CN106386482A - Rapid propagation method of high-quality seedlings of Dendrobium officinale with natural light cultivation for one-step seedling formation - Google Patents
Rapid propagation method of high-quality seedlings of Dendrobium officinale with natural light cultivation for one-step seedling formation Download PDFInfo
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- 239000004471 Glycine Substances 0.000 claims description 3
- 240000008790 Musa x paradisiaca Species 0.000 claims description 3
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a rapid propagation method of high-quality seedlings of Dendrobium officinale with natural light cultivation for one-step seedling formation. The method mainly comprises the following six steps in order: a Dendrobium officinale material is prepared, cultivation rooms only with natural light are established, a fluid suspension of seeds is prepared, aseptic sowing is carried out for seeds, one-step seedling formation cultivation, and transplanting of test-tube seedlings. The one-step seedling formation cultivation is further divided into three phases: induced cultivation of protocorm, cultivation of small plantlets, and cultivation of big seedlings. The invention also comprises a one-step seedling formation medium used in the rapid propagation method. A one-step seedling formation is carried out without artificial transplantation, the cultivation rooms only with natural light are used, positions for cultivation can be changed according to different demands of light in different growth stages, and produced seedlings are healthy and strong as well as are easy to survive; special seedling exercising treatment is not needed before transplantation, after the seedlings are out of bottles, the seedlings can be transplanted directly, and production cost is substantially reduced. The seeds with high germination rate, medium with abundant nutrition and cultivation rooms only with natural light are used, in order to realize large scale production of one-step seedling formation of Dendrobium officinale seedlings.
Description
Technical field
The invention belongs to by the plant regeneration technology of tissue culture technique and in particular to a kind of Herba Dendrobii high quality seedling
Natural optical culture once-seedling forming quick propagating method, and the once-seedling forming culture medium being adopted.
Background technology
Herba Dendrobii is orchid family Dendrobium Epiphyte, the rare Chinese medicine being just well recognized as since ancient times.Wild iron sheet stone
Environmental condition needed for dry measure used in former times growth promoter is extremely harsh, poor growth, and breeding potential is extremely low, and artificially predatory excavation has been directed at open country
Pig iron skin Dendrobium Plants are bordering on exhaustion.Current propagation method mainly divides two classes, and a class is tissue rapid propagation.Should before tissue cultured seedling cultivation
First carry out the seedling exercising of 14～21 days, concrete measure is that tissue cultured seedling is moved to seedling exercising room so as in the environment of open change gradually
Reform of nature environment, can bottle outlet plantation when tissue cultured seedling leaf color is dark green.First bottle cap is opened before bottle outlet, allow bottle seedling outdoor empty
Place 2～3 days in gas, allow its reform of nature humiture.Then carry out washing Seedling, wash and during Seedling, the seedling with culture medium is gently taken
Go out, be placed in cleaning in basin, pollution Seedling and bare-root seeding or few root should be separated.Tissue cultured seedling is first cleaned with tap water, mainly
Agar should be washed off, in order to avoid agar is mouldy causes rotten, then be rinsed with clear water again.Wash during Seedling preferably based on Seedling size and
Good and bad synchronization is classified, to cultivate, to manage, the survival rate improving tissue cultured seedling simultaneously makes it grow neatly.Another kind of is plant division
Cuttage, cardinal principle is selection life in 1 year or life in 2 years during plant division, color and luster is light green, healthy and strong, sprout many, well developed root system and no disease and pests harm
Plant make kind of a strain, cut off deadwood, disconnected branch, old branch and long fibrous root, strain clump cut, be divided into little Cong, every clump carries leaf
Stem strain 5～7, you can plantation.
