CN105638467A - Method for cultivating anoectochilus formosanus by using culture bag - Google Patents
Method for cultivating anoectochilus formosanus by using culture bag Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a method for cultivating anoectochilus formosanus by using a culture bag. A transparent sealing plastic bag is used for replacing a traditional glass culture bottle to serve as a culture container; and according to different growth phases of the anoectochilus formosanus, an induction culture medium, a proliferation culture medium, a strong seedling culture medium or a rooting culture medium is injected into the culture bag, so that anoectochilus formosanus explants are subjected to tissue culture and seedling culture. When the anoectochilus formosanus is subjected to the tissue culture by adopting the method, the pollution can be effectively controlled, and the contamination rate is smaller than 3%; an inoculation progress is simplified and damages to the seedlings are reduced; an inoculating speed is improved and anoectochilus formosanus tissue culture seedlings with strong disease resistance are obtained; and meanwhile, the operation of taking out seedlings from the culture bottle after bagged tissue culture also can be avoided, and integration of tissue culture, seedling growing and product transportation of the anoectochilus formosanus is realized, so that the production cost is greatly reduced, the yield is improved and the growth period is shortened.
Description
Technical field
The invention belongs to Chinese crude drug growing and cultivation technical field, be specifically related to a kind of method that culture bag cultivates Herba Anoectochili roxburghii.
Background technology
Herba Anoectochili roxburghii (Anoectochilusroxburghii (Wall.) Lindl.) is the orchid family Anoectochilus Blume rare medicinal plant, has another name called Anoectochilus nefiliforme (Nakai) hara, Anoectochilus Roxburghii etc., and it is distributed mainly on the SOUTHERN CHINA provinces such as Fujian. Herba Anoectochili roxburghii with all herbal medicine, its flat sweet in the mouth of property, there is effect of clearing away heat and cooling blood, removing dampness and detoxicating, in the good reputation having " king of medicine ", " gold grass " among the people, and it has to view and admire and waits many-sided value so that it is have wide DEVELOPMENT PROSPECT.
At present, the artificial culture of Herba Anoectochili roxburghii mainly adopts glass culture bottle to carry out tissue culture. But glass culture bottle is broken, not easily carry, and owing to glass culture bottle takes up room relatively big, when it is carried out disinfection, the culture bottle limited amount of single treatment, can reduce its production efficiency. The more important thing is, owing to culture bottle adopts spiral sealing with bottle cap, needing first bottleneck and bottle cap to be rotated calcination before inoculation operation, its disinfecting action is complicated, consuming time, and easily causes microbiological contamination because of misoperation, so that whole bottle is cultivated Seedling and gone out of use.
Summary of the invention
It is an object of the invention to provide a kind of method that culture bag cultivates Herba Anoectochili roxburghii, it adopts transparent sealing plastic bag to substitute traditional glass culture bottle and cultivates as culture vessel, can effectively control to pollute, simplify vaccine program, reduce Seedling damage, improve inoculation speed, obtain the Herba Anoectochili roxburghii tissue cultured seedling that disease resistance is strong, and seedling needs the operation of bottle outlet nursery after can avoiding bottled group of training, realize the group training of Herba Anoectochili roxburghii, nursery and Product transport integration, thus being substantially reduced production cost, improving yield, shortening growth cycle.
For achieving the above object, the present invention adopts the following technical scheme that
A kind of culture bag cultivates the method for Herba Anoectochili roxburghii, it adopts transparent sealing plastic bag as culture bag, different growth phases according to Herba Anoectochili roxburghii, culture bag is injected inducing culture, proliferated culture medium, strong seedling culture base or root media, thus the outer implant of Herba Anoectochili roxburghii is carried out group training and nursery.
