30 DEG CConstant temperature culture 1-30 days, gets in bacterium piece access liquid symbiosis fungi culture medium and carries out fermented and cultured at colony edge;Access amount is 1-10% by weight, vibration rotating speed 20-300 rev/min, the 20-30 DEG C of dark 1-60 days that cultivates;
Step 2: the cultivation of dendrobe tissue culture seedling
A. the induction of protocorm and Multiple Buds
Get the tender shoots that wild Dendrodium is given birth to then, with 0.1% mercuric chloride solution sterilizing 2-10 minute, aseptic water washingAfter 3-5 time, the stem section of clip 0.5-3 centimetre of band joint, is inoculated on inducing culture, in cultivation temperatureUnder 10-30 DEG C of condition, cultivate 3-30 days; Illuminance 1000～2500Lx, illumination 12 hours/day;
Described inducing culture is: MS, 6-benzyl purine 0.1-10mg/L, methyl α-naphthyl acetate 0.1-10mg/L, sugarcaneSugar 10-40g/L, sunglo juice 20-200g/L, agar 1-30g/L, pH4-9;
B. strengthening seedling and rooting is cultivated
Multiple Buds is forwarded on culture medium 1 and cultivates 5-50 days, and condition of culture is: intensity of illumination be 1000Lx～3000Lx, periodicity of illumination is illumination/dark=14h/10h, temperature 20-30 DEG C; Plant height reaches 2cm when aboveBe forwarded to culture medium 2 and cultivate 10-50 days, condition of culture is: intensity of illumination is 2000Lx～3000Lx,Periodicity of illumination is illumination/dark=14h/10h, temperature 20-30 DEG C;
Described culture medium 1 is: 1/2MS, sucrose 10-40g/L, sunglo juice 20-200g/L, banana 10-40g/L,Agar 1-30g/L, pH4-9;
Described culture medium 2 is: 1/2MS, sucrose 10-40g/L, sunglo juice 20-200g/L, banana 10-40g/L,Active carbon 0.1-10g/L, agar 1-30g/L, pH4-9;
Step 3: group training seedling and fungal component co-incubation
Get fungal component liquid spawn 1～30% and be inoculated in symbiotic culture medium, the dark 3-9 that cultivates at 20-30 DEG CAfter it, then the access of group training seedling is cultivated to 10-50 days altogether;
Condition of culture altogether: intensity of illumination is 1000-3000Lx, and periodicity of illumination is: illumination/dark=14h/10h,Temperature 20-30 DEG C;
Symbiotic culture medium is: 1/2MS culture medium, potato 100-300g/L, sucrose 20-40g/L, sungloJuice 20-200g/L, inositol 20-200mg/L, agar 1-30g/L, pH4.0-9.0;
Step 4: the hardening of Mycorrhizal group training seedling is cultivated
A. under natural lighting in bottle hardening 5-10 days;
B. Mycorrhizal is successfully organized to training seedling and taken out from vial, in 0.1% liquor potassic permanganate, soakAfter 3-9 minute, dry plantation in plastic tub, temperature 15-35 DEG C, humidity 40-80% and sunshade rate areUnder the condition of 50-70%, cultivate hardening phase 3-12 month; Cultivation matrix is through 121 DEG C of autoclaving 2h'sFresh liver moss is cool to being used for plantation after normal temperature until liver moss.
Described step 1 is further:
A. the isolation and purification of symbiosis fungi
Take the life root of wild Dendrodium, under flowing water, rinse 5-10 minute, and remove surface attachment with hairbrushThing, is then positioned on blotting paper, rinses with 72% alcohol, and after aseptic water washing 3-5 time, 0.1%Mercuric chloride solution soaks 2-5 minute, aseptic water washing 4 times; Thin following is cut to 0.05-0.3cm2Tissue segments connectPlant and cultivate to symbiosis fungi culture medium, condition of culture is: 20-30 DEG C, dark culturing; When notching edge longWhile going out hypha,hyphae, employing mycelia top method of purification progressively purifying obtains pure bacterial strain;
Described symbiosis fungi culture medium is: glucose or sucrose 10-40g/L, oatmeal 10-100g/L, pineTrailing plants juice 1-150g/L, chloramphenicol or penicillin 0.001-1g/L, potassium dihydrogen phosphate 0.01-10g/L, magnesium sulfate0.01-10g/L, yeast extract 1-10g/L, agar 1-30g/L, pH value is 4-9, sterilising conditions is: 121 DEG C,20 minutes;
B. the fermented and cultured of symbiosis fungi
By after the slant tube actication of culture of mycorrhizal fungi, transfer in symbiosis fungi culture medium, in 20-30 DEG CConstant temperature culture 1-30 days, gets at colony edge the training of fermenting in small bacteria block access liquid symbiosis fungi culture mediumSupport; Access amount is 1-10% by weight; Vibration rotating speed 20-300 rev/min, the 20-30 DEG C of dark 1-60 that cultivatesMy god.
