CN104429968B - A kind of method for tissue culture of Premna fulva Craib. - Google Patents
A kind of method for tissue culture of Premna fulva Craib. Download PDFInfo
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- CN104429968B CN104429968B CN201410788077.XA CN201410788077A CN104429968B CN 104429968 B CN104429968 B CN 104429968B CN 201410788077 A CN201410788077 A CN 201410788077A CN 104429968 B CN104429968 B CN 104429968B
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Abstract
The invention discloses the method for tissue culture of a kind of Premna fulva Craib., including: inducing culture, enrichment culture, strong seedling culture and root culture, wherein, inducing culture, enrichment culture, strong seedling culture and root culture carry out in solid medium. The method for tissue culture of Premna fulva Craib. provided by the invention can go out a large amount of excellent Premna fulva Craib. seedling being suitable for and transplanting by Fast-propagation, meets the needs on producing.
Description
Technical field
The present invention relates to a kind of method for propagation, particularly relate to the method for tissue culture of a kind of Premna fulva Craib..
Background technology
Premna fulva Craib., for Verbenaceae Radix Premnae fulvae, its stem or root are used as medicine title Folium premnae puberulae, acrid in the mouth, micro-sweet, slightly warm in nature; There is effect of promoting blood circulation to remove blood stasis, strengthening the tendons and bones, wind-expelling pain-stopping, for diseases such as rheumatic arthralgia, hypertrophy ridge synthetism vertebra inflammation, scapulohumeral periarthritis, impotence due to deficiency of the kidney, delayed periods.
The production of Premna fulva Craib. is mainly by wild resource, and Regional Distribution is scattered and reserves are rare. Along with the exploitation of series of products such as " JIANGU ZHUSHEYE ", " injury bone growing particle ", " Compound Premna Fulva Craib Liniments " that be primary raw material with Premna fulva Craib., its market demand rises year by year, and wild resource suffers excessively to excavate, and is on the verge of exhaustion. Under field conditions (factors), Premna fulva Craib. relies primarily on seed and breeds, but because its hip number is big, the Supply of photosynthate of blade is limited, causes fruit tiny, and seed development is imperfect, vitality is low, and production is difficult to commerial growing. Adopt biotechnology one tissue culture technique, it is possible to effectively and rapidly improve seedling quality and the breeding coefficient of Premna fulva Craib., it is achieved the industrial seedling rearing of Premna fulva Craib. high quality seedling, with the needs in satisfied production.
The outer implant major part of plant tissue culture takes from field, surface is attached with substantial amounts of microorganism, in order to obtain aseptic explant, necessary disinfection before implant inoculated and cultured outside, sterilization requires to kill the microorganism of outer planting surface, ensures that the damage of external implant is little as far as possible simultaneously. The sterilization of outer implant is the primary link in plant tissue culture, is also most important link. The problems such as existing disinfecting, mostly adopt hydrogen peroxide, sodium hypochlorite, antibiotic and mercuric chloride etc. to carry out disinfection, there is Disinfection Effect difference, disinfectant remains, and external implant and human injury are big, serious environment pollution.
The solid medium mummification of existing tissue culture is very fast, and culture periphery easily piles up metabolite, causes that the nutrition distribution pattern of culture medium is uneven, affects the growth of plant.
Summary of the invention
It is an object of the invention to solve at least the above and/or defect, and the advantage that at least will be described later is provided.
Technical scheme provided by the invention is:
A kind of method for tissue culture of Premna fulva Craib., including:
Inducing culture, enrichment culture, strong seedling culture and root culture, described inducing culture, enrichment culture, strong seedling culture and root culture carry out in solid medium.
Preferably, in the method for tissue culture of described Premna fulva Craib., described solid medium puts into gelfoam, described gelfoam is the cube of length of side 0.5cm, every liter of described solid medium is placed with 100-120 described gelfoam, when described solid medium solidifies, described gelfoam all uniformly immerses in described solid medium.
Preferably, in the method for tissue culture of described Premna fulva Craib., in described inducing culture, being 24-26 DEG C by outer implant in temperature, intensity of illumination is 1500lux, and cultivates 20d when light application time is 10-12h/d, evoking adventive bud is sprouted, to obtain in vitro cuttings;
Solid medium in described inducing culture comprises: MS, 30g L-1Sucrose, 1.0mg L-16-benzyladenine, 0.2mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8.
Preferably, in the method for tissue culture of described Premna fulva Craib., described enrichment culture is carried out after described inducing culture, the in vitro cuttings obtained through described inducing culture is 24-26 DEG C in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 10-12h/d, obtain a large amount of adventitious bud;
Solid medium in described enrichment culture comprises: MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 1.0-2.0mg L-16-benzyladenine, 0.1-1.0mg L-1Naphthalene acetic acid, 0.3-0.5mg L-1Heteroauxing and 3.5g L-1Agar, and to regulate original ph be 5.8.