Described culture bag adopts polypropylene material to make, its bag height 14cm ~ 18cm, the end long 5cm ~ 8cm, bottom width 4cm ~ 7cm; It is being respectively arranged with sealing strip from bag mouth 0.6cm ~ 0.8cm and 1.2cm ~ 1.5cm place, and on bag one sidewall, be provided with an air-permeative filter device from bag mouth 6cm ~ 10cm place;
Described air-permeative filter device comprises the tubular body connecting bag inner chamber with air, and it is highly 4mm ~ 6mm, and nozzle diameter is 3.5mm ~ 4.0mm; The outboard end of described tubular body is provided with the discoid body for being fixed on bag, and its diameter is 12mm ~ 14mm; Described discoid body is provided with the bacteriological filtration ventilated membrane that diameter is 8mm ~ 10mm at the connected entrance place of tubular body Yu air.
Described inducing culture is in the culture medium of MS+0.8mg/L��1.2mg/L6-BA+25g/L��30g/L sucrose+5.5g/L��7.5g/L carrageenan, add the rice any of several broadleaf plants juice of its weight 8%��12% and the mixed liquor of bananas juice, wherein, the addition weight of rice any of several broadleaf plants juice is 6%��10%;
Described proliferated culture medium is in the culture medium of MS+1.8mg/L��2.5mg/L6-BA+0.03mg/L��0.06mg/LNAA+30g/L��35g/L sucrose+5.5g/L��7.5g/L agar, add the rice any of several broadleaf plants juice of its weight 8%��12% and the mixed liquor of bananas juice, wherein, the addition weight of rice any of several broadleaf plants juice is 6%��10%;
Described strong seedling culture base is in the culture medium of MS+0.03mg/L��0.05mg/L6-BA+0.3mg/L��0.6mg/LNAA+30g/L��35g/L sucrose+5.5g/L��7.5g/L agar, add the rice any of several broadleaf plants juice of its weight 8%��12% and the mixed liquor of bananas juice, wherein, the addition weight of rice any of several broadleaf plants juice is 6%��10%;
Described root media is in the culture medium of 1/2MS+0.3mg/L��0.6mg/LNAA+0.3mg/L��0.6mg/LIBA+10g/L��15g/L sucrose+5.5g/L��7.5g/L agar, add the rice any of several broadleaf plants juice of its weight 8%��12% and the mixed liquor of bananas juice, wherein, the addition weight of rice any of several broadleaf plants juice is 6%��10%.
The injection rate of each culture medium is the 15%��20% of culture bag volume.
Described culture bag is cultivated the method for Herba Anoectochili roxburghii and is specifically included following steps:
1) the stem section of wild Herba Anoectochili roxburghii plant or wash seeds are removed surface dirt, with cleaning agent for fruit and vegetable soaking and washing 15min��20min, then clean 6min with the hymexazol of 0.08wt%��0.1wt% is high with the generation of 0.1wt%��0.12wt% by weight the 3:5 mixed liquid dipping being mixed, dry; 30min, sterile water wash 3��4 times is soaked again with sodium hypochlorite; Finally soak 3min��5min with the potassium permanganate of 0.15wt%��0.2wt%, blot with filter paper;
2) the stem section of clip belt segment or after being peeled off by seed hull with dissecting knife in superclean bench, it can be used as outer implant to be inoculated in the culture bag being injected with inducing culture, in 24 DEG C��25 DEG C, inducing culture callus 5d��7d under dark condition;
3) callus after inducing culture is transferred in the culture bag being injected with proliferated culture medium, in 23 DEG C��26 DEG C, cultivate 35d��45d under illumination condition, every 10d��14d subculture is once;
4) plant after propagation is transferred in the culture bag being injected with strong seedling culture base, in 22 DEG C��25 DEG C, cultivate 45d��55d under illumination condition;
5) by the stem section of the plant clip belt segment after strong sprout, be transferred in the culture bag being injected with root media, in 24 DEG C��26 DEG C, cultivate 45d��55d under illumination condition after, proceeding to seedling exercising 15d��25d in facility plastic greenhouse can emerge.
The present invention has the great advantage that
(1) present invention transparent plastic bag replaces traditional glass culture bottle to carry out the group training of Herba Anoectochili roxburghii, the use of disinfectant and fuel alcohol can be reduced in a large number, and can effectively control to pollute, microbiological contamination rate < 3%, and simplify vaccine program, alleviate workload in a large number, decrease Seedling damage, improve inoculation speed, thus obtaining the Herba Anoectochili roxburghii tissue cultured seedling that disease resistance is strong; Meanwhile, after adopting the inventive method also can avoid bottled group of training, seedling needs the operation of bottle outlet nursery, it is achieved the group training of Herba Anoectochili roxburghii, nursery and Product transport integration, thus being substantially reduced production cost, improving yield, shortening growth cycle.