The A of described step 2 is further:
A. the induction of protocorm and Multiple Buds: get the wild Dendrodium tender shoots of raw tool 3-5 joint then, remove leafAfter sheet, under flowing water, rinse 5-10 minute, on superclean bench with 70% alcohol-pickled 1-3 minute,Use again 0.1% mercuric chloride solution sterilizing 2-10 minute, after aseptic water washing 5 times, blot with aseptic blotting paper,The stem section of clip 0.5-3 centimetre of band joint, is inoculated on inducing culture, under cultivation temperature 10-30 DEG C condition,Cultivate 3-30 days; Illuminance 1000～2500Lx, illumination 12 hours/day;
Described inducing culture is: MS, 6-benzyl purine 0.1-10mg/L, methyl α-naphthyl acetate 0.1-10mg/L, sugarcaneSugar 10-40g/L, sunglo juice 20-200g/L, agar 1-30g/L, pH4-9.
The B of described step 2 is further:
B. strengthening seedling and rooting is cultivated: by after many adventitious buds proliferation that utilizes the induction of stem section generations, Multiple Buds can be cutBecome single bud to be forwarded on culture medium 1 and cultivate 5-50 days, condition of culture is: intensity of illumination be 1000Lx～3000Lx, periodicity of illumination is illumination/dark=14h/10h, temperature 20-30 DEG C; More than plant height can reach 2cmIn time, is forwarded to culture medium 2 and cultivates 10-50 days, and condition of culture is: intensity of illumination is 2000Lx～3000Lx,Periodicity of illumination is illumination/dark=14h/10h, temperature 20-30 DEG C;
Described culture medium 1 is: 1/2Ms, sucrose 10-40g/L, sunglo juice 20-200g/L, banana 10-40g/L,Agar 1-30g/L, pH4-9;
Described culture medium 2 is: 1/2MS, sucrose 10-40g/L, sunglo juice 20-200g/L, banana 10-40g/L,Active carbon 0.1-10g/L, agar 1-30g/L, pH4-9;
Described step 4 is further:
Selecting annual 3-5 month is the season that bottle outlet is transplanted, or similar 3-5 month natural conditions are providedGrowing environment carries out bottle outlet transplanting;
A. under natural lighting in bottle hardening 5-10 days;
B. Mycorrhizal is successfully organized to training seedling and taken out from vial, clean the culture medium of root, 0.1%In liquor potassic permanganate, soak after 3-9 minute and dry plantation in plastic tub, temperature 15-35 DEG C, humidityUnder the condition that 40-80% and sunshade rate are 50-70%, cultivate, second day starts foliage spray clear water, every day 2Inferior, every two weeks imposes nutrient solution once, hardening phase 3-12 month; With spraying process maintain air humidity andWater content, the composition of nutrient solution is the aqueous solution that contains potassium dihydrogen phosphate and ammonium nitrate, cultivation matrix is warpThe fresh liver moss of 121 DEG C of autoclaving 2h is cool to being used for plantation after normal temperature until liver moss; In the mistake of stem of noble dendrobium growthIn journey, can mend as required applying liquid mycorrhizal fungi.
Embodiment
Embodiment 1
Step 1: the separation of symbiosis fungi and cultivation
A. the isolation and purification of symbiosis fungi
Take the life root of wild Dendrodium, under flowing water, rinse 5-10 minute, and remove surface attachment with hairbrushThing, is then positioned on blotting paper, rinses with 72% alcohol, and after aseptic water washing 3-5 time, 0.1%Mercuric chloride solution soaks 2 minutes, aseptic water washing 4 times; Thin following is cut to 0.05cm2Tissue segments be inoculated into altogetherOn raw fungi culture medium, cultivate, condition of culture is: 20 DEG C, and dark culturing; When notching edge grows fungi bacteriumWhen silk, employing mycelia top method of purification progressively purifying obtains pure bacterial strain;
Described symbiosis fungi culture medium is: glucose 10g/L, oatmeal 10g/L, sunglo juice 1g/L, chlorine are mouldElement 0.001g/L, potassium dihydrogen phosphate 0.01g/L, magnesium sulfate 0.01g/L, yeast extract 1g/L, agar 1g/L,PH value is 4, and sterilising conditions is: 121 DEG C, 20 minutes;
B. the fermented and cultured of symbiosis fungi
By after the slant tube actication of culture of mycorrhizal fungi, transfer in symbiosis fungi culture medium, in 20 DEG C of perseverancesTemperature is cultivated 1 day, gets in small bacteria block access liquid symbiosis fungi culture medium and carries out fermented and cultured at colony edge;Access amount is 1% by weight; 20 revs/min of vibration rotating speeds, 20 DEG C of dark cultivations 1 day.
