CN105993961A - Plant tissue culture support - Google Patents
Plant tissue culture support Download PDFInfo
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- CN105993961A CN105993961A CN201610602204.1A CN201610602204A CN105993961A CN 105993961 A CN105993961 A CN 105993961A CN 201610602204 A CN201610602204 A CN 201610602204A CN 105993961 A CN105993961 A CN 105993961A
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- culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention belongs to the technical field of biology, and particularly relates to a plant tissue culture support. The support comprises dry sphagnum and de-ionized water, wherein the volume ratio of the dry sphagnum to the de-ionized water is (0.5-1):1. Compared with the prior art, the plant tissue culture support has the beneficial effects that the plant tissue culture support is easy to operate and low in tissue culture cost; energy resources are saved; the working time is shortened, the labor intensity is reduced, the inoculation efficiency is improved, and the pollution probability is reduced; an explant grows robustly, and is high in transplantation survival rate; good fixing effects are achieved; the air permeability is high; a culture medium is long in service cycle; a tissue culture environment can more meet physiological requirements of a plant.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of plant tissue culture holder.
Background technology
Culture medium physical state for plant tissue culture has liquid and solid two types.Fluid medium is fitted
Cultivate in cell, promote cell biological yield or the increase of metabolite.Solid medium is used for callus
Cultivate with test tube Seedling etc..Holder refers to the growth of the outer implant of beneficially plant, artificially adds culture medium to
In, external implant play some materials of supporting function.The multiplex agar of holder of culture medium at present.Agar is
Polysaccharide, itself does not provide any nutrition, and it is a kind of macromolecular compound extracted from Sargassum.Agar is only
Being dissolved in the hot water of more than 90 DEG C, become colloidal sol, less than 40 DEG C start solidification, become solid-like gel.Agar
The factor such as coagulation grade and the time of high temperature sterilize, temperature and Medium's PH Value also have relation, for a long time
High temperature sterilize can make the coagulation ability of agar decline, and alkali high temperature in addition crossed by peracid all can make agar hydrolyze, from
And lose coagulation ability, generally, the pH value of culture medium is when 5.8, and the coagulation result of agar is preferable.In recent years
Come, have some reports to mention employing agarose, polyurethane sponge, absorbent cotton, glass bead, glass cotton, quartz
Sand, Vermiculitum, perlite, loam, river sand, filter paper, gelatin, silica gel, acrylamide, foam plastics etc.,
Even Fructus Cucurbitae moschatae and Rhizoma Solani tuber osi etc. are as the holder of plant tissue culture.The feature of agarose is that it belongs to polysaccharide,
Preparation process is complicated, and cost is significantly larger than agar.The gel strength of agarose is high, is generally used for unicellular or former
Raw plastid is cultivated.Absorbent cotton is hygroscopic, air permeable effect good, and it does callus growth in the culture medium of holder
Situation is preferable, also just because of absorbent cotton good water absorption, often leads to cultivate the not enough phenomenon of liquid measure and make outer planting
Body late growing stage is bad；When polyurethane sponge does holder, outer planting bulk-growth is vigorous, and low cost, saving of labor province
Power, culture root without with water rinse can directly move into cool canopy, therefore survival rate is higher, but there is also by
Cause by force later stage nutritional solution not enough in absorbability and affect the situation of outer planting bulk-growth, and be found to have vitreous body
Seedling produces；Vermiculitum, perlite do that holder has low cost, simple to operate, outer planting bulk-growth is the best
Feature, but its deficiency to be the fixed effect of external implant fine, this point and glass bead, glass cotton, quartz
Sand, loam, river sand etc. are identical.Therefore, this area needs a kind of good permeability, the cultivation that transplanting survival rate is high badly
Base holder.
Summary of the invention
The present invention solves the deficiencies in the prior art, it is provided that a kind of plant tissue culture holder, use sphagna
Holder as the culture medium of plant tissue culture has: outer planting bulk-growth is healthy and strong, transplanting survival rate is high；Become
This is low；Simple to operate；Good fixing effect；The advantages such as good permeability.