Preferably, in the method for tissue culture of described Premna fulva Craib., described strong seedling culture is carried out after described enrichment culture, the adventitious bud obtained through described enrichment culture is 24-26 DEG C in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 10-12h/d, obtain healthy and strong plant;
Solid medium in described strong seedling culture comprises: MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 0.5-1.5mg L-16-benzyladenine, 0.1-2mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8.
Preferably, in the method for tissue culture of described Premna fulva Craib., described root culture is carried out after described strong seedling culture, the healthy and strong plant obtained through described strong seedling culture is 24-26 DEG C in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 10-12h/d, obtain band root;
Solid medium in described root culture comprises: 1/2MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 0.3-0.5mg L-1Root-inducing powder, 1.0-2.0mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8.
Preferably, in the method for tissue culture of described Premna fulva Craib., also including the step obtaining aseptic explant before described inducing culture, the step of described acquisition aseptic explant is:
Step one, take the stem section with axillalry bud as outer implant, it is positioned in beaker, add the liquid detergent aqueous solution soaking 5-10min that mass fraction is 0.05%-0.1%, immersion process is scrubbed gently described outer planting surface dirt with brush pen, use running water 15-20min after taking out described outer implant, move to superclean bench;
Step 2, outer implant after processing in described step one is soaked the 10-20 second in the ethanol that volume fraction is 70%-75%, then by described outer implant containing the surfactant that parts by weight are 0.01-0.1 part, the mixed solution of the antibacterial peptide of 0.1-1.0 part and the sterilized water of 70-100 part soaks 10-20min, recycling Low Temperature Plasma Treating is immersed in the outer implant 1-5min in described mixed solution, aseptic water washing is used 3-5 time after taking out described outer implant, 2-5min, finally blot described outer planting surface moisture with aseptic absorbent paper, it is cut into the stem section with an axillalry bud of 0.5-0.8cm, obtain aseptic explant.
Preferably, in the method for tissue culture of described Premna fulva Craib., surfactant described in described step 2 is any one or a few in tween 20, sucrose ester and hexadecyltrimethylammonium chloride.
Preferably, in the method for tissue culture of described Premna fulva Craib., described step 2 utilize the step of outer implant described in described Low Temperature Plasma Treating be: below the described mixed solution liquid level that submerged by the nozzle of atmos low-temperature plasma device, oxygen and mixing gas that helium mass ratio is 1: 20 is passed in described atmos low-temperature plasma device, then electric discharge device is opened, discharge process 1-5min allows the low temperature plasma that described atmos low-temperature plasma device produces contact with water, and the outer implant in described mixed solution is carried out sterilization processing.
Preferably, in the method for tissue culture of described Premna fulva Craib., also including the step of acclimatization and transplants after described root culture, the step of described acclimatization and transplants is:
Step (1), at temperature 23-25 DEG C, in the solid medium of described root culture add water make the solid medium of described root culture be entirely located in water, and continue cultivate 3-5d; And,
Step (2), clean the root culture medium of the test tube Seedling after processing in described step (1), be transplanted to peat soil with thin river sand mass ratio be 3: 1 mixed-matrix in cultivate 30-40d, then transplant to land for growing field crops.
The invention provides the method for tissue culture of a kind of Premna fulva Craib.. The invention have the advantage that
The first, in the present invention, external implant is first sterilized by soak with ethanol, the advantages such as it is very fast that ethanol has sterilization speed, and human body zest is little, nontoxic, external implant not damaged; Using antibacterial peptide soaking disinfection again, antibacterial peptide has Heat stability is good, broad-spectrum antiseptic, has no drug resistance, to advantages such as human body and outer implant not damageds; Finally with low-temperature plasma sterilization, high-voltage discharge low temperature plasma, combine Strong oxdiative chemical effect and the physical effects such as ultraviolet radiation, high intensity electromagnetic field such as free radical, ozone, hydrogen peroxide, destroy microbial cell body and hereditary material DNA simultaneously, can realize sterilizing fast and efficiently under room temperature, condition of normal pressure, outer implant and safety and environmental protection, noresidue will not be damaged.
The second, owing to solid medium mummification is very fast, culture periphery easily piles up metabolite, causes that the nutrition distribution pattern of culture medium is uneven. The present invention has put into gelfoam in solid medium, and gelfoam water absorption is very strong, and gelfoam is not when culture medium solidifies, a lot of culture medium can be absorbed, so, can effectively keep nutritional labeling, the distribution ratio making nutritional labeling is more uniform, promotes the growth of outer implant.
3rd, the present invention adds 6-benzyladenine and naphthalene acetic acid in inducing culture, it is possible to evoking adventive bud is sprouted; Proliferated culture medium adds 6-benzyladenine, naphthalene acetic acid and heteroauxing and can promote differentiation and the growth of adventitious bud;Strong seedling culture base adds 6-benzyladenine and naphthalene acetic acid can promote Seedling strong and the propagation again of bud; 1/2MS root media adds root-inducing powder and naphthalene acetic acid can obtain complete band root, can directly transplant to substrate after seedling exercising.
