CN112369325A - Safe, low-toxicity and high-efficiency sterilization method for plant explants - Google Patents
Safe, low-toxicity and high-efficiency sterilization method for plant explants Download PDFInfo
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- 230000001954 sterilising effect Effects 0.000 title claims abstract description 53
- 238000004659 sterilization and disinfection Methods 0.000 title claims abstract description 47
- 238000000034 method Methods 0.000 title claims abstract description 21
- 231100000053 low toxicity Toxicity 0.000 title claims abstract description 13
- OKNPHOXYVYNIDL-UHFFFAOYSA-N 1-bromo-3-chloro-1,3,5-triazinane-2,4,6-trione Chemical compound ClN1C(=O)NC(=O)N(Br)C1=O OKNPHOXYVYNIDL-UHFFFAOYSA-N 0.000 claims abstract description 27
- 239000000645 desinfectant Substances 0.000 claims abstract description 21
- TWFZGCMQGLPBSX-UHFFFAOYSA-N Carbendazim Natural products C1=CC=C2NC(NC(=O)OC)=NC2=C1 TWFZGCMQGLPBSX-UHFFFAOYSA-N 0.000 claims abstract description 17
- JNPZQRQPIHJYNM-UHFFFAOYSA-N carbendazim Chemical compound C1=C[CH]C2=NC(NC(=O)OC)=NC2=C1 JNPZQRQPIHJYNM-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000006013 carbendazim Substances 0.000 claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000004140 cleaning Methods 0.000 claims abstract description 10
- 238000002791 soaking Methods 0.000 claims abstract description 6
- 239000008223 sterile water Substances 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 4
- 238000005406 washing Methods 0.000 claims abstract description 3
- 238000011282 treatment Methods 0.000 claims description 15
- 239000000463 material Substances 0.000 claims description 10
- 239000008399 tap water Substances 0.000 claims description 4
- 235000020679 tap water Nutrition 0.000 claims description 4
- 239000003599 detergent Substances 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 21
- 230000007613 environmental effect Effects 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 description 33
- 238000011109 contamination Methods 0.000 description 18
- 230000001580 bacterial effect Effects 0.000 description 15
- 241000233866 Fungi Species 0.000 description 11
- OSVXSBDYLRYLIG-UHFFFAOYSA-N chlorine dioxide Inorganic materials O=Cl=O OSVXSBDYLRYLIG-UHFFFAOYSA-N 0.000 description 11
- 230000002538 fungal effect Effects 0.000 description 11
- 241000894006 Bacteria Species 0.000 description 8
- 241000234435 Lilium Species 0.000 description 7
- 244000061456 Solanum tuberosum Species 0.000 description 7
- 235000002595 Solanum tuberosum Nutrition 0.000 description 7
- 230000000844 anti-bacterial effect Effects 0.000 description 7
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 230000009885 systemic effect Effects 0.000 description 5
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 4
- 239000005842 Thiophanate-methyl Substances 0.000 description 4
- 238000010306 acid treatment Methods 0.000 description 4
- 239000003899 bactericide agent Substances 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- 125000001475 halogen functional group Chemical group 0.000 description 4
- 230000002147 killing effect Effects 0.000 description 4
- 239000012466 permeate Substances 0.000 description 4
- QGHREAKMXXNCOA-UHFFFAOYSA-N thiophanate-methyl Chemical compound COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC QGHREAKMXXNCOA-UHFFFAOYSA-N 0.000 description 4
- ZKQDCIXGCQPQNV-UHFFFAOYSA-N Calcium hypochlorite Chemical compound [Ca+2].Cl[O-].Cl[O-] ZKQDCIXGCQPQNV-UHFFFAOYSA-N 0.000 description 3
- CXCSSXVRAITCEA-UHFFFAOYSA-N OC(=O)C(Br)=CCl Chemical compound OC(=O)C(Br)=CCl CXCSSXVRAITCEA-UHFFFAOYSA-N 0.000 description 3
- 239000005708 Sodium hypochlorite Substances 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 229960002233 benzalkonium bromide Drugs 0.000 description 3
- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 description 3
- 230000000249 desinfective effect Effects 0.000 description 3
- 229960002523 mercuric chloride Drugs 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 3
- 239000003206 sterilizing agent Substances 0.000 description 3
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000007844 bleaching agent Substances 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- CUILPNURFADTPE-UHFFFAOYSA-N hypobromous acid Chemical compound BrO CUILPNURFADTPE-UHFFFAOYSA-N 0.000 description 2
- QWPPOHNGKGFGJK-UHFFFAOYSA-N hypochlorous acid Chemical compound ClO QWPPOHNGKGFGJK-UHFFFAOYSA-N 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 229910052753 mercury Inorganic materials 0.000 description 2
- 238000004161 plant tissue culture Methods 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- JYEUMXHLPRZUAT-UHFFFAOYSA-N 1,2,3-triazine Chemical compound C1=CN=NN=C1 JYEUMXHLPRZUAT-UHFFFAOYSA-N 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000008763 Mercury poisoning Diseases 0.000 description 1
- 206010027439 Metal poisoning Diseases 0.000 description 1
- 240000008790 Musa x paradisiaca Species 0.000 description 1