To achieve the object of the present invention, the technical scheme is that a kind of plant tissue culture holder, including
Solid carbon dioxide tongue and deionized water, solid carbon dioxide tongue is (0.5～1) with the volume ratio of deionized water: 1.Preferably, solid carbon dioxide tongue
It is 1:1 with the volume ratio of deionized water.
Above-mentioned holder is used to make culture medium, with the following method: by solid carbon dioxide tongue with deionized water by a certain percentage
Put into so that solid carbon dioxide tongue expands in container, standby；Expansion sphagna is positioned in culture bottle, adds culture fluid,
The addition of culture fluid was not there just to be sphagna.As one preferred embodiment of the present invention, described expansion
The addition of sphagna is 1/5th of culture bottle volume.
The step using above-mentioned culture medium holder to carry out plant tissue culture is as follows:
S1. take the outer implant of pre-incubated plant, use Bulbus Allii aqueous solution to carry out sterilization treatment；
S2. the outer implant after sterilizing is carried out initial culture, and carries out the screening of culture medium, determine optimal just generation
Culture medium；
S3. on the basis of initial culture, carry out successive transfer culture, and carry out the screening of culture medium, determine optimal
Subculture medium；
S4. on the basis of successive transfer culture, carry out root culture, and carry out the screening of culture medium, determine optimal
Root media；
S5. suitable rooting culture substrate and condition are screened；
All being added with expansion sphagna holder in above-mentioned just generation, subculture, root media, operating procedure is as follows:
A. prepare expansion sphagna holder, in culture bottle, first add the expansion sphagna of 1/5 volume；
Just generation, subculture, root media is added the most again in culture bottle；
C. the last outer implant of inoculation in culture medium, sealing, cultivate in culturing room.
Above-mentioned culture medium can be used for the tissue culture of dicotyledon, monocotyledonous tissue culture, Growing season
Make outer implant with the tender tip of the leaf bud in dormancy season, stem section, seed and Growing season and carry out tissue culture, use leaf
Sheet is made outer implant and is carried out tissue culture, the initial culture of outer implant, successive transfer culture, root culture, callus
Suspension culture.
Further, above-mentioned initial culture base or subculture medium or root media are at MS minimal medium
In middle interpolation 6-BA, NAA, IBA, GA, sucrose one or more.
Preferably, Bulbus Allii aqueous solution mass concentration is 30～50%.
In the scientific research and actual application of plant tissue culture, inventor finds that sphagna can also be as plant
The holder of culture medium during tissue culture, and achieve good effect.Sphagna is a kind of natural lichen, again
Name bog moss (Herba Sphagni), for Sphagnaceae plant.Use sphagna can carry as culture medium holder
Next following effect:
1. outer planting bulk-growth is healthy and strong, and transplanting survival rate is high.No matter initial culture, successive transfer culture or root culture,
During with sphagna for holder, the growth of outer implant is the most healthy and the strongest, and during transplanting, survival rate is the highest.
2. low cost.Two aspect reasons: one, commonly using with plant tissue culture compared with holder agar, sphagna price is low.
At present, the market price, the price of agar is 3～5 times of sphagna price.And Conventional solid culture medium, every 1000ml
Culture medium, the usage amount of sphagna is 15～about 20g, and the usage amount of agar is 7～about 10g.Two, sphagna
Can also repeatedly use.Agar has certain requirement, some fine jades to condition elements such as storage temperature and times
Fat can occur certain physicochemical property to change because of storage discomfort, and after High Temperature High Pressure, its acidity increases, and causes cultivating
Base does not solidifies, it is impossible to normally use, thus produces waste, and sphagna stable in physicochemical property, to storage temperature and
Time, after High Temperature High Pressure, pH was unchanged, is not result in the change of culture medium character without being strict with.Therefore may be used
Cost is trained with reduction group.