4th, the present invention adopts biotechnology that Premna fulva Craib. is carried out tissue-culturing quick-propagation, maintain the merit of original kind, cultivate the Premna fulva Craib. seedling being available for field production in a large number at short notice, significantly improve seedling quality and the breeding coefficient of Premna fulva Craib., suitable in factorial praluction, thus meeting the needs on producing.
5th, the simple bud growth coefficient that the present invention obtains reaches more than 6.5 times, and through expanding propagation, strong sprout and root culture, tissue cultured seedling rooting rate reaches more than 93.7%, and after transplanting medium, survival rate is more than 90.4%. The method for tissue culture of Premna fulva Craib. provided by the invention can go out a large amount of excellent Premna fulva Craib. seedling being suitable for and transplanting by Fast-propagation, meets the needs on producing.
Detailed description of the invention
Below in conjunction with embodiment, the present invention is described in further detail, to make those skilled in the art can implement according to this with reference to description word.
Embodiment 1:
The method for tissue culture of a kind of Premna fulva Craib., comprises the following steps:
Step one, the drawing materials and sterilization of outer implant: take the stem section with axillalry bud as outer implant, place in beaker, with the liquid detergent aqueous solution soaking 5min that mass fraction is 0.05%, immersion process cleans outer planting surface dirt with brush pen, then through running water 15min gently; Move to superclean bench, with the mercuric chloride that 100mL mass fraction the is 0.1% sterilization 8min that with the addition of 2-3 and drip tween 20, then with aseptic water washing 3 times, then blot surface moisture with aseptic absorbent paper, it is cut into the 0.5cm stem section with an axillalry bud, it is thus achieved that aseptic explant;
Step 2, in vitro cuttings acquisition: the aseptic explant obtained in described step one is inoculated in inducing culture, being 24 DEG C in cultivation temperature, intensity of illumination is 1500lux, cultivates 20d when light application time is 10h/d, evoking adventive bud is sprouted, and obtains in vitro cuttings; Wherein said inducing culture with MS for minimal medium, additional 30g L-1Sucrose, 1.0mg L-16-benzyladenine, 0.2mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8;
The breeding of step 3, test tube Seedling is cultivated: being placed in proliferated culture medium by the in vitro cuttings obtained in described step 2, be 24 DEG C in cultivation temperature, intensity of illumination is 1500lux, cultivates 30d, obtain a large amount of adventitious bud when light application time is 10h/d; Wherein said proliferated culture medium with MS for minimal medium, additional 30g L-1Sucrose, 1.0g L-1Activated carbon, 1.0mg L-16-benzyladenine, 0.1mg L-1Naphthalene acetic acid, 0.3mg L-1Heteroauxing and 3.5g L-1Agar, and to regulate original ph be 5.8;
Step 4, the growth of plants are cultivated: being placed in strong seedling culture base by the test tube Seedling adventitious bud obtained in described step 3, be 24 DEG C in cultivation temperature, intensity of illumination is 1500lux, cultivate 30d when light application time is 10h/d, obtain healthy and strong plant; Wherein said strong seedling culture base with MS for minimal medium, additional 30g L-1Sucrose, 1.0g L-1Activated carbon, 0.5mg L-16-benzyladenine, 0.1mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8;
Step 5, rooting of vitro seedling are cultivated: being placed in root media by the healthy and strong plant obtained in described step 4, be 24 DEG C in cultivation temperature, intensity of illumination is 1500lux, cultivate 30d, obtain complete band root when light application time is 10h/d;Wherein said root media with 1/2MS for minimal medium, additional 30g L-1Sucrose, 1.0g L-1Activated carbon, 0.3mg L-1Root-inducing powder, 1.0mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8;
Step 6, test tube Seedling acclimatization and transplants: after the process of described step 5 obtains complete band root, bottle cap is opened in the indoor that room temperature is 23 DEG C, bottle adds a small amount of tap water and floods culture medium, seedling exercising 3d, test tube Seedling is taken out with tweezers, clean root culture medium, be transplanted in the substrate that peat soil and thin river sand mass ratio are 3: 1 growth 30d, then transplant to land for growing field crops.