Although the technology that tissue culture breeds seedlings of Dendrobium officinale has succeeded, seedlings of Dendrobium officinale generally existing is cultivated
Cycle length, pollution rate height, complex operation, production cost higher the problems such as, thus govern the quick of Herba Dendrobii industry sending out
Exhibition.As Publication No. CN101564008A, entitled " a kind of no hormone culture of dendrobium candidum axenic seedlings and Fast-propagation side
The domestic patent of invention of method " discloses a kind of culture of no hormone and the method for quickly breeding of dendrobium candidum axenic seedlings, and content includes
The formation of the axenic germination of Seeds of Dendrobium Candidum, protocorm and seedling and preservation, the taking root, breed and strong seedling culture of aseptic seedling,
The process such as the experienced Seedling of aseptic seedling and transplanting.But this technology have the drawback that the Induction Process of protocorm and seedling be related to HONGGUANG,
The use of green glow, unglazed, gold-tinted and blue light, therefore process are extremely complex, and the process inducing is difficult to control, and is unfavorable in big
Technical scale production application
Content of the invention
The technical problem to be solved in the present invention is the cultivation cycle length of seedlings of Dendrobium officinale generally existing, pollution rate height, behaviour
The problems such as make complicated, production cost is higher problem, the present invention is by using the seed of high germination rate, nutritious culture
Base, full natural light culturing room realize seedlings of Dendrobium officinale once-seedling forming large-scale production, have low cost, efficient feature,
It is the High biotechnology of the large-scale production of seedling, the sustainable development of Herba Dendrobii industry can be promoted.
For solving the above problems, technical scheme proposed by the present invention is a kind of Herba Dendrobii high quality seedling nature optical culture one
Secondary seedling forming quick propagating method, comprises the steps of：
(1) prepare Herba Dendrobii material；
(2) build full natural light culturing room
Using light transmissive material, and it is equipped with shading system, cooling system and culturing rack, guarantee that culture is indoor by shading system
The intensity of illumination of top layer culturing rack is 3000～5000Lux, and intensity of illumination moves down and reduces with the number of plies of culturing rack, and bottom is trained
The intensity of illumination of foster frame is 100～1000Lux, and cooling system can be cultivated by the temperature control of glass culturing room at 20～30 DEG C
Depending on frame length can be according to the size of culturing room；
(3) make seed suspension liquid
In Herba Dendrobii field gene bank, the strong healthy strain of selection robust growth, germination rate height, resistance, disease and insect resistance,
Carry out artificial pollination bagging in season of flowers, the capsule choosing certain limit fruit age is explant, carries out disinfection, cleans and removes
Water, then uses aseptic operation knife to cut capsule, the seed in capsule is all eluted to one equipped with the bottle of sterilized water, each capsule
The seed correspondence of fruit is eluted to a bottle, makes seed suspension liquid, each capsule seed suspension liquid is carried out with the inspection of seed density
Survey, the inoculation that seed concentration is 10-15 grain/milliliter is diluted to further according to every bottle of suspension seed concentration sterilized water
Seed suspension liquid；
(4) aseptic seeding
The once-seedling forming extremely equipped with 150 milliliters for 2 milliliters of the seed suspension liquid inoculated is drawn with liquid-transfering gun under aseptic condition
In the Cymbidium ensifolium (L.) Sw. culture bottle of solid medium, the seed amount 20-30 grain of every bottle of Herba Dendrobii；
(5) once-seedling forming culture
Protocorm inducing culture：Seed suspension liquid is uniformly distributed in media surface, this culture bottle is placed entirely natural
On culturing rack in optical culture room, keep its intensity of illumination in 100～1000Lux, keep culture room temperature in (26 ± 2) DEG C,
Cultivate 60～90 days under the conditions of this, seed is sprouted and formed protocorm, and protocorm sends out roots and blade；
Seedling is cultivated：By the suitable layers of seedling movement to culturing rack, strengthen its natral light cahure, promote seedling growth,
This period incubation time is 90 days, and cultivation temperature is (26 ± 2) DEG C, and seedling grows up to seedlings, height of seedling 3～5cm, 3～5～leaf
Piece, 3～5 root hairs, 1～3 centimetre of root length；Seedlings base portion sprouts 1-2 new talent, forms tufted seedling；
Stock breeding：By the top layer of seedlings movement to culturing rack, cultivated under high natural light light intensity, both can be promoted
The growth of seedlings, can reach the effect of seedling exercising again, and this period cultivates 60 days, and cultivation temperature is (26 ± 2) DEG C, now iron sheet stone
Every clump of 2-5 Seedling of dry measure used in former times Seedling, height of seedling 5～8cm, have 4-6 save, 4 with blade, more than 3 roots, thick more than the 2.0mm of stem, reach
The standard of Seedling；
(6) transplanting of test tube seedling
During transplanting, take out Seedling of taking root from culture bottle, after cleaning the culture medium of attachment, molten with millesimal potassium permanganate
Immersion steeps 5min, and Seedling of then taking root dries and bleaches to root, and planting matrix, using sterile-processed bark, notes keeping suitable
Preferably humidity and temperature, is placed in cultivation at shady and cool ventilation, and the plant after surviving for 30 days about can produce new root system, now may move into
Booth carries out normal water, fertilizer, pencil reason.