The cultivation of step 2. dendrobe tissue culture seedling
A. the induction of protocorm and Multiple Buds: get the wild Dendrodium tender shoots of raw tool 3-5 joint then, remove leafAfter sheet, under flowing water, rinse 5-10 minute, on superclean bench with 70% alcohol-pickled 1 minute, thenWith 0.1% mercuric chloride solution sterilizing 2 minutes, after aseptic water washing 5 times, blot clip with aseptic blotting paperThe stem section of 0.5 centimetre of band joint, is inoculated on inducing culture, under 10 DEG C of conditions of cultivation temperature, cultivates 3My god; Illuminance 1000～2500Lx, illumination 12 hours/day;
Described inducing culture is: MS, 6-benzyl purine 0.1mg/L, methyl α-naphthyl acetate 0.1mg/L, sucrose 10g/L,Sunglo juice 20g/L, agar 1g/L, pH4;
B. strengthening seedling and rooting is cultivated: by after many adventitious buds proliferation that utilizes the induction of stem section generations, Multiple Buds can be cutBecome single bud to be forwarded on culture medium 1 and cultivate 5 days, condition of culture is: intensity of illumination is 1000Lx～3000Lx,Periodicity of illumination is illumination/dark=14h/10h, temperature 20-30 DEG C; Plant height can reach 2cm and be forwarded to when aboveCulture medium 2 is cultivated 10 days, and condition of culture is: intensity of illumination is 2000Lx～3000Lx, and periodicity of illumination isIllumination/dark=14h/10h, 20 DEG C of temperature;
Described culture medium 1 is: 1/2MS, sucrose 10g/L, sunglo juice 20g/L, banana 10g/L, agar1g／L，pH4；
Described culture medium 2 is: 1/2MS, sucrose 10g/L, sunglo juice 20g/L, banana 10g/L, active carbon0.1g/L, agar 1g/L, pH4;
3. group training seedling and fungal component co-incubation
Get fungal component liquid spawn 1% and be inoculated in symbiotic culture medium, dark cultivation after 3 days at 20 DEG C, thenThe access of group training seedling is cultivated 10 days altogether;
Condition of culture altogether: intensity of illumination is 1000-3000Lx, and periodicity of illumination is: illumination/dark=14h/10h,20 DEG C of temperature;
Symbiotic culture medium is: 1/2MS culture medium, potato 100g/L, sucrose 20g/L, sunglo juice 20g/L,Inositol 20mg/L, agar 1g/L, pH4;
4. the hardening of Mycorrhizal group training seedling is cultivated
Annual 3-5 month is the season that bottle outlet is transplanted;
A. under natural lighting in bottle hardening 5 days;
B. Mycorrhizal is successfully organized to training seedling and taken out from vial, clean the culture medium of root, 0.1%In liquor potassic permanganate, soak after 3 minutes and dry plantation in plastic tub, at 15 DEG C of temperature, humidity 40% andSunshade rate is to cultivate under 50% condition, within second day, starts foliage spray clear water, every day 2 times, every two weeksImpose nutrient solution once, 3 months hardening phases; Process and maintain air humidity and water content, nutrient solution with sprayingComposition be the aqueous solution that contains potassium dihydrogen phosphate and ammonium nitrate, cultivation matrix is through 121 DEG C of autoclaving 2hFresh liver moss, cool to being used for plantation after normal temperature until liver moss; Can be as required in the process of stem of noble dendrobium growthMend applying liquid mycorrhizal fungi.
Embodiment 2
Step 1: the separation of symbiosis fungi and cultivation
A. the isolation and purification of symbiosis fungi
Take the life root of wild Dendrodium, under flowing water, rinse 5-10 minute, and remove surface attachment with hairbrushThing, is then positioned on blotting paper, rinses with 72% alcohol, and after aseptic water washing 3-5 time, 0.1%Mercuric chloride solution soaks 5 minutes, aseptic water washing 4 times; Thin following is cut to 0.3cm2Tissue segments be inoculated into altogetherOn raw fungi culture medium, cultivate, condition of culture is: 30 DEG C, and dark culturing; When notching edge grows fungi bacteriumWhen silk, employing mycelia top method of purification progressively purifying obtains pure bacterial strain;
Described symbiosis fungi culture medium is: sucrose 10g/L, oatmeal 100g/L, sunglo juice 150g/L, green grass or young cropsMycin 1g/L, potassium dihydrogen phosphate 10g/L, magnesium sulfate 10g/L, yeast extract 10g/L, agar 30g/L,PH value is 9, and sterilising conditions is: 121 DEG C, 20 minutes;
B. the fermented and cultured of symbiosis fungi
By after the slant tube actication of culture of mycorrhizal fungi, transfer in symbiosis fungi culture medium, in 30 DEG C of perseverancesTemperature is cultivated 30 days, gets in small bacteria block access liquid symbiosis fungi culture medium and carries out fermented and cultured at colony edge;Access amount is 10% by weight; 300 revs/min of vibration rotating speeds, 30 DEG C of dark cultivations 60 days.
