CN103858764B - Centipeda minima tissue culture rapid propagation medium and centipeda minima one-time seedling formation tissue culture rapid propagation method - Google Patents
Centipeda minima tissue culture rapid propagation medium and centipeda minima one-time seedling formation tissue culture rapid propagation method Download PDFInfo
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Abstract
The invention discloses a centipeda minima tissue culture rapid propagation medium which is a medium formulation formed by culture solution with low salinity and plant growth regulators; after the tissue culture rapid propagation medium is used, the axillary bud stem section of an optimal plant is taken as an explant, the whole sterile culture process only needs one medium, and the cultivation process is simple and easy. In the medium, subculture and root induction are synchronously carried out, and the seedlings can be formed for once. The centipeda minima tissue culture seedling growth period is shortened, and industrial production can be better realized. The invention also discloses a centipeda minima one-time seedling formation tissue culture rapid propagation method; a way of propagating buds by using buds is adopted by the enrichment culture process, so that the multiplication coefficient is high, and the variation phenomenon is not caused; the callus is not formed in the rooting process, and cortex rooting is realized, so that the survival rate of transplanting is high.
Description
Technical field
The present invention relates to tissue culture medium (TCM), particularly relate to a kind of Centipeda minima culture medium for tissue culture.The present invention relates to tissue-culturing rapid propagation medium and the tissue culture and rapid propagation method of a Plants, particularly relate to Centipeda minima tissue-culturing rapid propagation medium and once-seedling forming tissue culture and rapid propagation method.
Background technology
Centipeda minima (Centipeda minina L) A.Br.etAschers. calls Myriogyne minuta, wild garden Sui, eritrichium pedumculare etc., for composite family Myriogyne minuta belongs to annual herb plant, and all herbal medicine.All include in " Chinese Pharmacopoeia " each version.Centipeda minima has that anti-inflammatory, antigen are biological, antibacterial, antiallergy, antitumor and cytotoxin, protect liver and eliminate the effects such as R-plasmid, is used for the treatment of the diseases such as nasopharyngeal carcinoma, rhinitis, calculosis, headache, traumatic injury and facial paralysis clinically.In recent years, due to the extensive use of weed killer herbicide, output is caused to fall sharply, add that Centipeda minima complete stool is medicinal, and single plant yield is low, Centipeda minima gathers with suffering predation formula, wild natural resources is subject to heavy damage, the market price is raised on the way, is of many uses simply, has the herbal species of development prospect.
Adopt method for tissue culture energy amount reproduction seedling, Centipeda minima tissue culture rarely has report, and conventional tissue culture method divides shoot proliferation and root induction two steps, and the medium of employing divides subculture medium and root media, and growing-seedling period is long.Centipeda minima is annual herb plant, and complete stool is medicinal, and bottle seedling of taking root can become clump to plant in seedbed, therefore if synchronously carry out Centipeda minima shoot proliferation and root induction in same medium, once-seedling forming, escapable cost and shortening plantlet in vitro growing-seedling period, be conducive to industrialization and produce.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, there is provided a kind of Centipeda minima tissue-culturing rapid propagation medium and once-seedling forming tissue culture and rapid propagation method, in this medium, shoot proliferation and root induction synchronously carry out, once-seedling forming, shorten the Centipeda minima tissue culture cycle, be conducive to industrialization and produce.
Technical scheme of the present invention is as follows:
One, a Centipeda minima tissue-culturing rapid propagation medium, kind and the concentration of material contained by during it often rises (L) are as follows:
The compound method of Centipeda minima tissue-culturing rapid propagation medium of the present invention is:
1 mother liquor
1.1, for ease of sampling, should first prepare various mother liquor, be divided into macroelement mother liquor, micro-mother liquor, organic matter mother liquor and each plant growth regulators mother liquor.Sucrose, agar should not be made into mother liquor, directly sample when needing;
1.2 macroelement mother liquors should become the multiple proportion of 10-100 with each concentration of component of organic matter mother liquor with the concentration of component of medium, micro-mother liquor becomes the multiple proportion of 200-500, and the concentration of plant-growing-help chemicals mother liquor should be made into 1mg/ml;
1.3 mother liquors select aseptic distilled water, deionized water or ultra-pure water to prepare, with the water boiled during a large amount of production;
During 1.4 preparation macroelement mother liquor, each component should be dissolved separately, mixes one by one afterwards, otherwise easily cause precipitation by the order of nitrogen, calcium, magnesium, phosphorus;
1.5 micro-mother liquors contain sees the labile potassium iodide of light and oxidizable ferrous sulfate, and application brown bottle is preserved;
1.6 plant growth regulator mother liquors and organic matter mother liquor should be placed in refrigerator and preserve;
Should use in time after 1.7 mother liquor, period of storage was no more than 1 month;
1.8 find that mother liquor has precipitation, or have growth of microorganism, or have algal grown, should pass into disuse.