The most simple to operate.Compared with doing holder with agar, when sphagna is cooked holder, it is possible to reduce dissolve agar
Link, also can reduce the link washing agar before transplanting from tissue cultured seedling root system.
The most time-consuming, reduce and pollute.The culture medium of holder is done with agar, during aseptic inoculation, need to be by outer planting
Body cuttage is in culture medium, and the time-consuming of cuttage work typically accounts for the 1/2～1/3 of whole inoculation time.Due to water
Tongue culture medium, between solid-state and liquid, during inoculation, only need to uniformly be placed on media surface by outer implant
, this is possible not only to save inoculation time, improves inoculation efficiency, it is also possible to reduce pollution probability.
5. save the energy, shorten working hours, alleviate working strength.When making the culture medium that agar is holder,
Agar needs temperature more than 95 DEG C, after infusion 15min, could preferably melt, and infusion process also takes
Carve and observe, the most easily.And when making the culture medium that sphagna is holder, it is not necessary to infusion, it is possible not only to
Save the energy, shorten working hours, it is also possible to alleviate working strength.
6. good fixing effect.Compared with glass bead, glass cotton, quartz sand, loam, river sand etc., sphagna props up
The fixed effect holding the external implant of thing is more preferable.Inventor is outer implant with Nicotiana tabacum L., the Radix Astragali, at glass bead, glass
Cotton, quartz sand, loam, river sand are to do experiment in the culture medium of holder, find inoculation after, from
When superclean bench moves on to culturing room, owing to slight rocks, with glass bead, glass cotton, quartz sand, loam,
River sand is that the outer planting body in holder defines inclination, and the outer implant with sphagna as holder is then fixed very
Good.7. good permeability.Compared with the holder such as agar, the breathability of sphagna is more preferable, is conducive to the life of outer implant
Long growth.Inventor is that outer implant is seeded in the culture medium as holder with sphagna and agar respectively with iris
On, when result is with sphagna for holder, iris growth potential agar to be much better than.Because Phalaenopsis is in aerobic
Plant, grows potential difference in the medium that breathability is bad, thus may determine that the good permeability of sphagna, be more suitable for
As holder.
8. the culture medium using sphagna to do holder uses the cycle long.Do compared with the culture medium of holder with agar,
Tissue cultured seedling is when preserving for a long time, and the culture medium dehydration of sphagna holder is slow, hence it is evident that be better than agar culture medium.
9. sphagna is cooked the culture medium of holder group training environment can be made closer to the psychological need of plant.Agar is cultivated
Base need to regulate pH value about 5.8, and pH does not solidifies less than 5.6 culture medium, so, some is grown
The plant of environmental requirement higher ph, when group training, utilizes agar culture medium, and its growing way can be affected.
And when utilizing sphagna culture medium, the pH value of culture medium according to the plant requirement to growing environment, can be regulated,
Make group training environment closer to the psychological need of plant.
10. during aseptic inoculation, agar culture medium, need to be by culture cuttage in culture medium, and under routine operation,
It is time-consuming that cuttage works, and typically accounts for the 1/2～1/3 of whole inoculation time.Due to sphagna culture medium, Jie Yugu
Between state and liquid, during inoculation, only culture need to uniformly be placed on media surface, this is possible not only to
Save inoculation time, improve inoculation efficiency, it is also possible to reduce pollution probability.
Compared with prior art the invention has the beneficial effects as follows: sphagna is a kind of natural bog moss, physics and chemistry
Matter is stable, good permeability, and the transplanting survival rate with sphagna as holder can reach more than 95%, and with agar
Survival rate for holder can only achieve about 90%.To non-phytotoxic, use sphagna as plant tissue culture
Culture medium holder there is comprehensive advantage, show themselves in that simple to operate；The low cost of tissue culture；Save energy
Source；Shorten working hours, alleviate working strength, improve inoculation efficiency, reduce pollution probability；Outer planting bulk-growth
Stalwartness, transplanting survival rate is high；Good fixing effect；Good permeability；Culture medium uses the cycle long；Group can be made to train ring
Border is closer to the psychological need of plant.