The cultivation of step 2. dendrobe tissue culture seedling
A. the induction of protocorm and Multiple Buds: get the wild Dendrodium tender shoots of raw tool 3-5 joint then, remove leafAfter sheet, under flowing water, rinse 5-10 minute, on superclean bench with 70% alcohol-pickled 3 minutes, thenWith 0.1% mercuric chloride solution sterilizing 10 minutes, after aseptic water washing 5 times, blot clip with aseptic blotting paperThe stem section of 0.5-3 centimetre of band joint, is inoculated on inducing culture, under 30 DEG C of conditions of cultivation temperature, cultivates30 days; Illuminance 1000～2500Lx, illumination 12 hours/day;
Described inducing culture is: MS, 6-benzyl purine 10mg/L, methyl α-naphthyl acetate 10mg/L, sucrose 40g/L,Sunglo juice 200g/L, agar 30g/L, pH9;
B. strengthening seedling and rooting is cultivated: by after many adventitious buds proliferation that utilizes the induction of stem section generations, Multiple Buds can be cutBecome single bud to be forwarded on culture medium 1 and cultivate 50 days, condition of culture is: intensity of illumination be 1000Lx～3000Lx, periodicity of illumination is illumination/dark=14h/10h, 30 DEG C of temperature; Plant height can reach 2cm and turn when aboveBe connected to culture medium 2 and cultivate 50 days, condition of culture is: intensity of illumination is 2000Lx～3000Lx, illumination weekPhase is illumination/dark=14h/10h, 30 DEG C of temperature;
Described culture medium 1 is: 1/2MS, sucrose 40g/L, sunglo juice 200g/L, banana 40g/L, agar30g／L，pH9；
Described culture medium 2 is: 1/2MS, sucrose 40g/L, sunglo juice 200g/L, banana 40g/L, activityCharcoal 10g/L, agar 30g/L, pH9;
3. group training seedling and fungal component co-incubation
Get fungal component liquid spawn 30% and be inoculated in symbiotic culture medium, dark cultivation after 9 days at 30 DEG C, thenThe access of group training seedling is cultivated 50 days altogether;
Condition of culture altogether: intensity of illumination is 1000-3000Lx, and periodicity of illumination is: illumination/dark=14h/10h,30 DEG C of temperature;
Symbiotic culture medium is: 1/2MS culture medium, potato 300g/L, sucrose 40g/L, sunglo juice 200g/L,Inositol 200mg/L, agar 30g/L, pH9;
4. the hardening of Mycorrhizal group training seedling is cultivated
Provide the growing environment of similar 3-5 month natural conditions to carry out bottle outlet transplanting;
A. under natural lighting in bottle hardening 10 days;
B. Mycorrhizal is successfully organized to training seedling and taken out from vial, clean the culture medium of root, 0.1%In liquor potassic permanganate, soak after 9 minutes and dry plantation in plastic tub, at 35 DEG C of temperature, humidity 80% andSunshade rate is to cultivate under 70% condition, within second day, starts foliage spray clear water, every day 2 times, every two weeksImpose nutrient solution once, 12 months hardening phases; Process and maintain air humidity and water content, nutrient solution with sprayingComposition be the aqueous solution that contains potassium dihydrogen phosphate and ammonium nitrate, cultivation matrix is through 121 DEG C of autoclaving 2hFresh liver moss, cool to being used for plantation after normal temperature until liver moss; Can be as required in the process of stem of noble dendrobium growthMend applying liquid mycorrhizal fungi.
Embodiment 3
Step 1: the separation of symbiosis fungi and cultivation
A. the isolation and purification of symbiosis fungi
Take the life root of wild Dendrodium, under flowing water, rinse 5-10 minute, and remove surface attachment with hairbrushThing, is then positioned on blotting paper, rinses with 72% alcohol, and after aseptic water washing 3-5 time, 0.1%Mercuric chloride solution soaks 3 minutes, aseptic water washing 4 times; Thin following is cut to 0.05-0.3cm2Tissue segments inoculationTo symbiosis fungi culture medium, cultivate, condition of culture is: 25 DEG C, and dark culturing; When notching edge grows veryWhen bacterium mycelia, employing mycelia top method of purification progressively purifying obtains pure bacterial strain;
Described symbiosis fungi culture medium is: sucrose 20g/L, oatmeal 20g/L, sunglo juice 10g/L, chlorine are mouldElement 0.01g/L, potassium dihydrogen phosphate 1g/L, magnesium sulfate 1g/L, yeast extract 2g/L, agar 5g/L, pH valueBe 5, sterilising conditions is: 121 DEG C, 20 minutes;
B. the fermented and cultured of symbiosis fungi
By after the slant tube actication of culture of mycorrhizal fungi, transfer in symbiosis fungi culture medium, in 25 DEG C of perseverancesTemperature is cultivated 10 days, gets in small bacteria block access liquid symbiosis fungi culture medium and carries out fermented and cultured at colony edge;Access amount is 2% by weight; 50 revs/min of vibration rotating speeds, 25 DEG C of dark cultivations 10 days.