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 241000589517 Pseudomonas aeruginosa Species 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 230000037358 bacterial metabolism Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 210000004666 bacterial spore Anatomy 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- QBWCMBCROVPCKQ-UHFFFAOYSA-N chlorous acid Chemical compound OCl=O QBWCMBCROVPCKQ-UHFFFAOYSA-N 0.000 description 1
- 238000011284 combination treatment Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 230000008029 eradication Effects 0.000 description 1
- 238000007689 inspection Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- -1 mercury ions Chemical class 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 210000004215 spore Anatomy 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 231100000167 toxic agent Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 239000003440 toxic substance Substances 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a safe, low-toxicity and high-efficiency sterilization method for a plant explant, which comprises the following steps: step 1: removing unused parts of the collected explants, and then cleaning; step 2: operating on a clean bench, soaking the explant in 70-75% alcohol solution for 10-30s, and performing primary sterilization; and step 3: performing secondary sterilization on the explant by using a disinfectant consisting of chlorobromoisocyanuric acid and carbendazim; and 4, step 4: washing the explant with sterile water; the invention adopts chlorobromoisocyanuric acid and carbendazim to sterilize the plant explants, which not only has good sterilization effect, but also has low toxicity and environmental protection.
Description
Technical Field
The invention relates to the technical field of plant tissue sterilization, in particular to a safe, low-toxicity and high-efficiency sterilization method for a plant explant.
Background
Because the surface of the explant and the interior of the plant tissue used for culturing the plant tissue taken from the outside are provided with microorganisms, if the microorganisms are not completely killed, once the microorganisms are brought into the culture medium, the culture medium is polluted, and particularly, germs existing in the plant tissue are difficult to eliminate, so that the method is a main pollution source.
Relevant researches on fungi and bacteria carried in plant tissues prove that Zhang Jianchun and the like carry out separation and identification on the endophytic fungi of banana tissues, and 9-genus 494 strains of endophytic fungi are separated together; perijunhui et al also reported that many endophytic bacteria exist inside plant tissues, and in plant tissue culture, because endophytic bacteria inside materials (inside cells or among cells) cannot be removed by a general surface disinfection method, the materials are brought into the culture process to cause pollution of the endophytic bacteria, and the harmfulness is very high.
The contamination existing in the primary culture or the previous generations of subcultures of some plants does not form obvious colonies but only forms 'filaments' and 'halos' inside the culture medium, and the 'halos' are not easy to be perceived by naked eyes, if the 'filaments' and 'halos' are not carefully observed and are easy to be ignored (the 'halos' are easy to be found in backlight inspection), the bacterial load is gradually accumulated along with the increase of the times of subcultures, and then the bacterial load is displayed on the culture medium, so that the culture fails.
In the invention of the same Huishu et al, although the cleaning and sterilization work are carried out synchronously, the microorganism washed from tap water or the explant is prevented from entering the interior of the plant trachea along the capillary of the explant, and the pollution rate of plant tissue culture is reduced to a certain extent, the method only reduces the invasion of external bacteria to the maximum extent in the explant sterilization process, but the endophyte in the tissue is not removed, and the explant still has a larger pollution rate in the culture process.
At present, the sterilization disinfectants which can be used for plant explant sterilization include alcohol, mercuric chloride, sodium hypochlorite, bleaching powder, hydrogen peroxide, benzalkonium bromide and the like.
Wherein, alcohol is the most commonly used surface disinfectant, and 70-75% alcohol has the best sterilization effect, because 95% or anhydrous alcohol can quickly dehydrate and solidify the protein on the surface of the thallus to form a layer of dry film, thereby preventing the continuous infiltration of the alcohol and greatly reducing the sterilization effect; in addition, the alcohol has stronger penetrating power, denaturalizes the mycoprotein, has good sterilization effect, has stronger moistening effect, can remove the air on the material and is beneficial to the infiltration of other disinfectants; but the alcohol has a great killing effect on the plant materials, the soaking time is too long, the growth of the plant materials can be influenced and even killed by the alcohol, and the time is strictly controlled when the plant materials are used; in addition, alcohol can not be used for thorough disinfection, is not used independently and is often used in combination with other disinfectants.