(2) present invention adopts inducing culture, proliferated culture medium, strong seedling culture base and root media successively according to the different growth phases of Herba Anoectochili roxburghii. Its inducing culture prepared by adding bananas juice and rice any of several broadleaf plants juice can be effectively improved inductivity, makes induction time shorten 3d��5d; And in proliferated culture medium, add the basic element of cell division (6-BA), can substantially expand proliferation times, promote tissue cultured seedling propagation; Rice any of several broadleaf plants has the feature of good anti-anthrax, droop, it is added in strong seedling culture base and plant can be made to grow fine, plant height 4cm��6cm, the thick 2.8mm��3.4mm of stem, the number of blade 3��5 after plant cultivation 45d��55d, explanation is processed effectively facilitating plant strain growth, and gained Herba Anoectochili roxburghii tissue cultured seedling resistance increases, there is after transplanting the characteristic of good anti-anthrax, droop, substantially increase the survival rate of seedling replanting; Adding class auxin NAA, IBA in root media and can significantly improve the quality of rooting rate and root, after making transplanting, survival rate of plant reaches more than 95%.
Accompanying drawing explanation
Fig. 1 is the unitary construction schematic diagram of cultivating system in the embodiment of the present invention.
Fig. 2 is the organigram of culture bag in the embodiment of the present invention.
Fig. 3 is the organigram of air-permeative filter device in culture bag.
In figure: 1-outer packaging bag, 2-supports framework, 3-culture bag, 31-sealing strip, 32-air-permeative filter device, 321-tubular body, 322-discoid body, 323-bacteriological filtration ventilated membrane.
Detailed description of the invention
In order to make content of the present invention easily facilitate understanding, below in conjunction with detailed description of the invention, technical solutions according to the invention are described further, but the present invention is not limited only to this.
Choose Fructus Musae or the rice any of several broadleaf plants of high-quality, put in pot, add the water infusion 3min��5min of weight such as its grade, be incubated 15min��20min, elimination any of several broadleaf plants slag, take its supernatant and obtain required bananas juice or rice any of several broadleaf plants juice.
Embodiment 1
1) adopt the transparent sealing plastic bag that polypropylene material is made as culture bag, its bag height 14cm, the end long 5cm, bottom width 4cm; It is being respectively arranged with a sealing strip from bag mouth 0.6cm and 1.2cm place, and on bag one sidewall, be provided with an air-permeative filter device from bag mouth 6cm place; This air-permeative filter device comprises the tubular body connecting bag inner chamber with air, and it is highly 4mm, and nozzle diameter is 3.5mm; The outboard end of tubular body is provided with the discoid body for being fixed on bag, and its diameter is 12mm; Discoid body is provided with the bacteriological filtration ventilated membrane that diameter is 8mm, 0.18 ��m of aperture at the connected entrance place of tubular body Yu air; This culture bag is positioned in support framework, reinstalls and outer packaging bag carries out high temperature sterilize (such as Fig. 1,2,3);
2) in the culture medium of MS+0.8mg/L6-BA+25g/L sucrose+5.5g/L carrageenan, add the rice any of several broadleaf plants juice of its weight 8% and the mixed liquor (wherein, the addition weight of rice any of several broadleaf plants juice is 6%) of bananas juice, be configured to inducing culture;In the culture medium of MS+1.8mg/L6-BA+0.03mg/LNAA+30g/L sucrose+5.5g/L agar, add the rice any of several broadleaf plants juice of its weight 8% and the mixed liquor (wherein, the addition weight of rice any of several broadleaf plants juice is 6%) of bananas juice, be configured to proliferated culture medium; In the culture medium of MS+0.03mg/L6-BA+0.3mg/LNAA+30g/L sucrose+5.5g/L agar, add the rice any of several broadleaf plants juice of its weight 8% and the mixed liquor (wherein, the addition weight of rice any of several broadleaf plants juice is 6%) of bananas juice, be configured to strong seedling culture base; In the culture medium of 1/2MS+0.3mg/LNAA+0.3mg/LIBA+10g/L sucrose+5.5g/L agar, add the rice any of several broadleaf plants juice of its weight 8% and the mixed liquor (wherein, the addition weight of rice any of several broadleaf plants juice is 6%) of bananas juice, be configured to state root media;
