CN104585029B - A kind of method suppressing the pollution of Dysosma versipellis explant and brown stain - Google Patents
A kind of method suppressing the pollution of Dysosma versipellis explant and brown stain Download PDFInfo
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- CN104585029B CN104585029B CN201410840157.5A CN201410840157A CN104585029B CN 104585029 B CN104585029 B CN 104585029B CN 201410840157 A CN201410840157 A CN 201410840157A CN 104585029 B CN104585029 B CN 104585029B
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Abstract
The invention discloses a kind of Dysosma versipellis explant that suppresses to pollute and the method for brown stain, comprise and choose life in 5 years wild Dysosma versipellis rhizome above and be transplanted in clean river sand matrix and carry out preculture; The stem section of robust growth is positioned in N-long-chain thiocarboxylic acid type betain again and soaks, then use distilled water flushing, and scrub described explant surface smut; Again explant is utilized negative ion photocatalyst process, then use clorox sterilizing, and with aseptic water washing, then the moisture on surface is blotted with aseptic filter paper, and cut into square, and adopt heavy metal complexing agent to soak; Then MS improved culture medium light culture 2 weeks are inoculated in; Finally carry out bright cultivation.Method of the present invention effectively can suppress the pollution of Dysosma versipellis explant and prevent the brown stain of Dysosma versipellis explant, Study on tissue culture for medicinal plant Dysosma versipellis provides sufficient aseptic explant, thus ensure that the success rate of follow-up tissue cultures, and cost is lower, is suitable for suitability for industrialized production.
Description
Technical field
The present invention relates to a kind of method suppressing explant pollution and brown stain, particularly relate to a kind of method for tissue culture suppressing the pollution of Dysosma versipellis explant and brown stain.
Background technology
Medicinal plant Dysosma versipellis is under the jurisdiction of Berberidaceae Podophyllum emodi var chinense and belongs to perennial herb, is one of traditional main plant source of the cancer therapy drug such as Etoposide and Teniposide synthesis precursor-podophyllotoxin.Owing to excessively excavating, habitat destruction and the reason such as plant own growth is slow, Dysosma versipellis wild resource is close to exhaustion, and species are in imminent danger, has been listed in national Precious, Rare, Endangered three-level protective plant at present.Therefore, by adopting medicinal plant tissue culture propagating technology to obtain Dysosma versipellis callus or aseptic seedling, both can alleviate the resource pressure of Dysosma versipellis, also can provide available sterilizable material for industrial production.
The great advantage of medicinal plant tissue cultures can breed a large amount of high quality seedling in a short time with less material, have the features such as few with material, the cycle is short, speed is subject to seasonal restrictions soon and not.Therefore, tissue culture technique fast seedling growing is utilized to become the important channel of many medicinal plant commercialization nursery.
About the research report of the group culturation rapid propagating technology of Dysosma versipellis is less, its subject matter easily produces serious germ contamination in the tissue culture procedures of Dysosma versipellis, and pollution rate reaches 100%; Meanwhile, because Dysosma versipellis contains a large amount of aldehydes matters, be very easy to produce browning phenomenon; Stem after cutting, leaf texture discharge brown material from surface to medium, now medium overstrike gradually, and explant also browning and dead gradually, can not survive.
Brown stain is the problem often run in plant tissue culture course, as solution is improper, often causes cell death.Research about Browning Mechanism shows, containing more phenolic compound in plant cell, quinones substance can be changed under the effect of polyphenol oxidase, accumulate in explant injury, and spread in other tissue and medium, destroy normal metabolic process, implant is grown and is suppressed even death.The research of recent years shows, polyphenol oxidase is a kind of protein of cupric, and its oxidation activity depends on the redox of copper.Metal-chelator, can Cu if EDTA is as inhibitor
2+from enzyme, chelating out, forms stable chelate, and polyphenol oxidase is lost activity, thus reach the effect of Restrain browning.
Therefore, in order to set up efficient Dysosma versipellis group culturation rapid propagating technology system, primary pollution and the browning solving Dysosma versipellis explant.
Summary of the invention
An object of the present invention is to solve at least the problems referred to above and/or defect, and the advantage will illustrated at least is below provided.