Further, it is assumed that culturing rack is four-layer structure in step (2), then top layer second and third layer of culturing rack illumination down
Intensity is 1000～3000Lux.
Preferably, certain limit fruit age described in step (3) is 120 days.
Further, sterilization described in step (3), cleaning and eliminating water detailed process are：70% ethanol soaks 30 seconds, then
Sterilized 15～20 minutes with 0.1% mercuric chloride solution, aseptic water washing 4～5 times, with aseptic under aseptic condition on the superclean bench
Filter paper blots the moisture on capsule surface.
Further, the detection of seed density described in above-mentioned steps (3) refers to each capsule seed suspension liquid is planted
The detection of sub- density, detailed process is to draw 20 microlitres with aseptic liquid-transfering gun in the case of shaking up to calculate it under biological microscope
Seed amount, every bottle of seed suspension liquid repeats to take 10 times and calculates its averagely every milliliter seed number.
In addition, the present invention also provides a kind of above-mentioned Herba Dendrobii high quality seedling nature optical culture once-seedling forming Fast-propagation side
Once-seedling forming culture medium used in method, it comprises following component：Containing spending precious No. 1 1～2g, spend precious No. 2 1～2g, albumen for every liter
Peptone 0.5～2g, mashed potatoes 40～80g, banana puree 50～100g, apple butter 10～20g, activated carbon 500～1000mg, inositol 80
～120mg, glycine 1.5～2.5mg, thiamine hydrochloride (VB1) 0.05～0.2mg, pyridoxine hydrochloride (VB6) 0.4～
0.8mg, 0.05～0.1 milligram of nicotinic acid 0.4～0.8mg, 6- benzyl purine, naphthalene acetic acid (NAA) 0.2～2mg, sucrose 15～30g,
Agar 6～7g, pH 5.4-5.6.
Compared with prior art, advantage of the invention is that：
1, switching can be not required to manually, adds using full natural light culturing room, according to different growth phases pair with once-seedling forming
The position that the demand of light is different and moves culture, the seedling stalwartness produced easily survives, and needs not move through special before transplanting
Hardening treatment, directly bottle outlet can transplant, significantly reduce production cost.
2, carry out seedlings of Dendrobium officinale using the seed of high germination rate, nutritious culture medium, full natural light culturing room
Once-seedling forming large-scale production, test tube seedling no disease pest robust growth, seedling has quality better, resistance, growth is rapid, be easy to life
Produce management and reduce the advantage such as production cost, it is possible to achieve regularly, determine that matter, quantitative offer specification be standardized, neat and consistent high-quality
Herba Dendrobii test tube seedling is to meet the market demand.
Brief description
Fig. 1 is the flow chart of the present invention.
Specific embodiment
In conjunction with accompanying drawing, the specific embodiment of the present invention is described in further detail.