Detailed description of the invention
Following non-limiting example can make those of ordinary skill in the art that the present invention is more fully understood, but
Limit the present invention never in any form.In following embodiment if no special instructions, the experimental technique used is
Conventional method, material therefor, reagent etc. all can chemically company be bought.
All being added with expansion sphagna holder in following just generation, subculture, root media, operating procedure is as follows:
A. prepare expansion sphagna holder, solid carbon dioxide tongue and deionized water are put in container for 1:1 by volume so that
Solid carbon dioxide tongue expands, standby；Expansion sphagna is positioned in culture bottle, addition be culture bottle volume five/
One；
Adding just generation, subculture, root media the most again in culture bottle, addition was not there just to be sphagna；
C. in the culture medium made, inoculate outer implant, sealing, cultivate in culturing room.
Embodiment 1:
Select the leaf bud of dicotyledon short stem Liana rosa indica, after flowing water rinses, peel off the scale outside leaf bud, by bud
It is placed on 1:1 (W/V) Bulbus Allii aqueous solution sterilizing 30min, is seeded in the suitableeest of short stem Liana rosa indica immediately after
In initial culture culture medium (MS+6-BA0.5mg/L+NAA0.05mg/L+3% sucrose).To sprout after 30d
The leaf bud stem segment cuttage gone out is at the suitableeest subculture medium (MS+6-BA1.0mg/L+IBA0.2mg/L+2% sugarcane
Sugar) middle cultivation.After 30d, successive transfer culture Seedling is moved into optimal root media (1/2MS+6-BA0.2mg/L+IBA
1.0mg/L+2% sucrose) in carry out root culture, after growing good root system, transfer to perlite and sandy soil
On tame, finally transplant in neutral loam.
Embodiment 2:
Select the leaf leaf stem section that has of dicotyledon " red light " Prunus avium, after flowing water rinses, peel off outside leaf bud
Scale and the epidermis of stem section, one section of stem with leaf bud is put into 1:1 (W/V) Bulbus Allii aqueous solution and goes out
Bacterium 30min, is seeded in (MS+6-BA in the suitableeest initial culture culture medium of " red light " Prunus avium immediately after
1.0mg/L+IBA0.1mg/L+3% sucrose).Leaf bud stem segment cuttage sprouting gone out after 30d is trained at the suitableeest subculture
Base (2/3MS+6-BA0.5mg/L+IBA0.3mg/L+GA1.5+2% sucrose) is cultivated.To continue after 40d
Culture Seedling moves in optimal root media (1/2MS+IBA0.5mg/L+2% sucrose) and carries out root culture,
Transfer to tame on perlite and sandy soil after growing good root system, finally transplant in neutral loam.
Embodiment 3:
Select the seed of dicotyledon Radix Glycyrrhizae, through 98% dense sulfuric acid treatment 1h, soften hard seed coat, then
Fully rinse well with tap water.Under aseptic operating platform with 0.1% mercuric chloride sterilize 10min, with aseptic
Water rinses 3～4 times, then with 75% ethanol disinfection 40s, then with aseptic water washing 3～4 times.Initial culture
The optimal medium of (or inoculation) is MS culture medium, cultivates 1～2 week, carry out under dark condition
Illumination cultivation one month.Cultivating directly the take root optimal medium of seedling of band base of leaf section is: MS+13.5mg/L
KH2PO4+ 3% sucrose.Seedling of taking root selects directly to grow on the basis of successive transfer culture, good to growing
Transfer to after root system tame on perlite and sandy soil, finally transplant in neutral loam.