The cultivation of step 2. dendrobe tissue culture seedling
A. the induction of protocorm and Multiple Buds: get the wild Dendrodium tender shoots of raw tool 3-5 joint then, remove leafAfter sheet, under flowing water, rinse 5-10 minute, on superclean bench with 70% alcohol-pickled 1.5 minutes,Use again 0.1% mercuric chloride solution sterilizing 4 minutes, after aseptic water washing 5 times, blot clip with aseptic blotting paperThe stem section of 0.5-3 centimetre of band joint, is inoculated on inducing culture, under 15 DEG C of conditions of cultivation temperature, cultivates10 days; Illuminance 1000～2500Lx, illumination 12 hours/day;
Described inducing culture is: MS, 6-benzyl purine 1mg/L, methyl α-naphthyl acetate 1mg/L, sucrose 15g/L,Sunglo juice 50g/L, agar 5g/L, pH5;
B. strengthening seedling and rooting is cultivated: by after many adventitious buds proliferation that utilizes the induction of stem section generations, Multiple Buds can be cutBecome single bud to be forwarded on culture medium 1 and cultivate 10 days, condition of culture is: intensity of illumination be 1000Lx～3000Lx, periodicity of illumination is illumination/dark=14h/10h, 25 DEG C of temperature; Plant height can reach 2cm and turn when aboveBe connected to culture medium 2 and cultivate 15 days, condition of culture is: intensity of illumination is 2000Lx～3000Lx, illumination weekPhase is illumination/dark=14h/10h, 25 DEG C of temperature;
Described culture medium 1 is: 1/2MS, sucrose 15g/L, sunglo juice 50g/L, banana 15g/L, agar5g／L，pH5；
Described culture medium 2 is: 1/2MS, sucrose 15g/L, sunglo juice 50g/L, banana 15g/L, active carbon1g/L, agar 5g/L, pH5;
3. group training seedling and fungal component co-incubation
Get fungal component liquid spawn 5% and be inoculated in symbiotic culture medium, dark cultivation after 4 days at 25 DEG C, thenThe access of group training seedling is cultivated 15 days altogether;
Condition of culture altogether: intensity of illumination is 1000-3000Lx, and periodicity of illumination is: illumination/dark=14h/10h,Temperature 20-30 DEG C;
Symbiotic culture medium is: 1/2MS culture medium, potato 150g/L, sucrose 25g/L, sunglo juice 50g/L,Inositol 100mg/L, agar 5g/L, pH4.5;
4. the hardening of Mycorrhizal group training seedling is cultivated
Annual 3-5 month is the season that bottle outlet is transplanted;
A. under natural lighting in bottle hardening 6 days;
B. Mycorrhizal is successfully organized to training seedling and taken out from vial, clean the culture medium of root, 0.1%In liquor potassic permanganate, soak after 5 minutes and dry plantation in plastic tub, in 205 DEG C of temperature, humidity 45%With under the sunshade rate condition that is 55%, cultivate, within second day, start foliage spray clear water, every day 2 times, every halfThe moon imposes nutrient solution once, 4 months hardening phases; Process and maintain air humidity and water content, nutrition with sprayingThe composition of liquid is the aqueous solution that contains potassium dihydrogen phosphate and ammonium nitrate, and cultivation matrix is through 121 DEG C of autoclavingsThe fresh liver moss of 2h is cool to being used for plantation after normal temperature until liver moss; Can be according to need in the process of stem of noble dendrobium growthMend applying liquid mycorrhizal fungi.
Embodiment 4
Step 1: the separation of symbiosis fungi and cultivation
A. the isolation and purification of symbiosis fungi
Take the life root of wild Dendrodium, under flowing water, rinse 5-10 minute, and remove surface attachment with hairbrushThing, is then positioned on blotting paper, rinses with 72% alcohol, and after aseptic water washing 3-5 time, 0.1%Mercuric chloride solution soaks 4 minutes, aseptic water washing 4 times; Thin following is cut to 0.05-0.3cm2Tissue segments inoculationTo symbiosis fungi culture medium, cultivate, condition of culture is: 28 DEG C, and dark culturing; When notching edge grows veryWhen bacterium mycelia, employing mycelia top method of purification progressively purifying obtains pure bacterial strain;
Described symbiosis fungi culture medium is: sucrose 30g/L, oatmeal 70g/L, sunglo juice 50g/L, mouldElement 0.05g/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate 0.05g/L, yeast extract 3g/L, agar 15g/L,PH value is 6, and sterilising conditions is: 121 DEG C, 20 minutes;
B. the fermented and cultured of symbiosis fungi
By after the slant tube actication of culture of mycorrhizal fungi, transfer in symbiosis fungi culture medium, in 28 DEG C of perseverancesTemperature is cultivated 20 days, gets in small bacteria block access liquid symbiosis fungi culture medium and carries out fermented and cultured at colony edge;Access amount is 8% by weight; 100 revs/min of vibration rotating speeds, 28 DEG C of dark cultivations 20 days.