The flow chart of the present invention is as shown in figure 1, be mainly made up of 6 steps.It is followed successively by preparation Herba Dendrobii material, build
Full natural light culturing room, makes seed suspension liquid, aseptic seeding, the transplanting of once-seedling forming culture and test tube seedling.Wherein once become
Seedling culture is further divided into protocorm inducing culture, seedling culture and stock breeding three phases.
For making those of ordinary skill in the art that the present invention may be better understood, now provide a specific enforcement
Example is as follows：
1. prepare Herba Dendrobii (Dendrobium officinale Kimura et Migo) material.
2. full natural light culturing room builds：Build up the culturing room of printing opacity using safety glass, be equipped with shading system, cooling sets
Standby, culturing rack.Shading system includes inside and outside sunshade net, makes the illumination of culture building overlay culturing rack by sunshade net inside and outside regulation and control
Intensity is 3000～5000Lux, and top layer second and third layer of culturing rack intensity of illumination down is 1000～3000Lux, and bottom pushes up
The lower 4th layer of intensity of illumination 100～1000Lux of layer.Cooling system adopts air conditioner for both cooling and heating, can be by the temperature of glass culturing room
Control at 20～30 DEG C.A width of 60 centimetres of culturing rack, a height of 200 centimetres, from the ground 10 centimetres be the 4th layer, layer away from 60 centimetres,
Totally four layers, depending on culturing rack length can be according to the size of culturing room.
3. the making of seed suspension liquid：In Herba Dendrobii field gene bank, choose robust growth, germination rate height, resistance, resist
The strong healthy strain of pest and disease damage, carries out artificial pollination bagging in season of flowers, and choosing the capsule that fruit age is 120 days is explant
Body, soaks 30 seconds in 70% ethanol, then is sterilized 15～20 minutes with 0.1% mercuric chloride solution, aseptic water washing 4～5 times, Yu Chao
Blot the moisture on capsule surface with aseptic filter paper under aseptic condition on net workbench.Then aseptic operation knife is used to cut capsule, will
Seed in capsule is all eluted in a triangular flask equipped with 200 milliliters of sterilized water, and the seed correspondence of each capsule is eluted to
The one bottled triangular flask having 200 milliliters of sterilized water, makes seed suspension liquid.Seed density is carried out to each capsule seed suspension liquid
Detection, in the case of shaking up with aseptic liquid-transfering gun draw 20 microlitres calculate its seed amount, every bottle of kind under biological microscope
Fullness over the chest during pregnancy supernatant liquid repeats to take 10 times and calculates its averagely every milliliter seed number.Further with sterilized water according to every bottle of suspension seed concentration
It is diluted to the seed suspension liquid of the inoculation that seed concentration is 10-15 grain/milliliter.
4. aseptic seeding：2 milliliters of the seed suspension liquid of inoculation is drawn to equipped with 150 milliliters with liquid-transfering gun under aseptic condition
The Cymbidium ensifolium (L.) Sw. culture bottle of once-seedling forming solid medium in, the seed amount 20-30 grain of every bottle of Herba Dendrobii.Once-seedling forming is cultivated
Base：Containing spending precious No. 1 1.5g, spend precious No. 2 1.5g, peptone 2g, mashed potatoes 40g, banana puree 80g, apple butter 10g, activity for every liter
Carbon 500～1000mg, inositol 100mg, glycine 2.0mg, thiamine hydrochloride (VB1) 0.2mg, pyridoxine hydrochloride (VB6)
0.5mg, 0.05 milligram of nicotinic acid 0.5mg, 6- benzyl purine, naphthalene acetic acid (NAA) 0.5mg, sucrose 20g, agar 6g, pH 5.4-
5.6.