Embodiment 4:
Select the tender tip of dicotyledon " late red pearl " Prunus avium, under flowing water, rinse 1h, water-soluble in 50% Bulbus Allii
Immersion bubble 15min, is then seeded in the suitableeest initial culture base (MS+6-BA0.5mg/L+IBA0.1mg/L
+ 3% sucrose) in, after 30d, the tender tip is sprouted the stem section and is seeded in the suitableeest subculture multiplication medium (2/3MS+
6-BA0.5mg/L+IBA 0.3mg/L+GA1.5+2% sucrose) on；After 40d, successive transfer culture Seedling is moved into optimal
Root media (MS+6-BA0.1mg/L+IBA0.5mg/L+2% sucrose) carries out root culture, to long
Transfer to after going out good root system tame on perlite and sandy soil, finally transplant in neutral loam.
Embodiment 5:
Selecting the blade of dicotyledon Nicotiana tabacum, take that leaf color is normal, the big blade of healthy growth, flowing water rinses
1h, is immersed in 5min in 50% garlic.In superclean bench, blade is cut into 12Square
Lamellar, and cut several wound (not cutting off), the outer implant will handled well afterwards with tweezers at middle vein position
Keep flat and be seeded on callus culture base (MS+6-BA 1.0mg/L+IBA0.2mg/L+3% sucrose), enter
Row callus proliferation is cultivated, and when callus growth arrives a certain size, cuts and continues to be inoculated into wound healing group
Knit further amplification culture in culture medium.
Embodiment 6:
The bennet lateral bud (i.e. leaf bud) of menu cotyledon plant iris, flowing water rinses 1h, is immersed in 50% Bulbus Allii
20min in solution, is seeded in suitable initial culture base (MS+6-BA1.0mg/L+IBA0.5mg/L+3%
Sucrose) on, bennet lateral bud sprouting gone out after 30d is seeded in suitable proliferated culture medium (1/2MS+6-BA
8.0mg/L+NAA0.2mg/L+ sucrose 30g/L+ banana puree 30g/L+ activated carbon 2g/L) on, connect again after 30d
Plant at suitable root media (1/2MS+6-BA4.0mg/L+NAA 0.8mg/L+ sucrose 30g/L+ Fructus Musae
Mud 30g/L+ activated carbon 2g/L) on.Transfer to sphagna substrate grows after growing good root system.
Embodiment 7:
The stem section of menu cotyledon plant Radix Dauci Sativae, after flowing water rinses, puts into 30% Bulbus Allii by tool leaf stem section water-soluble
Liquid sterilizing 10min, is seeded in (MS+6-BA 1.0mg/L+NAA in suitable initial culture culture medium immediately after
0.3mg/L+2% sucrose).Leaf bud stem segment cuttage sprouting gone out after 20d is at suitable subculture training base (MS+
6-BA0.5mg/L+NAA 0.05mg/L+2% sucrose) middle cultivation.It is suitable to be moved into by successive transfer culture Seedling after 20d
Root media (MS+6-BA0.1mg/L+NAA1.0mg/L+2% sucrose) carries out root culture, to long
Transfer to after going out good root system tame on perlite and sandy soil, finally transplant in neutral loam.
Embodiment 8:
Selecting the seed of monocotyledonous plant Zea mays, flowing water is immersed in 10min in 30% Bulbus Allii aqueous solution after rinsing, and connects
Kind in the sphagna culture medium containing MS culture fluid, to transfer to after growing good stem and leaf and root system perlite and
Tame on sandy soil, finally transplant in neutral loam.
Embodiment 9:
The tender tip of menu cotyledon plant Herba Apii graveolentis, flowing water rinses 1h, in 30% Bulbus Allii aqueous solution soaking 10min,
Then it is seeded in initial culture base (MS+6-BA0.5mg/L+NAA0.2mg/L+3% sucrose), 20d
After the tender tip sprouted the stem section be seeded in subculture multiplication medium (MS+6-BA 1.0mg/L+NAA
0.1mg/L+2% sucrose) on；After 20d, successive transfer culture Seedling is moved into root media (MS+6-BA0.1mg/L
+ NAA1.0mg/L+2% sucrose) in carry out root culture, after growing good root system, transfer to perlite
Tame with on sandy soil, finally transplant in neutral loam.