The cultivation of step 2. dendrobe tissue culture seedling
A. the induction of protocorm and Multiple Buds: get the wild Dendrodium tender shoots of raw tool 3-5 joint then, remove leafAfter sheet, under flowing water, rinse 5-10 minute, on superclean bench with 70% alcohol-pickled 2 minutes, thenWith 0.1% mercuric chloride solution sterilizing 4 minutes, after aseptic water washing 5 times, blot clip with aseptic blotting paperThe stem section of 0.5-3 centimetre of band joint, is inoculated on inducing culture, under 20 DEG C of conditions of cultivation temperature, cultivates20 days; Illuminance 1000～2500Lx, illumination 12 hours/day;
Described inducing culture is: MS, 6-benzyl purine 5mg/L, methyl α-naphthyl acetate 3mg/L, sucrose 30g/L,Sunglo juice 100g/L, agar 50g/L, pH6;
B. strengthening seedling and rooting is cultivated: by after many adventitious buds proliferation that utilizes the induction of stem section generations, Multiple Buds can be cutBecome single bud to be forwarded on culture medium 1 and cultivate 20 days, condition of culture is: intensity of illumination be 1000Lx～3000Lx, periodicity of illumination is illumination/dark=14h/10h, 28 DEG C of temperature; Plant height can reach 2cm and turn when aboveBe connected to culture medium 2 and cultivate 40 days, condition of culture is: intensity of illumination is 2000Lx～3000Lx, illumination weekPhase is illumination/dark=14h/10h, 28 DEG C of temperature;
Described culture medium 1 is: 1/2MS, sucrose 30g/L, sunglo juice 150g/L, banana 20g/L, agar20g／L，pH6：
Described culture medium 2 is: 1/2MS, sucrose 30g/L, sunglo juice 150g/L, banana 30g/L, activityCharcoal 7g/L, agar 3g/L, pH7;
3. group training seedling and fungal component co-incubation
Get fungal component liquid spawn 1～30% and be inoculated in symbiotic culture medium, dark cultivation after 8 days at 25 DEG C,Again the access of group training seedling is cultivated 40 days altogether;
Condition of culture altogether: intensity of illumination is 1000-3000Lx, and periodicity of illumination is: illumination/dark=14h/10h,28 DEG C of temperature;
Symbiotic culture medium is: 1/2MS culture medium, potato 200g/L, sucrose 30g/L, sunglo juice 50g/L,Inositol 80mg/L, agar 15g/L, pH7;
4. the hardening of Mycorrhizal group training seedling is cultivated
Provide the growing environment of similar 3-5 month natural conditions to carry out bottle outlet transplanting;
A. under natural lighting in bottle hardening 8 days;
B. Mycorrhizal is successfully organized to training seedling and taken out from vial, clean the culture medium of root, 0.1%In liquor potassic permanganate, soak after 7 minutes and dry plantation in plastic tub, at 25 DEG C of temperature, humidity 60% andSunshade rate is to cultivate under 65% condition, within second day, starts foliage spray clear water, every day 2 times, every two weeksImpose nutrient solution once, 5 months hardening phases; Process and maintain air humidity and water content, nutrient solution with sprayingComposition be the aqueous solution that contains potassium dihydrogen phosphate and ammonium nitrate, cultivation matrix is through 121 DEG C of autoclaving 2hFresh liver moss, cool to being used for plantation after normal temperature until liver moss; Can be as required in the process of stem of noble dendrobium growthMend applying liquid mycorrhizal fungi.
Embodiment 5
Step 1: the separation of symbiosis fungi and cultivation
A. the isolation and purification of symbiosis fungi
Take the life root of wild Dendrodium, under flowing water, rinse 5-10 minute, and remove surface attachment with hairbrushThing, is then positioned on blotting paper, rinses with 72% alcohol, and after aseptic water washing 3-5 time, 0.1%Mercuric chloride solution soaks 4.5 minutes, aseptic water washing 4 times; Thin following is cut to 0.05-0.3cm2Tissue segments connectPlant and cultivate to symbiosis fungi culture medium, condition of culture is: 23 DEG C, and dark culturing; When notching edge growsWhen hypha,hyphae, employing mycelia top method of purification progressively purifying obtains pure bacterial strain;
Described symbiosis fungi culture medium is: sucrose 15g/L, oatmeal 90g/L, sunglo juice 130g/L, chlorine are mouldElement 0.1g/L, potassium dihydrogen phosphate 9g/L, magnesium sulfate 1g/L, yeast extract 8g/L, agar 2g/L, pH value8, sterilising conditions is: 121 DEG C, 20 minutes;
B. the fermented and cultured of symbiosis fungi
By after the slant tube actication of culture of mycorrhizal fungi, transfer in symbiosis fungi culture medium, in 27 DEG C of perseverancesTemperature is cultivated 25 days, gets in small bacteria block access liquid symbiosis fungi culture medium and carries out fermented and cultured at colony edge;Access amount is 9% by weight; 250 revs/min of vibration rotating speeds, 29 DEG C of dark cultivations 50 days.