Embodiment 2:
The method for tissue culture of a kind of Premna fulva Craib., comprises the following steps:
Step one, the drawing materials and sterilization of outer implant: take the stem section with axillalry bud as outer implant, place in beaker, with the liquid detergent aqueous solution soaking 5min that mass fraction is 0.05%, immersion process cleans outer planting surface dirt with brush pen, then through running water 15min gently; Move to superclean bench, with the mercuric chloride that 100mL mass fraction the is 0.1% sterilization 10min that with the addition of 2-3 and drip tween 20, then with aseptic water washing 5 times, then blot surface moisture with aseptic absorbent paper, it is cut into the 0.8cm stem section with an axillalry bud, it is thus achieved that aseptic explant;
Step 2, in vitro cuttings acquisition: the aseptic explant obtained in described step one is inoculated in inducing culture, being 26 DEG C in cultivation temperature, intensity of illumination is 1500lux, cultivates 20d when light application time is 12h/d, evoking adventive bud is sprouted, and obtains in vitro cuttings; Wherein said inducing culture with MS for minimal medium, additional 30g L-1Sucrose, 1.0mg L-16-benzyladenine, 0.2mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8;
The breeding of step 3, test tube Seedling is cultivated: being placed in proliferated culture medium by the in vitro cuttings obtained in described step 2, be 26 DEG C in cultivation temperature, intensity of illumination is 1500lux, cultivates 30d, obtain a large amount of adventitious bud when light application time is 12h/d; Wherein said proliferated culture medium with MS for minimal medium, additional 30g L-1Sucrose, 1.0g L-1Activated carbon, 2.0mg L-16-benzyladenine, 1.0mg L-1Naphthalene acetic acid, 0.5mg L-1Heteroauxing and 3.5g L-1Agar, and to regulate original ph be 5.8;
Step 4, the growth of plants are cultivated: being placed in strong seedling culture base by the test tube Seedling adventitious bud obtained in described step 3, be 26 DEG C in cultivation temperature, intensity of illumination is 1500lux, cultivate 30d when light application time is 12h/d, obtain healthy and strong plant; Wherein said strong seedling culture base with MS for minimal medium, additional 30g L-1Sucrose, 1.0g L-1Activated carbon, 1.5mg L-16-benzyladenine, 2.0mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8;
Step 5, rooting of vitro seedling are cultivated: being placed in root media by the healthy and strong plant obtained in described step 4, be 26 DEG C in cultivation temperature, intensity of illumination is 1500lux, cultivate 30d, obtain complete band root when light application time is 12h/d; Wherein said root media with 1/2MS for minimal medium, additional 30g L-1Sucrose, 1.0g L-1Activated carbon, 0.5mg L-1Root-inducing powder, 2.0mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8;
Step 6, test tube Seedling acclimatization and transplants: after the process of described step 5 obtains complete band root, bottle cap is opened in the indoor that room temperature is 25 DEG C, bottle adds a small amount of tap water and floods culture medium, seedling exercising 5d, test tube Seedling is taken out with tweezers, clean root culture medium, be transplanted in the substrate that peat soil and thin river sand mass ratio are 3: 1 growth 40d, then transplant to land for growing field crops.
Embodiment 3:
The method for tissue culture of a kind of Premna fulva Craib., comprises the following steps:
Step one, the drawing materials and sterilization of outer implant: take the stem section with axillalry bud as outer implant, place in beaker, with the liquid detergent aqueous solution soaking 5min that mass fraction is 0.05%, immersion process cleans outer planting surface dirt with brush pen, then through running water 15min gently; Move to superclean bench, with the mercuric chloride that 100mL mass fraction the is 0.1% sterilization 9min that with the addition of 2-3 and drip tween 20, then with aseptic water washing 4 times, then blot surface moisture with aseptic absorbent paper, it is cut into the 0.6cm stem section with an axillalry bud, it is thus achieved that aseptic explant;
Step 2, in vitro cuttings acquisition: the aseptic explant obtained in described step one is inoculated in inducing culture, being 25 DEG C in cultivation temperature, intensity of illumination is 1500lux, cultivates 20d when light application time is 11h/d, evoking adventive bud is sprouted, and obtains in vitro cuttings; Wherein said inducing culture with MS for minimal medium, additional 30g L-1Sucrose, 1.0mg L-16-benzyladenine, 0.2mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8;
The breeding of step 3, test tube Seedling is cultivated: being placed in proliferated culture medium by the in vitro cuttings obtained in described step 2, be 25 DEG C in cultivation temperature, intensity of illumination is 1500lux, cultivates 30d, obtain a large amount of adventitious bud when light application time is 11h/d; Wherein said proliferated culture medium with MS for minimal medium, additional 30g L-1Sucrose, 1.0g L-1Activated carbon, 1.5mg L-16-benzyladenine, 0.5mg L-1Naphthalene acetic acid, 0.4mg L-1Heteroauxing and 3.5g L-1Agar, and to regulate original ph be 5.8;
Step 4, the growth of plants are cultivated: being placed in strong seedling culture base by the test tube Seedling adventitious bud obtained in described step 3, be 25 DEG C in cultivation temperature, intensity of illumination is 1500lux, cultivate 30d when light application time is 11h/d, obtain healthy and strong plant; Wherein said strong seedling culture base with MS for minimal medium, additional 30g L-1Sucrose, 1.0g L-1Activated carbon, 1.0mg L-16-benzyladenine, 1.0mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8;
Step 5, rooting of vitro seedling are cultivated: being placed in root media by the healthy and strong plant obtained in described step 4, be 25 DEG C in cultivation temperature, intensity of illumination is 1500lux, cultivate 30d, obtain complete band root when light application time is 11h/d; Wherein said root media with 1/2MS for minimal medium, additional 30g L-1Sucrose, 1.0g L-1Activated carbon, 0.4mg L-1Root-inducing powder, 1.5mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8;
Step 6, test tube Seedling acclimatization and transplants: after the process of described step 5 obtains complete band root, bottle cap is opened in the indoor that room temperature is 24 DEG C, bottle adds a small amount of tap water and floods culture medium, seedling exercising 4d, test tube Seedling is taken out with tweezers, cleaning root culture medium, being transplanted to peat soil with thin river sand mass ratio is 3��The substrate of 1 grows 35d, then transplants to land for growing field crops.