Technical scheme provided by the invention is:
Suppress a method for the pollution of Dysosma versipellis explant and brown stain, comprise the following steps:
(1) choose the above wild Dysosma versipellis rhizome of life in 5 years as maternal plant, maternal plant is transplanted to and clean river sand matrix carries out preculture 1 month to growing new stem and leaf;
(2) take steps (1) robust growth, without the stem section of damage by disease and insect or young leaflet tablet, first be positioned in N-long-chain thiocarboxylic acid type betain and soak 5min, use distilled water flushing again 1 hour, in flushing process, scrub described explant surface smut with banister brush;
(3) explant after step (2) being processed utilizes negative ion photocatalyst process 10-15min, be the clorox sterilizing 4.5min-7min of 2.5% again by concentration, and with aseptic water washing 3 times, each washing time is 2min, blot the moisture on described explant surface again with aseptic filter paper, material is cut into the square of 1cm × 1cm or the stem section of 1cm length;
(4) concentration is adopted to be the stem section that cuts of the heavy metal complexing agent soaking step (3) of 0.1%-1% or square 1 hour;
(5) material after step (4) being processed is inoculated in MS improved culture medium, and pH value is adjusted to 5.8, at the temperature of 25 DEG C ± 1 DEG C, and light culture 2 weeks under 1.5 atmospheric conditions;
(6) it is 1000-2000Lux that the explant after step (5) being processed forwards intensity of illumination to, and light application time is 10h/d, and temperature is cultivate under the condition of culture of 25 DEG C ± 1 DEG C;
Described in step (3), negative ion photocatalyst is by TiO
2with the neutral liquid that the nanometer grade powder of piezo-electric crystal tourmaline is made.
Utilize in step (3) step of explant described in negative ion photocatalyst process for explant is positioned over scribble negative ion photocatalyst tank body in 10-15min, nitrogen is passed into and ozone mixing ratio is the mist of 10: 1 in tank body, negative ion photocatalyst produces the hydroxy plasma in a large number with bactericidal action by extraneous light excites, thus carries out sterilization processing to explant.
Described heavy metal complexing agent is phytic acid and diethylene triamine pentamethylene phosphonic 1: 3 solution mixed in mass ratio.
Utilize the step of heavy metal complexing agent process explant in concentration explant the being positioned over 10-15 DEG C heavy metal complexing agent that is 0.1%-1% in step (4), within every 15 minutes, change a heavy metal complexing agent, shading cultivates 1 hour.
The composition of MS improved culture medium described in step (5) comprises MS medium, 2mg/L caseinhydrolysate, 2mg/L active carbon, 0.02mg/L vitamin h and the 0.8mg/L Yan acid not containing copper ion.
Preferably, described in step (1), the humidity of river sand is 65%-85%.
Preferably, adopt in step (6) red LED light and blue LED light ratio be 4: 1 mixed light throw light on.
The invention provides a kind of method suppressing the pollution of Dysosma versipellis explant and brown stain.Advantage of the present invention is:
The first, the present invention Dysosma versipellis plant of selecting life in 5 years above, be to provide high-quality explant, reduce the basis of browning in incubation, compare the Dysosma versipellis of life in 3 years, the rhizome of the Dysosma versipellis plant that life in 5 years is above is larger, and the explant formed is also strongr.
The second, the present invention selects N-long-chain thiocarboxylic acid type betain to soak, N-long-chain thiocarboxylic acid type betain has good antibiotic property, the growth and breeding of Gram-positive bacteria, Gram-negative bacteria and some fungi can be suppressed, N-long-chain thiocarboxylic acid type betain surface-active is high simultaneously, excitant is very low, and wet performance is very outstanding, can effectively take out stains, can not cause damage to explant, and safe noresidue.
3rd, the present invention adopts negative ion photocatalyst process explant to explant, negative ion photocatalyst is under the irradiation of light, similar photosynthetic light-catalyzed reaction can be produced, produce the extremely strong free hydroxy of oxidability and active oxygen, there is very strong photoredox function, the various organic compound of oxidable decomposition and part inorganic matter, the cell membrane of bacterium and the protein of solidification virus can be destroyed, can kill bacteria Sum decomposition organic pollutant, organic pollutant is resolved into free of contamination water and carbonic acid gas, there is extremely strong sterilization, the function of anti-fouling and self-cleaning, at normal temperature, realize fast under condition of normal pressure, sterilization efficiently, explant can not be damaged, and safety and environmental protection.