5. once-seedling forming culture：
Protocorm inducing culture：Seed suspension liquid is uniformly distributed in media surface, this culture bottle is placed entirely natural
On the 4th floor culturing rack in optical culture room, keep its intensity of illumination in 100～1000Lux, keep culture room temperature (25 ±
2)℃.Cultivate 60～90 days under the conditions of this, seed is sprouted and formed protocorm, and protocorm sends out roots and blade, cultivation temperature is (26
±2)℃.
Seedling is cultivated：By the second of seedling movement to culturing rack or third layer, strengthen its natral light cahure, promote seedling
Growth, this period incubation time is 90 days, and seedling grows up to seedlings, height of seedling 3～5cm, 3～5～blade, 3～5 root hairs, root
Long 1～3 centimetre；Seedlings base portion sprouts 1-2 new talent, forms tufted seedling, and cultivation temperature is (26 ± 2) DEG C.
Stock breeding：By the ground floor of seedlings movement to culturing rack, cultivated under high natural light light intensity, both can be promoted
Enter the growth of seedlings, the effect of seedling exercising can be reached again.This period cultivates 60 days, now every clump of 2-5 Seedling of Herba Dendrobii Seedling, Seedling
High 5～8cm, have 4-6 save, 4 with blade, more than 3 roots, thick more than the 2.0mm of stem, reach the standard emerged, cultivation temperature
For (26 ± 2) DEG C.
6. the transplanting of test tube seedling：During transplanting, take out Seedling of taking root from culture bottle, after cleaning the culture medium of attachment, with thousand points
One of potassium permanganate solution soak 5min, Seedling of then taking root dries and bleaches to root, and planting matrix is using sterile-processed
Bark, note keeping suitable humidity and temperature, be placed in cultivation at shady and cool ventilation, survival rate is up to more than 98%.30 days about
Plant after surviving can produce new root system, now may move into booth and carry out normal water, fertilizer, pencil reason.
The foregoing is only a specific embodiment of the present invention, not in order to limit the present invention, all essences in the present invention
Within god and principle, any modification, equivalent substitution and improvement made etc., should be included within the scope of the present invention.
Claims (6)
1. Herba Dendrobii high quality seedling nature optical culture once-seedling forming quick propagating method is it is characterised in that comprise the steps of：
(1) prepare Herba Dendrobii material；
(2) build full natural light culturing room
Using light transmissive material, and it is equipped with shading system, cooling system and culturing rack, guarantee to cultivate building overlay by shading system
The intensity of illumination of culturing rack is 3000～5000Lux, and intensity of illumination moves down and reduces, bottom culturing rack with the number of plies of culturing rack
Intensity of illumination be 100～1000Lux, cooling system can be by the temperature control of glass culturing room at 20～30 DEG C, and culturing rack is long
Depending on degree can be according to the size of culturing room；
(3) make seed suspension liquid
In Herba Dendrobii field gene bank, the strong healthy strain of selection robust growth, germination rate height, resistance, disease and insect resistance, Yu Kai
Adolescence carries out artificial pollination bagging, and the capsule choosing certain limit fruit age is explant, carries out disinfection, cleans and eliminating water,
Then use aseptic operation knife to cut capsule, the seed in capsule is all eluted to one equipped with the bottle of sterilized water, each capsule
Seed correspondence be eluted to a bottle, make seed suspension liquid, each capsule seed suspension liquid carried out with the detection of seed density,
It is diluted to the kind of the inoculation that seed concentration is 10-15 grain/milliliter according to every bottle of suspension seed concentration sterilized water further
Fullness over the chest during pregnancy supernatant liquid；
(4) aseptic seeding
The once-seedling forming solid extremely equipped with 150 milliliters for 2 milliliters of the seed suspension liquid inoculated is drawn with liquid-transfering gun under aseptic condition
In the Cymbidium ensifolium (L.) Sw. culture bottle of culture medium, the seed amount 20-30 grain of every bottle of Herba Dendrobii；