Embodiment 4:
A kind of method for tissue culture of Premna fulva Craib., including:
Inducing culture, enrichment culture, strong seedling culture and root culture, described inducing culture, enrichment culture, strong seedling culture and root culture carry out in solid medium.
In the method for tissue culture of described Premna fulva Craib., described solid medium puts into gelfoam, described gelfoam is the cube of length of side 0.5cm, every liter of described solid medium is placed with 110 described gelfoam, when described solid medium solidifies, described gelfoam all uniformly immerses in described solid medium.
In the method for tissue culture of described Premna fulva Craib., in described inducing culture, being 25 DEG C by outer implant in temperature, intensity of illumination is 1500lux, and cultivates 20d when light application time is 11h/d, and evoking adventive bud is sprouted, to obtain in vitro cuttings;
Solid medium in described inducing culture comprises: MS, 30g L-1Sucrose, 1.0mg L-16-benzyladenine, 0.2mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8.
In the method for tissue culture of described Premna fulva Craib., carrying out described enrichment culture after described inducing culture, be 25 DEG C by the in vitro cuttings obtained through described inducing culture in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 11h/d, obtain a large amount of adventitious bud;
Solid medium in described enrichment culture comprises: MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 1.5mg L-16-benzyladenine, 0.5mg L-1Naphthalene acetic acid, 0.4mg L-1Heteroauxing and 3.5g L-1Agar, and to regulate original ph be 5.8.
In the method for tissue culture of described Premna fulva Craib., carrying out described strong seedling culture after described enrichment culture, be 25 DEG C by the adventitious bud obtained through described enrichment culture in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 11h/d, obtain healthy and strong plant;
Solid medium in described strong seedling culture comprises: MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 1.0mg L-16-benzyladenine, 1.0mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8.
In the method for tissue culture of described Premna fulva Craib., carrying out described root culture after described strong seedling culture, be 25 DEG C by the healthy and strong plant obtained through described strong seedling culture in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 11h/d, obtain band root;
Solid medium in described root culture comprises: 1/2MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 0.4mg L-1Root-inducing powder, 1.5mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8.
In the method for tissue culture of described Premna fulva Craib., also including the step obtaining aseptic explant before described inducing culture, the step of described acquisition aseptic explant is:
Step one, take the stem section with axillalry bud as outer implant, it is positioned in beaker, add the liquid detergent aqueous solution soaking 7min that mass fraction is 0.75%, immersion process is scrubbed gently described outer planting surface dirt with brush pen, use running water 17min after taking out described outer implant, move to superclean bench;
Step 2, outer implant after processing in described step one is soaked 15 seconds in the ethanol that volume fraction is 73%, then by described outer implant containing the hexadecyltrimethylammonium chloride that parts by weight are 0.05 part, the mixed solution of the sterilized water of the antibacterial peptide of 0.5 part and 85 parts soaks 15min, recycling Low Temperature Plasma Treating is immersed in the outer implant 3min in described mixed solution, aseptic water washing is used 4 times after taking out described outer implant, 4min, finally blot described outer planting surface moisture with aseptic absorbent paper, it is cut into the stem section with an axillalry bud of 0.6cm, obtain aseptic explant.
In the method for tissue culture of described Premna fulva Craib., described step 2 utilize the step of outer implant described in described Low Temperature Plasma Treating be: below the described mixed solution liquid level that submerged by the nozzle of atmos low-temperature plasma device, oxygen and mixing gas that helium mass ratio is 1: 20 is passed in described atmos low-temperature plasma device, then electric discharge device is opened, discharge process 3min allows the low temperature plasma that described atmos low-temperature plasma device produces contact with water, and the outer implant in described mixed solution is carried out sterilization processing.