Four, the present invention adopts and soaks explant by the solution of phytic acid and diethylene triamine pentamethylene phosphonic 1: 3 mixing in mass ratio, due in phytic acid containing multiple phosphate group, the complexing of phytic acid is than the wider model of EDTA and stable; Diaminoethanes tetraacethyl sodium chelating ability is all more eager to excel in whatever one does than EDTA class, phosphoric acid salt etc., and complexing capacity is high, and complexing stability constant is large, and metal ion etc. are not easy after complexing to dissociate, and chemical-resistant stability is good, nontoxic and easily biological-degradable, nuisanceless pollution.Therefore by adding phytic acid and diaminoethanes tetraacethyl sodium, by Cu
2+from polyphenol oxidase, chelating out, forms stable chelate, and polyphenol oxidase is lost activity, thus reach the effect of Restrain browning.
Five, the present invention adds caseinhydrolysate, active carbon, vitamin h and Yan acid in inducing culture, hydrolysis network albumen is a kind of Organic additives matter, it has facilitation to growing of Dysosma versipellis plant cell, and contributes to the formation suppressing brownization in explant During Callus Induction; Activated carbon uses as a kind of adsorbent, adsorbable micro substance and molecule, has positive effect for preventing Brown; Vitamin h is the multiple metabolic activity of considerable nutriment in growth and development of plants, its involved in plant, contributes to the Dysosma versipellis seedling cultivating high-quality.
Six, adopt in step (6) in the present invention red LED light and blue LED light ratio be 4: 1 mixed light throw light on, LED has that volume is little, lightweight, solid-state, the life-span is long, wavelength is special, driving voltage compared with low, photoeffect is high, energy consumption is little, safe, reliability and durability, be not easy the advantage that look declines, when proportion of red blue light is 4: 1, the inductivity of Dysosma versipellis explant is the highest.
Seven, the aseptic explant that obtains of the present invention, reaches more than 3.5 times through induction simple bud growth coefficient, and by expanding numerous, strong sprout and culture of rootage further, plantlet in vitro rooting rate reaches more than 85%, and after transplanting medium, survival rate is more than 90%.The method for tissue culture of Dysosma versipellis provided by the invention can go out a large amount of excellent Dysosma versipellis seedling being applicable to transplanting by Fast-propagation, meets the needs on producing.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, can implement according to this with reference to specification word to make those skilled in the art.
Embodiment 1:
Suppress a method for the pollution of Dysosma versipellis explant and brown stain, comprise the following steps:
(1) choose the above wild Dysosma versipellis rhizome of life in 5 years as maternal plant, maternal plant is transplanted to humidity be 65% river sand matrix on carry out preculture 1 month to growing new stem and leaf;
(2) take steps (1) robust growth, without the stem section of damage by disease and insect or young leaflet tablet, first be positioned in N-long-chain thiocarboxylic acid type betain and soak 5min, use distilled water flushing again 1 hour, in flushing process, scrub described explant surface smut with banister brush;
(3) explant after step (2) being processed is positioned over and scribbles TiO
210min in the tank body of the neutral coating made with the nanometer grade powder of piezo-electric crystal tourmaline, and in tank body, pass into nitrogen and ozone mixing ratio is the mist of 10: 1, be the clorox sterilizing 4.5min of 2.5% again by concentration, and with aseptic water washing 3 times, each washing time is 2min, blot the moisture on described explant surface again with aseptic filter paper, material is cut into the square of 1cm × 1cm or the stem section of 1cm length;
(4) concentration explant being positioned over 10-15 DEG C is in the heavy metal complexing agent of 0.1%, within every 15 minutes, change a heavy metal complexing agent, shading cultivates 1 hour, and described heavy metal complexing agent is phytic acid and diethylene triamine pentamethylene phosphonic 1: 3 solution mixed in mass ratio;
(5) material after step (4) being processed is inoculated in MS improved culture medium, and pH value is adjusted to 5.8, at the temperature of 25 DEG C ± 1 DEG C, and light culture 2 weeks under 1.5 atmospheric conditions; The composition of described MS improved culture medium comprises MS medium, 2mg/L caseinhydrolysate, 2mg/L active carbon, 0.02mg/L vitamin h and the 0.8mg/L Yan acid not containing copper ion.