The cultivation of step 2. dendrobe tissue culture seedling
A. the induction of protocorm and Multiple Buds: get the wild Dendrodium tender shoots of raw tool 3-5 joint then, remove leafAfter sheet, under flowing water, rinse 5-10 minute, on superclean bench with 70% alcohol-pickled 2.5 minutes,Use again 0.1% mercuric chloride solution sterilizing 9 minutes, after aseptic water washing 5 times, blot clip with aseptic blotting paperThe stem section of 0.5-3 centimetre of band joint, is inoculated on inducing culture, under 25 DEG C of conditions of cultivation temperature, cultivates25 days; Illuminance 1000～2500Lx, illumination 12 hours/day;
Described inducing culture is: MS, 6-benzyl purine 0.5mg/L, methyl α-naphthyl acetate 0.5mg/L, sucrose 35g/L,Sunglo juice 150g/L, agar 25g/L, pH8;
B. strengthening seedling and rooting is cultivated: by after many adventitious buds proliferation that utilizes the induction of stem section generations, Multiple Buds can be cutBecome single bud to be forwarded on culture medium 1 and cultivate 30 days, condition of culture is: intensity of illumination be 1000Lx～3000Lx, periodicity of illumination is illumination/dark=14h/10h, 29 DEG C of temperature; Plant height can reach 2cm and turn when aboveBe connected to culture medium 2 and cultivate 45 days, condition of culture is: intensity of illumination is 2000Lx～3000Lx, illumination weekPhase is illumination/dark=14h/10h, 28 DEG C of temperature;
Described culture medium 1 is: 1/2MS, sucrose 35g/L, sunglo juice 150g/L, banana 35g/L, agar20g／L，pH8；
Described culture medium 2 is: 1/2MS, sucrose 30g/L, sunglo juice 180g/L, banana 30g/L, activityCharcoal 8g/L, agar 25g/L, pH8;
3. group training seedling and fungal component co-incubation
Get fungal component liquid spawn 25% and be inoculated in symbiotic culture medium, dark cultivation after 7 days at 29 DEG C, thenThe access of group training seedling is cultivated 45 days altogether;
Condition of culture altogether: intensity of illumination is 1000-3000Lx, and periodicity of illumination is: illumination/dark=14h/10h,23 DEG C of temperature;
Symbiotic culture medium is: 1/2MS culture medium, potato 150g/L, sucrose 30g/L, sunglo juice 100g/L,Inositol 250mg/L, agar 18g/L, pH9;
4. the hardening of Mycorrhizal group training seedling is cultivated
Provide the growing environment of similar 3-5 month natural conditions to carry out bottle outlet transplanting;
A. under natural lighting in bottle hardening 8 days;
B. Mycorrhizal is successfully organized to training seedling and taken out from vial, clean the culture medium of root, 0.1%In liquor potassic permanganate, soak after 8 minutes and dry plantation in plastic tub, at 25 DEG C of temperature, humidity 70% andSunshade rate is to cultivate under 65% condition, within second day, starts foliage spray clear water, every day 2 times, every two weeksImpose nutrient solution once, 10 months hardening phases; Process and maintain air humidity and water content, nutrient solution with sprayingComposition be the aqueous solution that contains potassium dihydrogen phosphate and ammonium nitrate, cultivation matrix is through 121 DEG C of autoclaving 2hFresh liver moss, cool to being used for plantation after normal temperature until liver moss; Can be as required in the process of stem of noble dendrobium growthMend applying liquid mycorrhizal fungi.
Test example
Get each 1000 strains of gained stem of noble dendrobium seedling of embodiment 1～5 by method kind described in ZL200810233727.9Plant, be calculated to be motility rate, as following table 1:
| Embodiment | Survive strain number (strain) | Survival rate (%) |
| 1 | 985 | 98.5 |
| 2 | 980 | 98.0 |
| 3 | 993 | 99.3 |
| 4 | 989 | 98.9 |
| 5 | 100 | 100 |
The stem of noble dendrobium kind shoot survival percent that method of the present invention makes is as seen from the above table higher than 98%.