3) the stem section of wild Herba Anoectochili roxburghii plant or wash seeds are removed surface dirt, with cleaning agent for fruit and vegetable soaking and washing 15min, then clean 6min with the hymexazol of 0.08wt% is high with the generation of 0.1wt% by weight the 3:5 mixed liquid dipping being mixed, dry; 30min, sterile water wash 3 times is soaked again with sodium hypochlorite; Finally soak 3min with the potassium permanganate of 0.15wt%, blot with filter paper;
4) the stem section of clip belt segment in superclean bench, takes 10 sections and is inoculated in the culture bag being injected with inducing culture as outer implant, in 24 DEG C, inducing culture callus 5d under dark condition;
5) callus after inducing culture is transferred in the culture bag being injected with proliferated culture medium, in 23 DEG C, cultivate 35d under illumination condition, every 10d subculture is once;
6) plant after propagation is transferred in the culture bag being injected with strong seedling culture base, in 22 DEG C, cultivate 45d under illumination condition;
7) by the stem section of the plant clip belt segment after strong sprout, be transferred in the culture bag being injected with root media, in 24 DEG C, cultivate 45d under illumination condition after, proceeding to seedling exercising 15d in facility plastic greenhouse can emerge.
Embodiment 2
1) adopt the transparent sealing plastic bag that polypropylene material is made as culture bag, its bag height 16cm, the end long 7cm, bottom width 5cm; It is being respectively arranged with two sealing strips from bag mouth 0.7cm and 1.3cm place, and on bag one sidewall, be provided with an air-permeative filter device from bag mouth 8cm place; This air-permeative filter device comprises the tubular body connecting bag inner chamber with air, and it is highly 5mm, and nozzle diameter is 3.8mm; The outboard end of tubular body is provided with the discoid body for being fixed on bag, and its diameter is 13mm; Discoid body is provided with the bacteriological filtration ventilated membrane that diameter is 9mm, 0.2 ��m of aperture at the connected entrance place of tubular body Yu air; This culture bag is positioned in support framework, reinstalls and outer packaging bag carries out high temperature sterilize;
2) in the culture medium of MS+1.0mg/L6-BA+27.5g/L sucrose+6.5g/L carrageenan, add the rice any of several broadleaf plants juice of its weight 10% and the mixed liquor (wherein, the addition weight of rice any of several broadleaf plants juice is 8%) of bananas juice, be configured to inducing culture; In the culture medium of MS+2.2mg/L6-BA+0.05mg/LNAA+32.5g/L sucrose+6.5g/L agar, add the rice any of several broadleaf plants juice of its weight 10% and the mixed liquor (wherein, the addition weight of rice any of several broadleaf plants juice is 8%) of bananas juice, be configured to proliferated culture medium; In the culture medium of MS+0.04mg/L6-BA+0.5mg/LNAA+32.5g/L sucrose+6.5g/L agar, add the rice any of several broadleaf plants juice of its weight 10% and the mixed liquor (wherein, the addition weight of rice any of several broadleaf plants juice is 8%) of bananas juice, be configured to strong seedling culture base; In the culture medium of 1/2MS+0.4mg/LNAA+0.4mg/LIBA+12.5g/L sucrose+6.5g/L agar, add the rice any of several broadleaf plants juice of its weight 10% and the mixed liquor (wherein, the addition weight of rice any of several broadleaf plants juice is 8%) of bananas juice, be configured to state root media;
3) the stem section of wild Herba Anoectochili roxburghii plant or wash seeds are removed surface dirt, with cleaning agent for fruit and vegetable soaking and washing 18min, then clean 6min with the hymexazol of 0.09wt% is high with the generation of 0.11wt% by weight the 3:5 mixed liquid dipping being mixed, dry; 30min, sterile water wash 3 times is soaked again with sodium hypochlorite; Finally soak 4min with the potassium permanganate of 0.18wt%, blot with filter paper;
4) after seed hull being peeled off with dissecting knife in superclean bench, take 11 and be inoculated in the culture bag being injected with inducing culture as outer implant, in 24 DEG C, inducing culture callus 6d under dark condition;
5) callus after inducing culture is transferred in the culture bag being injected with proliferated culture medium, in 24 DEG C, cultivate 40d under illumination condition, every 12d subculture is once;
6) plant after propagation is transferred in the culture bag being injected with strong seedling culture base, in 24 DEG C, cultivate 50d under illumination condition;
7) by the stem section of the plant clip belt segment after strong sprout, be transferred in the culture bag being injected with root media, in 25 DEG C, cultivate 50d under illumination condition after, proceeding to seedling exercising 20d in facility plastic greenhouse can emerge.