(6) mixed light that the explant after step (5) being processed is 4: 1 by red LED light and blue LED light ratio throws light on, intensity of illumination is 1000Lux, light application time is 10h/d, and temperature is cultivate under the condition of culture of 25 DEG C ± 1 DEG C, browning rate 0.8%.
Embodiment 2:
Suppress a method for the pollution of Dysosma versipellis explant and brown stain, comprise the following steps:
(1) choose the above wild Dysosma versipellis rhizome of life in 5 years as maternal plant, maternal plant is transplanted to humidity be 85% river sand matrix on carry out preculture 1 month to growing new stem and leaf;
(2) take steps (1) robust growth, without the stem section of damage by disease and insect or young leaflet tablet, first be positioned in N-long-chain thiocarboxylic acid type betain and soak 5min, use distilled water flushing again 1 hour, in flushing process, scrub described explant surface smut with banister brush;
(3) explant after step (2) being processed is positioned over and scribbles TiO
215min in the tank body of the neutral coating made with the nanometer grade powder of piezo-electric crystal tourmaline, and in tank body, pass into nitrogen and ozone mixing ratio is the mist of 10: 1, be the clorox sterilizing 7min of 2.5% again by concentration, and with aseptic water washing 3 times, each washing time is 2min, blot the moisture on described explant surface again with aseptic filter paper, material is cut into the square of 1cm × 1cm or the stem section of 1cm length;
(4) concentration explant being positioned over 10-15 DEG C is in the heavy metal complexing agent of 1%, within every 15 minutes, change a heavy metal complexing agent, shading cultivates 1 hour, and described heavy metal complexing agent is phytic acid and diethylene triamine pentamethylene phosphonic 1: 3 solution mixed in mass ratio;
(5) material after step (4) being processed is inoculated in MS improved culture medium, and pH value is adjusted to 5.8, at the temperature of 25 DEG C ± 1 DEG C, and light culture 2 weeks under 1.5 atmospheric conditions; The composition of described MS improved culture medium comprises MS medium, 2mg/L caseinhydrolysate, 2mg/L active carbon, 0.02mg/L vitamin h and the 0.8mg/L Yan acid not containing copper ion.
(6) mixed light that the explant after step (5) being processed is 4: 1 by red LED light and blue LED light ratio throws light on, intensity of illumination is 2000Lux, light application time is 10h/d, and temperature is cultivate under the condition of culture of 25 DEG C ± 1 DEG C, browning rate 1.3%.
Embodiment 3:
Suppress a method for the pollution of Dysosma versipellis explant and brown stain, comprise the following steps:
(1) choose the above wild Dysosma versipellis rhizome of life in 5 years as maternal plant, maternal plant is transplanted to humidity be 75% river sand matrix on carry out preculture 1 month to growing new stem and leaf;
(2) take steps (1) robust growth, without the stem section of damage by disease and insect or young leaflet tablet, first be positioned in N-long-chain thiocarboxylic acid type betain and soak 5min, use distilled water flushing again 1 hour, in flushing process, scrub described explant surface smut with banister brush;
(3) explant after step (2) being processed is positioned over and scribbles TiO
212min in the tank body of the neutral coating made with the nanometer grade powder of piezo-electric crystal tourmaline, and in tank body, pass into nitrogen and ozone mixing ratio is the mist of 10: 1, be the clorox sterilizing 6min of 2.5% again by concentration, and with aseptic water washing 3 times, each washing time is 2min, blot the moisture on described explant surface again with aseptic filter paper, material is cut into the square of 1cm × 1cm or the stem section of 1cm length;
(4) concentration explant being positioned over 10-15 DEG C is in the heavy metal complexing agent of 0.7%, within every 15 minutes, change a heavy metal complexing agent, shading cultivates 1 hour, and described heavy metal complexing agent is phytic acid and diethylene triamine pentamethylene phosphonic 1: 3 solution mixed in mass ratio;
(5) material after step (4) being processed is inoculated in MS improved culture medium, and pH value is adjusted to 5.8, at the temperature of 25 DEG C ± 1 DEG C, and light culture 2 weeks under 1.5 atmospheric conditions; The composition of described MS improved culture medium comprises MS medium, 2mg/L caseinhydrolysate, 2mg/L active carbon, 0.02mg/L vitamin h and the 0.8mg/L Yan acid not containing copper ion.