In the method for tissue culture of described Premna fulva Craib., also including the step of acclimatization and transplants after described root culture, the step of described acclimatization and transplants is:
Step (1), at temperature 24 DEG C, in the solid medium of described root culture add water make the solid medium of described root culture be entirely located in water, and continue cultivate 4d; And,
Step (2), clean the root culture medium of the test tube Seedling after processing in described step (1), be transplanted to peat soil with thin river sand mass ratio be 3: 1 mixed-matrix in cultivate 35d, then transplant to land for growing field crops.
Embodiment 5:
A kind of method for tissue culture of Premna fulva Craib., including:
Inducing culture, enrichment culture, strong seedling culture and root culture, described inducing culture, enrichment culture, strong seedling culture and root culture carry out in solid medium.
In the method for tissue culture of described Premna fulva Craib., described solid medium puts into gelfoam, described gelfoam is the cube of length of side 0.5cm, every liter of described solid medium is placed with 100 described gelfoam, when described solid medium solidifies, described gelfoam all uniformly immerses in described solid medium.
In the method for tissue culture of described Premna fulva Craib., in described inducing culture, being 24 DEG C by outer implant in temperature, intensity of illumination is 1500lux, and cultivates 20d when light application time is 10h/d, and evoking adventive bud is sprouted, to obtain in vitro cuttings;
Solid medium in described inducing culture comprises: MS, 30g L-1Sucrose, 1.0mg L-16-benzyladenine, 0.2mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8.
In the method for tissue culture of described Premna fulva Craib., carrying out described enrichment culture after described inducing culture, be 24 DEG C by the in vitro cuttings obtained through described inducing culture in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 10h/d, obtain a large amount of adventitious bud;
Solid medium in described enrichment culture comprises: MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 1.0mg L-16-benzyladenine, 0.1mg L-1Naphthalene acetic acid, 0.3mg L-1Heteroauxing and 3.5g L-1Agar, and to regulate original ph be 5.8.
In the method for tissue culture of described Premna fulva Craib., carrying out described strong seedling culture after described enrichment culture, be 24 DEG C by the adventitious bud obtained through described enrichment culture in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 10h/d, obtain healthy and strong plant;
Solid medium in described strong seedling culture comprises: MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 0.5mg L-16-benzyladenine, 0.1mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8.
In the method for tissue culture of described Premna fulva Craib., carrying out described root culture after described strong seedling culture, be 24 DEG C by the healthy and strong plant obtained through described strong seedling culture in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 10h/d, obtain band root;
Solid medium in described root culture comprises: 1/2MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 0.3mg L-1Root-inducing powder, 1.0mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8.
In the method for tissue culture of described Premna fulva Craib., also including the step obtaining aseptic explant before described inducing culture, the step of described acquisition aseptic explant is:
Step one, take the stem section with axillalry bud as outer implant, it is positioned in beaker, add the liquid detergent aqueous solution soaking 5min that mass fraction is 0.05%, immersion process is scrubbed gently described outer planting surface dirt with brush pen, use running water 15min after taking out described outer implant, move to superclean bench;
Step 2, outer implant after processing in described step one is soaked the 10-20 second in the ethanol that volume fraction is 70%, then by described outer implant containing the tween 20 that parts by weight are 0.01 part, the mixed solution of the sterilized water of the antibacterial peptide of 0.1 part and 70 parts soaks 10min, recycling Low Temperature Plasma Treating is immersed in the outer implant 1.0min in described mixed solution, aseptic water washing is used 3 times after taking out described outer implant, 2.0min, finally blot described outer planting surface moisture with aseptic absorbent paper, it is cut into the stem section with an axillalry bud of 0.5cm, obtain aseptic explant.
In the method for tissue culture of described Premna fulva Craib., described step 2 utilize the step of outer implant described in described Low Temperature Plasma Treating be: below the described mixed solution liquid level that submerged by the nozzle of atmos low-temperature plasma device, oxygen and mixing gas that helium mass ratio is 1: 20 is passed in described atmos low-temperature plasma device, then electric discharge device is opened, discharge process 1.0min allows the low temperature plasma that described atmos low-temperature plasma device produces contact with water, and the outer implant in described mixed solution is carried out sterilization processing.
In the method for tissue culture of described Premna fulva Craib., also including the step of acclimatization and transplants after described root culture, the step of described acclimatization and transplants is:
Step (1), at temperature 23 DEG C, in the solid medium of described root culture add water make the solid medium of described root culture be entirely located in water, and continue cultivate 3d; And,
Step (2), clean the root culture medium of the test tube Seedling after processing in described step (1), be transplanted to peat soil with thin river sand mass ratio be 3: 1 mixed-matrix in cultivate 30d, then transplant to land for growing field crops.
Embodiment 6:
A kind of method for tissue culture of Premna fulva Craib., including:
Inducing culture, enrichment culture, strong seedling culture and root culture, described inducing culture, enrichment culture, strong seedling culture and root culture carry out in solid medium.