2 medium preparations
2.1 according to culture medium prescription, measures mother liquor in proportion, goes in the containers such as stainless-steel pan after the constant volume that adds water;
2.2 add appropriate agar and sugar, and stirring makes coagulating agent fully disperse, and sugar fully dissolves;
2.3 are heated to firm boiling, and coagulating agent is dissolved completely.During the self-reacting device preparation medium dividing rim to stir according to limit, the process heating and coagulating agent is dissolved can be saved;
2.4 are adjusted to pH to 5.8-6.0 with 1.0N hydrochloric acid or 1.0N sodium hydroxide;
Medium is dispensed in wide-mouth bottle by 2.5 as early as possible, in order to avoid culture medium solidifying or retrogradation and be difficult to packing;
Medium should be avoided during 2.6 packing medium to adhere to bottleneck, once be stained with, should wipe clean, otherwise easily cause pollution.
3 medium sterilizations
Point medium installed is put into autoclave sterilizer by 3.1 sterilizes;
At 3.2 heating initial stages, when the air pressure of sterilizing pan disinfection room reaches 0.05Mpa, open condensation trap, drain sterilization cold air inside;
3.3 when disinfection room internal gas pressure reach 0.11Mpa, temperature reach 121 DEG C time, start timing, keep temperature and pressure sterilization 15 ~ 20 minutes;
3.4 close heating power supply switch, by slow exhaust mode discharge hot gas, open sterilization pot cover or door, take out medium when the air pressure of indoor to be sterilized is down to atmospheric pressure;
3.5 medium should be placed in the clean environment cooling of little air flowing and few dust, otherwise cause mycotic spore to enter culture vessel in cooling intake process, produce mould contamination.Or cool with the thick cloth parcel that high pressure-temperature was sterilized.
4 medium storages
4.1 medium should be as far as possible now with the current.
4.2 can at the clean and immobilising environment short time storage medium of air, but the medium of storage more than 1 month should be passed into disuse.
Two, Centipeda minima once-seedling forming tissue culture and rapid propagation method, comprises the following steps:
Step 1. explant is sterilized: in fine day choose robust growth, without the Centipeda minima plant of damage by disease and insect, clip stem section and terminal bud are as explant, cut off blade (staying petiole), clean 1-2 time with the water being added with a little liquid detergent, clean with running water again, then sterilize 6-7 minute with the mercury chloride of 0.1% on superclean bench and shake for several times, finally using sterile water wash 4-5 time.
The induction of step 2. bud: shear the explant that disinfected into about the long section of 2cm, every section is stayed 1-2 axillalry bud, then oblique cutting or be vertically inserted in the medium prepared is housed vial in.When explant is cultivated for the first time, need full light culture 10-15 days axillalry bud can start to sprout, and then move to the place cultivation seeing light, the preferred wide-mouth bottle of vial.
The propagation of step 3. bud and culture of rootage: when axillalry bud sprout grow to about 1cm time, cut axillalry bud to be seeded in and to be equipped with in the vial of medium, the preferred wide-mouth bottle of vial, carries out shoot proliferation and the culture of rootage of bud, culturing room's temperature is (26 ± 2) DEG C, illumination 12h.d
-1, illuminance 1500-3000lx.Squamous subculture about 20 days buds grow to 2-3 ㎝, start to sprout root system, within 30 days, grow to 3-5 ㎝, the long 2-3 ㎝ of root, form whole plant.After 30 days, then shear the axillary bud stem section of aseptic seedling from some bottle seedling whole plants of taking root, cultivate, continuous shoot proliferation, cultivate about 20 days, growth coefficient reaches 6.0-8.5.Bottle seedling of another part being taken root directly moves to outdoor hardening booth and cultivate 5-7 days under natural scattering illumination.