Mercuric chloride is also the most commonly used sterilizing agent for sterilizing the surfaces of plant explants; it is an inorganic mercurial agent, which inhibits the activity of bacterial mercaptol enzyme, makes the bacterial metabolism generate obstacle, and mercury ions can combine with mycoprotein to denature the protein, thereby killing the thallus, and has strong bactericidal power, larger effect than phenol, but no effect on spores and viruses; the compound is also a highly toxic compound, has a certain toxic action on plant tissues, cannot be treated for a long time to disinfect the interior of the plant tissues, has slight volatility at room temperature, and can possibly cause mercury poisoning of laboratory workers.
Therefore, a sterilizing agent for replacing mercuric chloride to disinfect the surfaces of plant explants is found, the use amount of mercury is reduced, and the sterilizing agent has important significance for safe use and environmental protection.
Sodium hypochlorite can release active chloride ions to kill thalli; the disinfectant has strong disinfection capability, is not easy to remain and is harmless to the environment; but the sodium hypochlorite solution is very alkaline and has certain destructive effect on plant materials.
The bleaching powder contains calcium hypochlorite as effective component, has high disinfecting effect and no environmental pollution, and is easy to absorb moisture to dissipate available chlorine and to lose efficacy.
The hydrogen peroxide has good disinfection effect, is easy to remove, can not damage explants, can only be used for disinfecting leaves, and can not permeate into tissues for sterilization.
Benzalkonium bromide, named benzalkonium bromide, is a surface active disinfectant, and the surface active agent changes the permeability of bacterial cytoplasmic membranes, so that bacterial cytoplasmic substances are leaked out, and the metabolism of the bacterial cytoplasmic substances is blocked, thereby playing a role in sterilization; has little damage to most plant explants and good bactericidal effect, but has no effect on pseudomonas aeruginosa, acid-fast bacillus and bacterial spores.
In conclusion, the advantages and the disadvantages of the disinfectants are compared, and no disinfectant can permeate into tissues to sterilize and has little damage to plant tissues, so that the disinfectant has great significance for finding a safe, low-toxicity and high-cost-performance disinfectant capable of killing microorganisms and endophytes on the surfaces of plant explants.
Disclosure of Invention
The invention aims to: aiming at the problem that no disinfectant can permeate into tissues to sterilize and has small damage to plant tissues at present, the method for safely, low-toxicity and high-efficiency sterilization of the plant explants is provided, and the problems that the disinfectant can permeate into the tissues to sterilize and has small damage to the plant tissues are solved.
The technical scheme of the invention is as follows:
a safe, low-toxicity and high-efficiency sterilization method for plant explants comprises the following steps:
step 1: removing unused parts of the collected explants, and then cleaning;
step 2: operating on a clean bench, soaking the explant in 70-75% alcohol solution for 10-30s, and performing primary sterilization;
and step 3: performing secondary sterilization on the explant by using a disinfectant consisting of chlorobromoisocyanuric acid and carbendazim;
and 4, step 4: the explants were washed with sterile water.
Further, the cleaning process in step 1 includes: after being cleaned by liquid detergent, the mixture is washed for 30min by a large amount of tap water.
Further, the concentration of the chlorobromoisocyanuric acid in the step 3 is 3 g/L; the concentration of carbendazim is 1 g/L.
Further, the secondary sterilization in the step 3 is continuously carried out by shaking, so that the plant material is fully contacted with the disinfectant.
Further, the number of times of washing in the step 4 is 3-5 times.
In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:
1. a method for sterilizing plant explant with safety, low toxicity and high efficiency comprises sterilizing explant with disinfectant comprising chlorobromoisocyanuric acid and carbendazim, mixing chlorobromoisocyanuric acid and carbendazim, and sterilizing fungi and bacteria with conventional HgCl2Has equivalent sterilization effect and can completely replace HgCl2Sterilizing and disinfecting the plant explant; meanwhile, the chlorobromoisocyanuric acid is used as a novel systemic bactericide which is efficient, broad-spectrum and convenient to store and transport, has the characteristics of safe and convenient use, small using amount, good bactericidal effect on the surface and the inside of an explant and the like, can slowly release hypobromous acid (HOBr) and hypochlorous acid (HOCL) on the surface of a crop, can kill various bacteria, algae, fungi and viruses, has the unique effects of protection, treatment, eradication and nutrition, is strong in systemic property, releases hypobromous acid through the systemic conduction effect to form triazine Diketone (DHT) and triazine (ADHT), has strong effects of killing bacteria, fungi and viruses in the tissue, has no interactive resistance, is safe for the crop, environment-friendly and nuisanceless, and is a first-choice bactericide for developing green agriculture. The carbendazim serving as a high-efficiency low-toxicity systemic bactericide with systemic treatment and protection effects is mixed with chlorobromoisocyanuric acid to replace HgCl2And the sterilization of the plant explants is carried out, so that the sterilization effect is good, and the low toxicity and the environmental protection are realized.
Detailed description of the preferred embodiments
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments.