In the method for tissue culture of described Premna fulva Craib., described solid medium puts into gelfoam, described gelfoam is the cube of length of side 0.5cm, every liter of described solid medium is placed with 120 described gelfoam, when described solid medium solidifies, described gelfoam all uniformly immerses in described solid medium.
In the method for tissue culture of described Premna fulva Craib., in described inducing culture, being 26 DEG C by outer implant in temperature, intensity of illumination is 1500lux, and cultivates 20d when light application time is 12h/d, and evoking adventive bud is sprouted, to obtain in vitro cuttings;
Solid medium in described inducing culture comprises: MS, 30g L-1Sucrose, 1.0mg L-16-benzyladenine, 0.2mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8.
In the method for tissue culture of described Premna fulva Craib., carrying out described enrichment culture after described inducing culture, be 26 DEG C by the in vitro cuttings obtained through described inducing culture in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 12h/d, obtain a large amount of adventitious bud;
Solid medium in described enrichment culture comprises: MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 2.0mg L-16-benzyladenine, 1.0mg L-1Naphthalene acetic acid, 0.5mg L-1Heteroauxing and 3.5g L-1Agar, and to regulate original ph be 5.8.
In the method for tissue culture of described Premna fulva Craib., carrying out described strong seedling culture after described enrichment culture, be 26 DEG C by the adventitious bud obtained through described enrichment culture in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 12h/d, obtain healthy and strong plant;
Solid medium in described strong seedling culture comprises: MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 1.5mg L-16-benzyladenine, 2.0mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8.
In the method for tissue culture of described Premna fulva Craib., carrying out described root culture after described strong seedling culture, be 26 DEG C by the healthy and strong plant obtained through described strong seedling culture in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 12h/d, obtain band root;
Solid medium in described root culture comprises: 1/2MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 0.5mg L-1Root-inducing powder, 2.0mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8.
In the method for tissue culture of described Premna fulva Craib., also including the step obtaining aseptic explant before described inducing culture, the step of described acquisition aseptic explant is:
Step one, take the stem section with axillalry bud as outer implant, it is positioned in beaker, add the liquid detergent aqueous solution soaking 10min that mass fraction is 0.1%, immersion process is scrubbed gently described outer planting surface dirt with brush pen, use running water 20min after taking out described outer implant, move to superclean bench;
Step 2, outer implant after processing in described step one is soaked 20 seconds in the ethanol that volume fraction is 75%, then by described outer implant containing the sucrose ester that parts by weight are 0.1 part, the mixed solution of the sterilized water of the antibacterial peptide of 1.0 parts and 100 parts soaks 20min, recycling Low Temperature Plasma Treating is immersed in the outer implant 5min in described mixed solution, aseptic water washing is used 5 times after taking out described outer implant, 5min, finally blot described outer planting surface moisture with aseptic absorbent paper, it is cut into the stem section with an axillalry bud of 0.8cm, obtain aseptic explant.
In the method for tissue culture of described Premna fulva Craib., described step 2 utilize the step of outer implant described in described Low Temperature Plasma Treating be: below the described mixed solution liquid level that submerged by the nozzle of atmos low-temperature plasma device, oxygen and mixing gas that helium mass ratio is 1: 20 is passed in described atmos low-temperature plasma device, then electric discharge device is opened, discharge process 5min allows the low temperature plasma that described atmos low-temperature plasma device produces contact with water, and the outer implant in described mixed solution is carried out sterilization processing.
In the method for tissue culture of described Premna fulva Craib., also including the step of acclimatization and transplants after described root culture, the step of described acclimatization and transplants is:
Step (1), at temperature 25 DEG C, in the solid medium of described root culture add water make the solid medium of described root culture be entirely located in water, and continue cultivate 5d; And,
Step (2), clean the root culture medium of the test tube Seedling after processing in described step (1), be transplanted to peat soil with thin river sand mass ratio be 3: 1 mixed-matrix in cultivate 40d, then transplant to land for growing field crops.
Although embodiment of the present invention are disclosed as above, but listed utilization that it is not restricted in description and embodiment, it can be applied to various applicable the field of the invention completely, for those skilled in the art, it is easily achieved other amendment, therefore, under the general concept limited without departing substantially from claim and equivalency range, the present invention is not limited to specific details and shown here as the embodiment with description.