Step 4. take root seedling transplanting and manage and protect: seedling blade to be taken root is unfolded, and leaf look dark green, and seedling stem is dark green, caulom extend, can transplant during height of seedling about 4cm.Medium is poured out during transplanting, take out seedling, wash the medium remained on root, and clip long root system, retain 2-2.5cm long, transplant in the seedbed of sterilizing through 0.2% potassium permanganate, medium of seedling bed is yellow soil: thin river sand: peat soil, and volume ratio is (3:1:1), to drench water, covered plastic film shed and black net, keep certain temperature and humidity, and will avoid direct sunlight.After transplanting one week, can open part film breathable, the area later opening film can strengthen gradually, alternately sprays bactericide at interval of 7 days, executes foliage fertilizer and composite fertilizer.Within 10-20 days, survive, survival rate more than 92%.
The invention has the advantages that, adopt the culture fluid of low salt concn of the present invention, be added with several plant combination of regulators and become a kind of culture medium prescription, use tissue-culturing rapid propagation medium of the present invention, excellent strain axillary bud stem section is explant, whole aseptic culture process only needs a kind of medium, and incubation is simple.Multiplying culture process is that growth coefficient is high, without variation phenomenon in bud numerous bud mode; Without Callus formation in rooting process, for skin zone takes root, transplanting survival rate is high.
Compared with prior art, Centipeda minima once-seedling forming tissue culture and rapid propagation method of the present invention, only needs a kind of medium in whole aseptic culture process, in this medium, squamous subculture and root induction synchronously carry out, once-seedling forming, shortens Centipeda minima plantlet in vitro growing-seedling period, is conducive to industrialization and produces.
Compared with the Centipeda minima tissue-culturing rapid propagation medium of currently available technology and tissue culture and rapid propagation method, advantage of the present invention is:
Accompanying drawing explanation
Fig. 1 is aseptic bud inducement.
Fig. 2 is the propagation seedling cultivated after the aseptic bud of inoculation 20 days.
Fig. 3 is the grown cultures growth of cereal crop seedlings condition of taking root of 30 days after the aseptic bud of inoculation.
Fig. 4 is that bottle outlet transplants little seedling rooting situation.
Fig. 5 is seedbed Centipeda minima growing state after transplant survival.
Fig. 6 is seedbed Centipeda minima growing state after transplant survival.
Embodiment
With embodiment, the invention will be further described below, but the present invention is not limited to these enforcement
Example.Embodiment 1:
Centipeda minima tissue-culturing rapid propagation medium, the kind of material contained by wherein often liter (L) and concentration
As follows:
Embodiment 2.
Centipeda minima tissue-culturing rapid propagation medium, kind and the concentration of material contained by wherein often liter (L) are as follows:
Embodiment 3.
Centipeda minima tissue-culturing rapid propagation medium, kind and the concentration of material contained by wherein often liter (L) are as follows:
Embodiment 4.
The once-seedling forming tissue-culturing rapid propagation of Centipeda minima:
Explant is sterilized: in fine day choose robust growth, without the Centipeda minima plant of damage by disease and insect, clip stem section and terminal bud are as explant, cut off blade (staying petiole), clean 1-2 time with the water being added with a little liquid detergent, clean with running water again, then shake for several times with mercury chloride sterilization 6-7 minute of 0.1% on superclean bench, finally use sterile water wash 4-5 time '
The induction of bud: shear the explant that disinfected into about the long section of 2cm, every section is stayed 1-2 axillalry bud, then oblique cutting or be vertically inserted in the medium that above-described embodiment 1 prepares is housed wide-mouth bottle in.When explant is cultivated for the first time, full light culture 10-15 days axillalry bud can start to sprout, and then moves to the place cultivation seeing light.
The propagation of bud and culture of rootage: when axillalry bud sprout grow to about 1cm time, cut axillalry bud and be seeded in and be equipped with in the wide-mouth bottle of medium, carry out shoot proliferation and the culture of rootage of bud, culturing room's temperature is (26 ± 2) DEG C, illumination 12h.d
-1, illuminance 1500-3000lx.Squamous subculture about 20 days buds grow to 2-3 ㎝, start to sprout root system, within 30 days, grow to 3-5 ㎝, the long 2-3 ㎝ of root, form whole plant.After 30 days, then shear the axillary bud stem section of aseptic seedling from some bottle seedling whole plants of taking root, cultivate, continuous shoot proliferation, cultivate about 20 days, growth coefficient reaches 6.0-8.5.Bottle seedling of another part being taken root directly moves to outdoor hardening booth and cultivate 5-7 days under natural scattering illumination.