(5) once-seedling forming culture
Protocorm inducing culture：Seed suspension liquid is uniformly distributed in media surface, this culture bottle is placed full natural light training
On culturing rack in foster room, keep its intensity of illumination in 100～1000Lux, keep culture room temperature in (26 ± 2) DEG C, this
Cultivate 60～90 days under part, seed is sprouted and formed protocorm, and protocorm sends out roots and blade；
Seedling is cultivated：By the suitable layers of seedling movement to culturing rack, strengthen its natral light cahure, promote seedling growth, now
Phase incubation time is 90 days, and cultivation temperature is (26 ± 2) DEG C, and seedling grows up to seedlings, height of seedling 3～5cm, 3～5～blade, 3～
Article 5, root hair, 1～3 centimetre of root length；Seedlings base portion sprouts 1-2 new talent, forms tufted seedling；
Stock breeding：By the top layer of seedlings movement to culturing rack, cultivated under high natural light light intensity, both can have been promoted seedlings
Growth, the effect of seedling exercising can be reached again, this period cultivate 60 days, cultivation temperature be (26 ± 2) DEG C, now Herba Dendrobii Seedling
Every clump of 2-5 Seedling, height of seedling 5～8cm, have 4-6 to save, 4 with blade, more than 3 roots, stem thick more than 2.0mm, reach and emerge
Standard；
(6) transplanting of test tube seedling
During transplanting, take out Seedling of taking root from culture bottle, after cleaning the culture medium of attachment, with the leaching of millesimal potassium permanganate solution
Bubble 5min, Seedling of then taking root dries and bleaches to root, and planting matrix, using sterile-processed bark, notes keeping suitably wet
Degree and temperature, are placed in cultivation at shady and cool ventilation, and the plant after surviving for 30 days about can produce new root system, now may move into booth
Carry out normal water, fertilizer, pencil reason.
2. Herba Dendrobii high quality seedling nature optical culture once-seedling forming quick propagating method according to claim 1, it is special
Levy and be in step (2), top layer second and third layer of culturing rack intensity of illumination down is 1000～3000Lux.
3. Herba Dendrobii high quality seedling nature optical culture once-seedling forming quick propagating method according to claim 1, it is special
Levy and be that certain limit fruit age described in step (3) is preferably 120 days.
4. Herba Dendrobii high quality seedling nature optical culture once-seedling forming quick propagating method according to claim 1, it is special
Levy and be that sterilization described in step (3), cleaning and eliminating water are specially immersion 30 seconds in 70% ethanol, more molten with 0.1% mercuric chloride
Liquid disinfectant 15～20 minutes, aseptic water washing 4～5 times, blots capsule with aseptic filter paper under aseptic condition on the superclean bench
The moisture on surface.
5. Herba Dendrobii high quality seedling nature optical culture once-seedling forming quick propagating method according to claim 1, it is special
Levy and be that the detection of seed density described in step (3) refers to each capsule seed suspension liquid is carried out with the detection of seed density,
Detailed process is to draw 20 microlitres with aseptic liquid-transfering gun in the case of shaking up to calculate its seed amount under biological microscope, every bottle
Seed suspension liquid repeats to take 10 times and calculates its averagely every milliliter seed number.
6. use in the Herba Dendrobii high quality seedling nature optical culture once-seedling forming quick propagating method described in a kind of claim 1
Once-seedling forming culture medium it is characterised in that comprising following component：Containing spending precious No. 1 1～2g, spend precious No. 2 1～2g, albumen for every liter
Peptone 0.5～2g, mashed potatoes 40～80g, banana puree 50～100g, apple butter 10～20g, activated carbon 500～1000mg, inositol 80
～120mg, glycine 1.5～2.5mg, thiamine hydrochloride (VB1) 0.05～0.2mg, pyridoxine hydrochloride (VB6) 0.4～
0.8mg, 0.05～0.1 milligram of nicotinic acid 0.4～0.8mg, 6- benzyl purine, naphthalene acetic acid (NAA) 0.2～2mg, sucrose 15～30g,
Agar 6～7g, pH 5.4-5.6.
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