Claims (9)
1. the method for tissue culture of a Premna fulva Craib., including inducing culture, enrichment culture, strong seedling culture and root culture, wherein, using the stem section with axillalry bud as outer implant, described inducing culture, enrichment culture, strong seedling culture and root culture carry out in solid medium; Consisting of of solid medium in described inducing culture: MS, 30g L-1Sucrose, 1.0mg L-16-benzyladenine, 0.2mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8; Consisting of of solid medium in described enrichment culture: MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 1.0-2.0mg L-16-benzyladenine, 0.1-1.0mg L-1Naphthalene acetic acid, 0.3-0.5mg L-1Heteroauxing and 3.5g L-1Agar, and to regulate original ph be 5.8; Consisting of of solid medium in described strong seedling culture: MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 0.5-1.5mg L-16-benzyladenine, 0.1-2mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8; Consisting of of solid medium in described root culture: 1/2MS, 30g L-1Sucrose, 1.0g L-1Activated carbon, 0.3-0.5mg L-1Root-inducing powder, 1.0-2.0mg L-1Naphthalene acetic acid and 3.5g L-1Agar, and to regulate original ph be 5.8, solid medium in described inducing culture, solid medium in described enrichment culture, solid medium in described strong seedling culture and all put into gelfoam in the solid medium in described root culture, described gelfoam is the cube of length of side 0.5cm, solid medium in every liter of described inducing culture, solid medium in described enrichment culture, solid medium in described strong seedling culture and be all placed with 100-120 described gelfoam in the solid medium in described root culture, solid medium in described inducing culture, solid medium in described enrichment culture, when the solid medium in described strong seedling culture and the solidification of the solid medium in described root culture, described gelfoam all uniformly immerses the solid medium in described inducing culture, solid medium in described enrichment culture, solid medium in described strong seedling culture and in the solid medium in described root culture.
2. the method for tissue culture of Premna fulva Craib. as claimed in claim 1, wherein, in described inducing culture, being 24-26 DEG C by outer implant in temperature, intensity of illumination is 1500lux, and cultivates 20d when light application time is 10-12h/d, evoking adventive bud is sprouted, to obtain in vitro cuttings.
3. the method for tissue culture of Premna fulva Craib. as claimed in claim 1, wherein, described enrichment culture is carried out after described inducing culture, the in vitro cuttings obtained through described inducing culture is 24-26 DEG C in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 10-12h/d, obtain a large amount of adventitious bud.
4. the method for tissue culture of Premna fulva Craib. as claimed in claim 1, wherein, described strong seedling culture is carried out after described enrichment culture, the adventitious bud obtained through described enrichment culture is 24-26 DEG C in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 10-12h/d, obtain healthy and strong plant.
5. the method for tissue culture of the Premna fulva Craib. as described in any one of claim 1-4, wherein, described root culture is carried out after described strong seedling culture, the healthy and strong plant obtained through described strong seedling culture is 24-26 DEG C in temperature, intensity of illumination is 1500lux, and light application time cultivates 30d when being 10-12h/d, obtain band root.
6. the method for tissue culture of Premna fulva Craib. as claimed in claim 1, also included the step obtaining aseptic explant before described inducing culture, and wherein, the step of described acquisition aseptic explant is:
Step one, take the stem section with axillalry bud as outer implant, it is positioned in beaker, add the liquid detergent aqueous solution soaking 5-10min that mass fraction is 0.05%-0.1%, immersion process is scrubbed gently described outer planting surface dirt with brush pen, use running water 15-20min after taking out described outer implant, move to superclean bench;
Step 2, outer implant after processing in described step one is soaked the 10-20 second in the ethanol that volume fraction is 70%-75%, then by described outer implant containing the surfactant that parts by weight are 0.01-0.1 part, the mixed solution of the antibacterial peptide of 0.1-1.0 part and the sterilized water of 70-100 part soaks 10-20min, recycling Low Temperature Plasma Treating is immersed in the outer implant 1-5min in described mixed solution, aseptic water washing is used 3-5 time after taking out described outer implant, 2-5min, finally blot described outer planting surface moisture with aseptic absorbent paper, it is cut into the stem section with an axillalry bud of 0.5-0.8cm, obtain aseptic explant.
7. the method for tissue culture of Premna fulva Craib. as claimed in claim 6, wherein, surfactant described in described step 2 is any one or a few in tween 20, sucrose ester and hexadecyltrimethylammonium chloride.
8. the method for tissue culture of Premna fulva Craib. as claimed in claim 6, wherein, described step 2 utilize the step of outer implant described in described Low Temperature Plasma Treating be: below the described mixed solution liquid level that submerged by the nozzle of atmos low-temperature plasma device, oxygen and helium mass is passed into than the mixing gas for 1:20 in described atmos low-temperature plasma device, then electric discharge device is opened, discharge process 1-5min allows the low temperature plasma that described atmos low-temperature plasma device produces contact with water, outer implant in described mixed solution is carried out sterilization processing.
9. the method for tissue culture of Premna fulva Craib. as claimed in claim 1, also includes the step of acclimatization and transplants after described root culture, and wherein, the step of described acclimatization and transplants is:
Step (1), at temperature 23-25 DEG C, in the solid medium of described root culture add water make the solid medium of described root culture be entirely located in water, and continue cultivate 3-5d; And,
The root culture medium of the test tube Seedling after processing in step (2), clean described step (1), is transplanted in the mixed-matrix that peat soil is 3:1 with thin river sand mass ratio and cultivates 30-40d, then transplant to land for growing field crops.
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