Embodiment 10:
The blade of menu cotyledon plant iris, flowing water rinses 1h, is immersed in 5min in 30% garlic.
In superclean bench, blade is cut into 12Square, then keep flat with by the square blade handled well
It is seeded on callus culture base (MS+6-BA 1.5mg/L+IBA0.4mg/L+2% sucrose), carries out more
Injured tissue enrichment culture, when callus growth arrives a certain size, cuts and continues to be inoculated into callus training
Support further amplification culture on base.
Embodiment 11
The blade selecting dicotyledon " Herba Seu Radix Peperomiae Tetraphyllae " rinses 1h as outer implant, flowing water, is immersed in 30% big
5min in Bulbus Allii solution.In superclean bench, blade is cut into 12Square, then with handling well
Square blade keeps flat and is seeded in initial culture base (MS+6-BA0.3mg/L+NAA 0.5mg/L+3% sucrose)
On, after turning out callus to, callus is forwarded subculture medium (2/3MS+6-BA 0.5mg/L+IBA
0.3mg/L+GA1.5+2% sucrose) in, carry out successive transfer culture.After successive transfer culture induction is sprouted, it is transferred to
In root media (1/2MS+NAA0.5mg/L+ sucrose 3%), after growing good root system, transfer to treasure
Tame on Zhu Yan and sandy soil, finally transplant in neutral loam.
Embodiment 12
Selecting the blade of dicotyledon Nicotiana tabacum, take that leaf color is normal, the big blade of healthy growth, flowing water rinses
1h, is immersed in 5min in 50% garlic.In superclean bench, blade is cut into the square of 12
Lamellar, and cut several wound (not cutting off), the outer implant will handled well afterwards with tweezers at middle vein position
Keep flat and be seeded on callus culture base (MS+6-BA 1.0mg/L+IBA0.2mg/L+3% sucrose), enter
Row callus culture, until callus induction out after, be transferred to subculture medium and root media
(MS+6-BA 1.0mg/L+IBA0.2mg/L+ sucrose 3%, note: subculture medium is identical with root media)
On, transfer to tame on perlite and sandy soil after growing good bud and root system, finally transplant in neutrality
In loam.
List of references
[1] Jiang Changyang, Wang Hongqing. a kind of new holder [J] in plant tissue culture. Scientia Agricultura Sinica, 1987,1:96.
[2] Zhao Lihong, Yan Xiao, Liu Hua, etc. application polyurethane sponge fritter makees the holder [J] of plant tissue culture. plant
Physiology's communication, 1988,1:46.
[3] Guo Dongwei, Li Chunlian, Guo Yuexia, etc. different holders are to kobold (StachysfloridanaSchuttl.exBenth)
The impact [J] of rooting of vitro seedling. Journal of Northwest Sci Tech University of Agriculture and Forestry (natural science edition), 2004,10:25-28.
[4] Zhao Jianping, Bi Kehua, Wang Xiuqiang, etc. the research that " Ai Xisi " Fructus Cucurbitae moschatae rooting of vitro seedling is affected by different holders
[J]. biotechnology, 1998,3:27-29.
[5] Guo Yanhua, Yu Shuijing, Li Xiaoli. the holder impact [J] on Chinese rose callus induction. Agriculture in Jiangxi
Report, 2008,1:118-119.
[6] Han Yanghai, Zhao Haihong, Chen Dexiang. the different holder impacts [J] on Vitro Rapid Propagation of Virus-free Potato. Heilungkiang
Agricultural sciences, 2008,5:22-24.