Embodiment 3
1) adopt the transparent sealing plastic bag that polypropylene material is made as culture bag, its bag height 18cm, the end long 8cm, bottom width 7cm; It is being respectively arranged with two sealing strips from bag mouth 0.8cm and 1.5cm place, and on bag one sidewall, be provided with an air-permeative filter device from bag mouth 10cm place; This air-permeative filter device comprises the tubular body connecting bag inner chamber with air, and it is highly 6mm, and nozzle diameter is 4.0mm; The outboard end of tubular body is provided with the discoid body for being fixed on bag, and its diameter is 14mm; Discoid body is provided with the bacteriological filtration ventilated membrane that diameter is 10mm, 0.22 ��m of aperture at the connected entrance place of tubular body Yu air; This culture bag is positioned in support framework, reinstalls and outer packaging bag carries out high temperature sterilize;
2) in the culture medium of MS+1.2mg/L6-BA+30g/L sucrose+7.5g/L carrageenan, add the rice any of several broadleaf plants juice of its weight 12% and the mixed liquor (wherein, the addition weight of rice any of several broadleaf plants juice is 10%) of bananas juice, be configured to inducing culture; In the culture medium of MS+2.5mg/L6-BA+0.06mg/LNAA+35g/L sucrose+7.5g/L agar, add the rice any of several broadleaf plants juice of its weight 12% and the mixed liquor (wherein, the addition weight of rice any of several broadleaf plants juice is 10%) of bananas juice, be configured to proliferated culture medium; In the culture medium of MS+0.05mg/L6-BA+0.6mg/LNAA+35g/L sucrose+7.5g/L agar, add the rice any of several broadleaf plants juice of its weight 12% and the mixed liquor (wherein, the addition weight of rice any of several broadleaf plants juice is 10%) of bananas juice, be configured to strong seedling culture base; In the culture medium of 1/2MS+0.6mg/LNAA+0.6mg/LIBA+15g/L sucrose+7.5g/L agar, add the rice any of several broadleaf plants juice of its weight 12% and the mixed liquor (wherein, the addition weight of rice any of several broadleaf plants juice is 10%) of bananas juice, be configured to state root media;
3) the stem section of wild Herba Anoectochili roxburghii plant or wash seeds are removed surface dirt, with cleaning agent for fruit and vegetable soaking and washing 20min, then clean 6min with the hymexazol of 0.1wt% is high with the generation of 0.12wt% by weight the 3:5 mixed liquid dipping being mixed, dry; 30min, sterile water wash 4 times is soaked again with sodium hypochlorite; Finally soak 5min with the potassium permanganate of 0.2wt%, blot with filter paper;
4) in superclean bench after the stem section of clip belt segment, take 12 sections and be inoculated in the culture bag being injected with inducing culture as outer implant, in 25 DEG C, inducing culture callus 7d under dark condition;
5) callus after inducing culture is transferred in the culture bag being injected with proliferated culture medium, in 26 DEG C, cultivate 45d under illumination condition, every 14d subculture is once;
6) plant after propagation is transferred in the culture bag being injected with strong seedling culture base, in 25 DEG C, cultivate 55d under illumination condition;
7) by the stem section of the plant clip belt segment after strong sprout, be transferred in the culture bag being injected with root media, in 26 DEG C, cultivate 55d under illumination condition after, proceeding to seedling exercising 25d in facility plastic greenhouse can emerge.