Take root seedling transplanting and manage and protect: seedling blade to be taken root is unfolded, and leaf look dark green, and seedling stem is dark green, caulom extend, can transplant during height of seedling about 4cm.Medium is poured out during transplanting, take out seedling, wash the medium remained on root, and clip long root system, retain 2-2.5cm long, transplant in the seedbed of sterilizing through 0.2% potassium permanganate, medium of seedling bed volume ratio is yellow soil: thin river sand: peat soil (3:1:1), to drench water, covered plastic film shed and black net, keep certain temperature and humidity, and to direct sunlight be avoided, after transplanting one week, part film breathable can be opened, the area later opening film can strengthen gradually, alternately bactericide is sprayed at interval of 7 days, execute foliage fertilizer and composite fertilizer.Within 10-20 days, survive, survival rate 92.5%.
Embodiment 5.
The once-seedling forming tissue-culturing rapid propagation of Centipeda minima:
Explant is sterilized: in fine day choose robust growth, without the Centipeda minima plant of damage by disease and insect, clip stem section and terminal bud are as explant, cut off blade (staying petiole), clean 1-2 time with the water being added with a little liquid detergent, clean with running water again, then shake for several times with mercury chloride sterilization 6-7 minute of 0.1% on superclean bench, finally use sterile water wash 4-5 time '
The induction of bud: shear the explant that disinfected into about the long section of 2cm, every section is stayed 1-2 axillalry bud, then oblique cutting or be vertically inserted in the medium that above-described embodiment 2 prepares is housed wide-mouth bottle in.When explant is cultivated for the first time, full light culture 10-15 days axillalry bud can start to sprout, and then moves to the place cultivation seeing light.
The propagation of bud and culture of rootage: when axillalry bud sprout grow to about 1cm time, cut axillalry bud and be seeded in and be equipped with in the wide-mouth bottle of medium, carry out shoot proliferation and the culture of rootage of bud, culturing room's temperature is (26 ± 2) DEG C, illumination 12h.d
-1, illuminance 1500-3000lx.Squamous subculture about 20 days buds grow to 2-3 ㎝, start to sprout root system, within 30 days, grow to 3-5 ㎝, the long 2-3 ㎝ of root, form whole plant.After 30 days, then shear the axillary bud stem section of aseptic seedling from some bottle seedling whole plants of taking root, cultivate, continuous shoot proliferation, cultivate about 20 days, growth coefficient reaches 6.0-8.5.Bottle seedling of another part being taken root directly moves to outdoor hardening booth and cultivate 5-7 days under natural scattering illumination.
Take root seedling transplanting and manage and protect: seedling blade to be taken root is unfolded, and leaf look dark green, and seedling stem is dark green, caulom extend, can transplant during height of seedling about 4cm.Medium is poured out during transplanting, take out seedling, wash the medium remained on root, and clip long root system, retain 2-2.5cm long, transplant in the seedbed of sterilizing through 0.2% potassium permanganate, medium of seedling bed volume ratio is yellow soil: thin river sand: peat soil (3:1:1), to drench water, covered plastic film shed and black net, keep certain temperature and humidity, and to direct sunlight be avoided, after transplanting one week, part film breathable can be opened, the area later opening film can strengthen gradually, alternately bactericide is sprayed at interval of 7 days, execute foliage fertilizer and composite fertilizer.Within 10-20 days, survive, survival rate 92.6%.
Embodiment 6.
The once-seedling forming tissue-culturing rapid propagation of Centipeda minima:
Explant is sterilized: in fine day choose robust growth, without the Centipeda minima plant of damage by disease and insect, clip stem section and terminal bud are as explant, cut off blade (staying petiole), clean 1-2 time with the water being added with a little liquid detergent, clean with running water again, then shake for several times with mercury chloride sterilization 6-7 minute of 0.1% on superclean bench, finally use sterile water wash 4-5 time '
The induction of bud: shear the explant that disinfected into about the long section of 2cm, every section is stayed 1-2 axillalry bud, then oblique cutting or be vertically inserted in the medium that above-described embodiment 3 prepares is housed wide-mouth bottle in.When explant is cultivated for the first time, full light culture 10-15 days axillalry bud can start to sprout, and then moves to the place cultivation seeing light.
The propagation of bud and culture of rootage: when axillalry bud sprout grow to about 1cm time, cut axillalry bud and be seeded in and be equipped with in the wide-mouth bottle of medium, carry out shoot proliferation and the culture of rootage of bud, culturing room's temperature is (26 ± 2) DEG C, illumination 12h.d
-1, illuminance 1500-3000lx.Squamous subculture about 20 days buds grow to 2-3 ㎝, start to sprout root system, within 30 days, grow to 3-5 ㎝, the long 2-3 ㎝ of root, form whole plant.After 30 days, then shear the axillary bud stem section of aseptic seedling from some bottle seedling whole plants of taking root, cultivate, continuous shoot proliferation, cultivate about 20 days, growth coefficient reaches 6.0-8.5.Bottle seedling of another part being taken root directly moves to outdoor hardening booth and cultivate 5-7 days under natural scattering illumination.