Claims (1)
1. the method that tissue is cultivated stem of noble dendrobium seedling fast, comprises the steps:
Step 1: the separation of symbiosis fungi and cultivation
A. the isolation and purification of symbiosis fungi
Take the life root of wild Dendrodium, under flowing water, rinse 5-10 minute, and use hairbrushRemove surface attachments, be then positioned on blotting paper, rinse with 72% alcohol,After aseptic water washing 3-5 time, 0.1% mercuric chloride solution soaks 2-5 minute, aseptic water washing 4Inferior; Radicula is cut to 0.05-0.3cm2Tissue segments be inoculated on symbiosis fungi culture medium and trainSupport, condition of culture is: 20-30 DEG C, dark culturing; When notching edge grows hypha,hyphaeTime, employing mycelia top method of purification progressively purifying obtains pure bacterial strain;
Described symbiosis fungi culture medium is: glucose or sucrose 10-40g/L, oatmeal10-100g/L, sunglo juice 1-150g/L, chloramphenicol or penicillin 0.001-1g/L, phosphoric acidPotassium dihydrogen 0.01-10g/L, magnesium sulfate 0.01-10g/L, yeast extract 1-10g/L, agar1-30g/L, pH4-9, sterilising conditions is: 121 DEG C, 20 minutes;
B. the fermented and cultured of symbiosis fungi
By after the slant tube actication of culture of mycorrhizal fungi, transfer in symbiosis fungi culture mediumIn, in 20-30 DEG C of constant temperature culture 1-30 days, get small bacteria block access liquid altogether at colony edgeIn raw fungi culture medium, carry out fermented and cultured; Access amount is 1-10% by weight; VibrationRotating speed 20-300 rev/min, the 20-30 DEG C of dark 1-60 days that cultivates;
Step 2: the cultivation of dendrobe tissue culture seedling
A. the induction of protocorm and Multiple Buds: get wild Dendrodium raw tool 3-5 save tender thenBud, removes after blade, under flowing water, rinses 5-10 minute, on superclean bench with 70%Alcohol-pickled 1-3 minute, then use 0.1% mercuric chloride solution sterilizing 2-10 minute, sterilized waterRinse after 5 times, blot with aseptic blotting paper, the stem section of clip 0.5-3 centimetre of band joint, connectsPlant to inducing culture, under cultivation temperature 10-30 DEG C condition, cultivate 3-30 days; LightIllumination 1000～2500Lx, illumination 12 hours/day;
Described inducing culture is: MS, 6-benzyl purine 0.1-10mg/L, methyl α-naphthyl acetate0.1-10mg/L, sucrose 10-40g/L, sunglo juice 20-200g/L, agar 1-30g/L,pH4-9；
B. strengthening seedling and rooting is cultivated: by after many adventitious buds proliferation that utilizes the induction of stem section generations, by clumpSprouting cuts into single bud and is forwarded on culture medium 1 and cultivates 5-50 days, and condition of culture is:Intensity of illumination is 1000Lx～3000Lx, and periodicity of illumination is illumination/dark=14h/10h,Temperature 20-30 DEG C; Plant height reaches 2cm and is forwarded to culture medium 2 when above and cultivates 10-50 days,Condition of culture is: intensity of illumination is 2000Lx～3000Lx, and periodicity of illumination is illumination/dark=14h/10h, temperature 20-30 DEG C;
Described culture medium 1 is: 1/2MS, sucrose 10-40g/L, sunglo juice 20-200g/L,Banana 10-40g/L, agar 1-30g/L, pH4-9;
Described culture medium 2 is: 1/2MS, sucrose 10-40g/L, sunglo juice 20-200g/L,Banana 10-40g/L, active carbon 0.1-10g/L, agar 1-30g/L, pH4-9;
Step 3: group training seedling and fungal component co-incubation
Get symbiosis liquid fungus strain 1～30% and be inoculated in symbiotic culture medium, at 20-30 DEG CLower dark cultivation after 3-9 days, then the access of group training seedling is cultivated to 10-50 days altogether;
Condition of culture altogether: intensity of illumination is 1000-3000Lx, and periodicity of illumination is: illumination/Dark=14h/10h, temperature 20-30 DEG C;
Symbiotic culture medium is: 1/2MS culture medium, potato 100-300g/L, sucrose20-40g/L, sunglo juice 20-200g/L, inositol 20-200mg/L, agar 1-30g/L,pH4-9；
Step 4: the hardening of Mycorrhizal group training seedling is cultivated
Selecting annual 3-5 month is the season that bottle outlet is transplanted, or similar 3-5 month is providedThe growing environment of natural conditions carries out bottle outlet transplanting;
A. under natural lighting in bottle hardening 5-10 days;
B. Mycorrhizal is successfully organized to training seedling and taken out from vial, clean the cultivation of rootBase soaks after 3-9 minute and dries plantation to plastic tub in 0.1% liquor potassic permanganateIn, under the condition that is 50-70% in temperature 15-35 DEG C, humidity 40-80% and sunshade rate, trainSupport, second day starts foliage spray clear water, and every day 2 times, every two weeks imposes nutrient solution oneInferior, hardening phase 3-12 month; Process and maintain air humidity and water content, nutrition with sprayingThe composition of liquid is the aqueous solution that contains potassium dihydrogen phosphate and ammonium nitrate, and cultivation matrix is warpThe fresh liver moss of 121 DEG C of autoclaving 2h is cool to being used for plantation after normal temperature until liver moss; At stoneIn the process of dry measure used in former times growth, mend as required applying liquid mycorrhizal fungi.
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| CN104904462B (en) * | 2015-05-25 | 2017-10-03 | 浙江欧银农业发展有限公司 | A kind of live broadcasting method of dendrobium candidum embryo |
| KR101816647B1 (en) * | 2015-09-23 | 2018-01-09 | 코웨이 주식회사 | Cosmetic composition comprising extracts of fermented usnea longissima using hypha of cauliflower mushroom as active ingredient |
| CN107535339A (en) * | 2017-09-27 | 2018-01-05 | 西双版纳银海福林石斛有限公司 | A kind of imitating wild planting process of the stem of noble dendrobium |
| CN107646656A (en) * | 2017-11-13 | 2018-02-02 | 广西******旧塘家庭农场 | A kind of implantation methods of dendrobium candidum |
| CN108834889B (en) * | 2018-05-31 | 2021-11-30 | 贵州盛达生植物发展有限公司 | Tissue culture seedling cultivation method for improving disease resistance of dendrobium officinale |
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