Adopting the inventive method to carry out Herba Anoectochili roxburghii tissue culture, work efficiency can be made to improve, plant grows fine, plant height 4cm��6cm, the thick 2.8mm��3.4mm of stem, the number of blade 3��5 after cultivation 45d��55d; Seedling pollution rate is below 3%, and planting percent reaches more than 95%, and 180d��200d can dispatch from the factory, and production cost can reduce 30%��50%.
The foregoing is only presently preferred embodiments of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to the covering scope of the present invention.
Claims (5)
1. the method cultivating Herba Anoectochili roxburghii by culture bag, it is characterized in that: adopt transparent sealing plastic bag as culture bag, different growth phases according to Herba Anoectochili roxburghii, culture bag is injected inducing culture, proliferated culture medium, strong seedling culture base or root media, thus the outer implant of Herba Anoectochili roxburghii is carried out group training and nursery.
2. the method cultivating Herba Anoectochili roxburghii by culture bag according to claim 1, it is characterised in that: described culture bag adopts polypropylene material to make, its bag height 14cm ~ 18cm, the end long 5cm ~ 8cm, bottom width 4cm ~ 7cm; It is being respectively arranged with sealing strip from bag mouth 0.6cm ~ 0.8cm and 1.2cm ~ 1.5cm place, and on bag one sidewall, be provided with an air-permeative filter device from bag mouth 6cm ~ 10cm place;
Described air-permeative filter device comprises the tubular body connecting bag inner chamber with air, and it is highly 4mm ~ 6mm, and nozzle diameter is 3.5mm ~ 4.0mm; The outboard end of described tubular body is provided with the discoid body for being fixed on bag, and its diameter is 12mm ~ 14mm; Described discoid body is provided with the bacteriological filtration ventilated membrane that diameter is 8mm ~ 10mm at the connected entrance place of tubular body Yu air.
3. the method cultivating Herba Anoectochili roxburghii by culture bag according to claim 1, it is characterized in that: described inducing culture is in the culture medium of MS+0.8mg/L��1.2mg/L6-BA+25g/L��30g/L sucrose+5.5g/L��7.5g/L carrageenan, add the rice any of several broadleaf plants juice of its weight 8%��12% and the mixed liquor of bananas juice, wherein, the addition weight of rice any of several broadleaf plants juice is 6%��10%;
Described proliferated culture medium is in the culture medium of MS+1.8mg/L��2.5mg/L6-BA+0.03mg/L��0.06mg/LNAA+30g/L��35g/L sucrose+5.5g/L��7.5g/L agar, add the rice any of several broadleaf plants juice of its weight 8%��12% and the mixed liquor of bananas juice, wherein, the addition weight of rice any of several broadleaf plants juice is 6%��10%;
Described strong seedling culture base is in the culture medium of MS+0.03mg/L��0.05mg/L6-BA+0.3mg/L��0.6mg/LNAA+30g/L��35g/L sucrose+5.5g/L��7.5g/L agar, add the rice any of several broadleaf plants juice of its weight 8%��12% and the mixed liquor of bananas juice, wherein, the addition weight of rice any of several broadleaf plants juice is 6%��10%;
Described root media is in the culture medium of 1/2MS+0.3mg/L��0.6mg/LNAA+0.3mg/L��0.6mg/LIBA+10g/L��15g/L sucrose+5.5g/L��7.5g/L agar, add the rice any of several broadleaf plants juice of its weight 8%��12% and the mixed liquor of bananas juice, wherein, the addition weight of rice any of several broadleaf plants juice is 6%��10%.
4. the method cultivating Herba Anoectochili roxburghii by culture bag according to claim 1, it is characterised in that: the injection rate of each culture medium is the 15%��20% of culture bag volume.