(6) mixed light that the explant after step (5) being processed is 4: 1 by red LED light and blue LED light ratio throws light on, intensity of illumination is 1500Lux, light application time is 10h/d, and temperature is cultivate under the condition of culture of 25 DEG C ± 1 DEG C, browning rate 1%.
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the embodiment described.
Claims (3)
1. suppress a method for the pollution of Dysosma versipellis explant and brown stain, it is characterized in that, comprise the following steps:
(1) choose the above wild Dysosma versipellis rhizome of life in 5 years as maternal plant, maternal plant is transplanted to and clean river sand matrix carries out preculture 1 month to growing new stem and leaf;
(2) take steps (1) robust growth, without the stem section of damage by disease and insect or young leaflet tablet, first be positioned in N-long-chain thiocarboxylic acid type betain and soak 5min, use distilled water flushing again 1 hour, in flushing process, scrub described explant surface smut with banister brush;
(3) explant after step (2) being processed utilizes negative ion photocatalyst process 10-15min, be the clorox sterilizing 4.5min-7min of 2.5% again by concentration, and with aseptic water washing 3 times, each washing time is 2min, blot the moisture on described explant surface again with aseptic filter paper, material is cut into the square of 1cm × 1cm or the stem section of 1cm length;
(4) concentration is adopted to be the stem section that cuts of the heavy metal complexing agent soaking step (3) of 0.1%-1% or square 1 hour;
(5) material after step (4) being processed is inoculated in MS improved culture medium, and pH value is adjusted to 5.8, at the temperature of 25 DEG C ± 1 DEG C, and light culture 2 weeks under 1.5 atmospheric conditions;
(6) it is 1000-2000Lux that the explant after step (5) being processed forwards intensity of illumination to, and light application time is 10h/d, and temperature is cultivate under the condition of culture of 25 DEG C ± 1 DEG C;
Wherein, described in step (3), negative ion photocatalyst is by TiO
2with the neutral liquid that the nanometer grade powder of piezo-electric crystal tourmaline is made;
Wherein, utilize in step (3) step of explant described in negative ion photocatalyst process for explant is positioned over scribble negative ion photocatalyst tank body in 10-15min, nitrogen is passed into and ozone mixing ratio is the mist of 10: 1 in tank body, negative ion photocatalyst produces the hydroxy plasma in a large number with bactericidal action by extraneous light excites, thus carries out sterilization processing to explant;
Wherein, described heavy metal complexing agent is phytic acid and diethylene triamine pentamethylene phosphonic 1: 3 solution mixed in mass ratio;
Wherein, utilize the step of heavy metal complexing agent process explant in concentration explant the being positioned over 10-15 DEG C heavy metal complexing agent that is 0.1%-1% in step (4), within every 15 minutes, change a heavy metal complexing agent, shading cultivates 1 hour;
Wherein, the composition of MS improved culture medium described in step (5) comprises MS medium, 2mg/L caseinhydrolysate, 2mg/L active carbon, 0.02mg/L vitamin h and the 0.8mg/L Yan acid not containing copper ion.
2. the method suppressing the pollution of Dysosma versipellis explant and brown stain as claimed in claim 1, it is characterized in that, described in step (1), the humidity of river sand is 65%-85%.
3. as claimed in claim 2 suppress Dysosma versipellis explant to pollute and the method for brown stain, it is characterized in that, in step (6) employing red LED light and blue LED light ratio be 4: 1 mixed light throw light on.
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| CN105010146B (en) * | 2015-08-14 | 2018-02-16 | 广西壮族自治区药用植物园 | The rapid propagation method of Dysosma versipellis |
| CN107278889A (en) * | 2017-06-21 | 2017-10-24 | 河池市智汇科技咨询有限公司 | Jerusalem artichoke adventitious bud inducing method |
| CN107155893A (en) * | 2017-06-21 | 2017-09-15 | 河池市智汇科技咨询有限公司 | Suppress the method for hickory nut Explant browning |
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