Take root seedling transplanting and manage and protect: seedling blade to be taken root is unfolded, and leaf look dark green, and seedling stem is dark green, caulom extend, can transplant during height of seedling about 4cm.Medium is poured out during transplanting, take out seedling, wash the medium remained on root, and clip long root system, retain 2-2.5cm long, transplant in the seedbed of sterilizing through 0.2% potassium permanganate, medium of seedling bed volume ratio is yellow soil: thin river sand: peat soil (3:1:1), to drench water, covered plastic film shed and black net, keep certain temperature and humidity, and to direct sunlight be avoided, after transplanting one week, part film breathable can be opened, the area later opening film can strengthen gradually, alternately bactericide is sprayed at interval of 7 days, execute foliage fertilizer and composite fertilizer.Within 10-20 days, survive, survival rate 94.1%.
Claims (4)
1. a Centipeda minima tissue-culturing rapid propagation medium, is characterized in that:
Kind and the concentration of material contained by during it often rises (L) are as follows:
2. a Centipeda minima once-seedling forming tissue culture and rapid propagation method, is characterized in that comprising the following steps:
Step 1. explant is sterilized: in fine day choose robust growth, without the Centipeda minima plant of damage by disease and insect, clip stem section and terminal bud, as explant, cut off blade, cleaning, sterilize;
The induction of step 2. bud: shear the explant that disinfected into about the long section of 2cm, every section is stayed 1-2 axillalry bud, then oblique cutting or be vertically inserted in medium according to claim 1 is housed vial in; When explant is cultivated for the first time, a full light culture 10-15 days axillalry bud is sprouted, then moves to the place cultivation seeing light;
The propagation of step 3. bud and culture of rootage: when axillalry bud sprout grow to 1cm time, cut axillalry bud and be seeded in and be equipped with in the vial of medium, carry out shoot proliferation and the culture of rootage of bud, culturing room's temperature is 26 ± 2 DEG C, illumination 12h.d
-1, illuminance 1500-3000lx; Squamous subculture about 20 days buds grow to 2-3 ㎝, sprout root system, within 30 days, grow to 3-5 ㎝, the long 2-3 ㎝ of root, form whole plant, after 30 days, then shear the axillary bud stem section of aseptic seedling from some bottle seedling whole plants of taking root, cultivate, continuous shoot proliferation, cultivate 20 days, growth coefficient reaches 6.0-8.5, and bottle seedling of another part being taken root directly moves to outdoor hardening booth and cultivate 5-7 days under natural scattering illumination;
Step 4. take root seedling transplanting and manage and protect: seedling blade to be taken root is unfolded, leaf look dark green, seedling stem is dark green, caulom extends, can transplant during height of seedling 4cm, medium is poured out during transplanting, take out seedling, wash the medium remained on root, and clip long root system, retain 2-2.5cm long, transplant in the seedbed of sterilizing through 0.2% potassium permanganate, medium of seedling bed is yellow soil: thin river sand: peat soil, volume ratio is 3:1:1, to drench water, covered plastic film shed and black net, avoid direct sunlight, after transplanting one week, open part film breathable, alternately bactericide is sprayed at interval of 7 days, execute foliage fertilizer and composite fertilizer.
3. Centipeda minima once-seedling forming tissue culture and rapid propagation method according to claim 2, is characterized in that: described sterilization is the mercury chloride sterilization 6-7 minute of 0.1%.
4. Centipeda minima once-seedling forming tissue culture and rapid propagation method according to claim 2, is characterized in that: vial is wide-mouth bottle.
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| CN1907312A (en) * | 2006-08-22 | 2007-02-07 | 湖北中医学院 | Use of centipeda volatile oil in preparation of tumor growth and proliferation inhibitor |
| CN103040891A (en) * | 2013-01-08 | 2013-04-17 | 施慧达药业集团(吉林)有限公司 | Centipeda minima volatile oil and preparation method and application thereof |
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| CN103040891A (en) * | 2013-01-08 | 2013-04-17 | 施慧达药业集团(吉林)有限公司 | Centipeda minima volatile oil and preparation method and application thereof |
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