[7] yellow bright cloud, Zhang Yueqin, Shao Mingwen. application polyurethane sponge fritter makees the holder [J] that Sugarbeet is cultivated. in
State's beet sugar industry, 1991,6:20-22.
[8] Ma Chunyan, outstanding heroes, Yuan's handle is deposited. hormone and the holder impact [J] on Rosa floribunda Fast-propagation. and Jilin forestry
Science and technology, 2005,2:4-6.
Claims (7)
1. a plant tissue culture holder, it is characterised in that include solid carbon dioxide tongue and deionized water, solid carbon dioxide
Tongue is (0.5～1) with the volume ratio of deionized water: 1.
2. one kind makes culture medium with the holder described in claim 1, it is characterised in that method is as follows:
Solid carbon dioxide tongue and deionized water are put into by a certain percentage so that solid carbon dioxide tongue expands in container, standby；Sphagna will be expanded
Being positioned in culture bottle, add culture fluid, the addition of culture fluid was not there just to be sphagna.
Culture medium the most according to claim 2, it is characterised in that the described addition expanding sphagna
For culture bottle volume 1/5th.
Culture medium the most according to claim 2, it is characterised in that this culture medium can be used for dicotyledonous planting
The tissue culture of thing, monocotyledonous tissue culture, Growing season and the leaf bud in dormancy season, stem section, seed with
And the tender tip outer implant of work of Growing season carries out tissue culture, use blade to make outer implant and carry out tissue culture, outer planting
The suspension culture of the initial culture of body, successive transfer culture, root culture and callus.
5. the method carrying out plant tissue culture with the culture medium holder described in claim 1, it is special
Levy and be, comprise the steps:
S1. take the outer implant of pre-incubated plant, use Bulbus Allii aqueous solution to carry out sterilization treatment；
S2. the outer implant after sterilizing is carried out initial culture, and carries out the screening of culture medium, determine optimal just generation
Culture medium；
S3. on the basis of initial culture, carry out successive transfer culture, and carry out the screening of culture medium, determine optimal
Subculture medium；
S4. on the basis of successive transfer culture, carry out root culture, and carry out the screening of culture medium, determine optimal
Root media；
S5. screening rooting culture substrate and transplanting condition；
Above-mentioned just generation, subculture, root media are all added with expansion sphagna holder, specific as follows:
A. prepare expansion sphagna holder, in culture bottle, first add the expansion sphagna of 1/5 volume；
Just generation, subculture, root media is added the most again in culture bottle；
C. the last outer implant of inoculation in culture medium, sealing, cultivate in culturing room.
The method of plant tissue culture the most according to claim 5, it is characterised in that described first generation
Culture medium or subculture medium or root media be in MS minimal medium add 6-BA, NAA, IBA,
In GA, sucrose one or more.
The method of plant tissue culture the most according to claim 5, it is characterised in that described step S1
In, Bulbus Allii aqueous solution mass concentration is 30～50%.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107372105A (en) * | 2017-06-30 | 2017-11-24 | 广西现代园林绿化工程种苗有限公司 | A kind of tissue culture and rapid propagation method of flower plants and nursery stock |
| CN110771509A (en) * | 2019-11-26 | 2020-02-11 | 大连大学 | Licorice artificial seed and culture method thereof |
| CN115595288A (en) * | 2022-11-28 | 2023-01-13 | 深圳市脉唐生物科技有限公司（Cn） | Microcarrier for cell culture and preparation method thereof |
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| CN110771509A (en) * | 2019-11-26 | 2020-02-11 | 大连大学 | Licorice artificial seed and culture method thereof |
| CN115595288A (en) * | 2022-11-28 | 2023-01-13 | 深圳市脉唐生物科技有限公司（Cn） | Microcarrier for cell culture and preparation method thereof |
| CN115595288B (en) * | 2022-11-28 | 2023-03-31 | 深圳市脉唐生物科技有限公司 | Microcarrier for cell culture and preparation method thereof |
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