5. the method cultivating Herba Anoectochili roxburghii by culture bag according to claim 1, it is characterised in that: it specifically includes following steps:
1) the stem section of wild Herba Anoectochili roxburghii plant or wash seeds are removed surface dirt, with cleaning agent for fruit and vegetable soaking and washing 15min��20min, then clean 6min with the hymexazol of 0.08wt%��0.1wt% is high with the generation of 0.1wt%��0.12wt% by weight the 3:5 mixed liquid dipping being mixed, dry; 30min, sterile water wash 3��4 times is soaked again with sodium hypochlorite; Finally soak 3min��5min with the potassium permanganate of 0.15wt%��0.2wt%, blot with filter paper;
2) the stem section of clip belt segment or after being peeled off by seed hull with dissecting knife in superclean bench, it can be used as outer implant to be inoculated in the culture bag being injected with inducing culture, in 24 DEG C��25 DEG C, inducing culture callus 5d��7d under dark condition;
3) callus after inducing culture is transferred in the culture bag being injected with proliferated culture medium, in 23 DEG C��26 DEG C, cultivate 35d��45d under illumination condition, every 10d��14d subculture is once;
4) plant after propagation is transferred in the culture bag being injected with strong seedling culture base, in 22 DEG C��25 DEG C, cultivate 45d��55d under illumination condition;
5) by the stem section of the plant clip belt segment after strong sprout, be transferred in the culture bag being injected with root media, in 24 DEG C��26 DEG C, cultivate 45d��55d under illumination condition after, proceeding to seedling exercising 15d��25d in facility plastic greenhouse can emerge.
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| CN201610002630.1A CN105638467B (en) | 2016-01-06 | 2016-01-06 | A kind of method that roxburgh anoectochilus terminal bud is cultivated with culture bag |
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| CN107333558A (en) * | 2017-08-30 | 2017-11-10 | 广东茂丰源农业科技有限公司 | A kind of packed roxburgh anoectochilus terminal bud implantation methods of sealing |
| CN109452330A (en) * | 2017-11-21 | 2019-03-12 | 浙江省嘉兴市农业科学研究院（所） | A kind of Plant Tissue Breeding bacteriostatic agent and its application in roxburgh anoectochilus terminal bud tissue cultures |
| CN109644851A (en) * | 2019-01-29 | 2019-04-19 | 福建农林大学 | A kind of method of factory culture roxburgh anoectochilus terminal bud |
| CN109769675A (en) * | 2019-03-11 | 2019-05-21 | 李甲辉 | Roxburgh anoectochilus terminal bud implantation methods |
| CN112772412A (en) * | 2020-12-30 | 2021-05-11 | 吴有光 | Method for performing open type plant tissue culture and rapid propagation by utilizing self-standing self-sealing film bag |
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| CN107333558A (en) * | 2017-08-30 | 2017-11-10 | 广东茂丰源农业科技有限公司 | A kind of packed roxburgh anoectochilus terminal bud implantation methods of sealing |
| CN109452330A (en) * | 2017-11-21 | 2019-03-12 | 浙江省嘉兴市农业科学研究院（所） | A kind of Plant Tissue Breeding bacteriostatic agent and its application in roxburgh anoectochilus terminal bud tissue cultures |
| CN109452330B (en) * | 2017-11-21 | 2020-10-20 | 浙江省嘉兴市农业科学研究院（所） | Bacteriostatic agent for plant tissue culture and application of bacteriostatic agent in anoectochilus formosanus tissue culture |
| CN109644851A (en) * | 2019-01-29 | 2019-04-19 | 福建农林大学 | A kind of method of factory culture roxburgh anoectochilus terminal bud |
| CN109769675A (en) * | 2019-03-11 | 2019-05-21 | 李甲辉 | Roxburgh anoectochilus terminal bud implantation methods |
| CN112772412A (en) * | 2020-12-30 | 2021-05-11 | 吴有光 | Method for performing open type plant tissue culture and rapid propagation by utilizing self-standing self-sealing film bag |
| CN114467731A (en) * | 2022-02-25 | 2022-05-13 | 金华市农业科学研究院(浙江省农业机械研究院) | Organic cultivation method for anoectochilus formosanus in sealed environment |
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