A safe, low-toxicity and high-efficiency sterilization method for plant explants comprises the following steps:
step 1: removing unused parts of the collected explants, and then cleaning;
step 2: operating on a clean bench, soaking the cleaned explant in 70-75% alcohol solution for 10-30s, and performing primary sterilization;
and step 3: using a disinfectant consisting of chlorobromoisocyanuric acid and carbendazim to perform secondary sterilization on the explants after primary sterilization;
and 4, step 4: the explants after the secondary sterilization are washed with sterile water.
The cleaning treatment in the step 1 comprises: after being cleaned by liquid detergent, the mixture is washed for 30min by a large amount of tap water.
The concentration of the chlorobromoisocyanuric acid in the step 3 is 3 g/L; the concentration of carbendazim is 1 g/L.
And 3, continuously shaking during secondary sterilization in the step 3 to ensure that the plant material is fully contacted with the disinfectant.
The number of times of cleaning in the step 4 is 3-5 times.
Firstly, removing unused parts of collected Lanzhou lily scales and potato stem sections, and then cleaning; then operating on a clean bench, soaking the explant in 70-75% alcohol solution for 10-30s, and performing primary sterilization.
Selection of chlorobromoisocyanuric acid and ClO2Setting different concentration gradients and time gradients, and performing secondary sterilization on the Lanzhou lily scales and the potato stem segments respectively with the HgCl concentration of 1.0g/L2(treatment for 10min) to determine chlorobromoisocyanuric acid and ClO2Sterilization effect on plant explants.
Chlorobromoisocyanuric acid and ClO2Respectively setting 5 concentration gradients and 5 time gradients, namely respectively treating chlorobromoisocyanuric acid for 4, 6, 8, 10 and 12 hours under the condition that the concentration is 1, 2, 3, 4 and 5g/L, and totally combining 25 treatments; ClO2(Xiuba) was treated at concentrations of 100, 200, 300, 400, 500mg/L for 1, 2, 3, 4, 5h, respectively, for a total of 25 treatment combinations.
The experimental data are as follows:
TABLE 1-1 Chlorobromoacrylic acid treatment and ClO2Statistics of bacterial contamination rate (%) for treatment of "Lily Scale
TABLE 1-2 Chlorobromoacrylic acid treatment and ClO2Treatment of bacterial contamination statistics for "potato Stem segments" (%)
TABLE 2-1 Chlorobromoacrylic acid treatment and ClO2Statistics of fungal contamination rate (%) "Lily Scale" treatment
TABLE 2 Chlorobromocyanuric acid treatment and ClO2Treatment of "Potato Stem segments" fungal contamination Rate statistics (%)
TABLE 3-1HgCl2Statistics of fungal contamination rate and bacterial contamination rate for processing lily scales
| Treatment time (min) | HgCl2(g/L) | Fungal contamination Rate (%) | Bacterial contamination ratio (%) |
| 10 | 1 | 16.3 | 0.34 |
TABLE 3-2HgCl2Statistics of fungal contamination rate and bacterial contamination rate for processing of potato stem segments
| Treatment time (min) | HgCl2(g/L) | Fungal contamination Rate (%) | Bacterial contamination ratio (%) |
| 10 | 1 | 10.4 | 0.17 |
The results show chlorobromoisocyanuric acid and ClO2The better the bacterial and fungal elimination with increasing concentration and with increasing time.
From tables 1-1 to 3-2, chlorobromoisocyanuric acid, ClO2And HgCl2The sterilizing effects of the Lanzhou lily scales and the potato stem sections are mutually compared, and the sterilizing effect of the chlorobromoisocyanuric acid is found to be obviously better than that of ClO2Good sterilization effect compared with HgCl when the treatment concentration of chlorobromoisocyanuric acid is 3g/L and the treatment time is 8h2Better still; but part of the endophytic fungi are not eliminated, and during the culture process, the endophytic fungi are mixedA small amount of fungal contamination ensues.
Therefore, the bactericide against the fungi and the chlorobromoisocyanuric acid are compounded for testing.
Namely, combination 1: chlorobromoisocyanuric acid (3g/L) + carbendazim (1, 1.5g/L) + Tween-20 (500. mu.l/L) for 8 h.
And (3) combination 2: chlorobromoisocyanuric acid (3g/L) + thiophanate-methyl (1.0, 1.5g/L) + tween-20 (500 mul/L) for 8 h.
TABLE 4-1 comparison of fungal and bacterial contamination rates for "Lily scales" treated after blending of chlorobromoisocyanuric acid with carbendazim and thiophanate-methyl
TABLE 4 comparison of fungal contamination rate and bacterial contamination rate of "potato stem" treated after mixing of chlorobromoisocyanuric acid with carbendazim and thiophanate methyl
In tables 4-1 and 4-2, the two combinations were compared with each other to select the combination having the best sterilization effect against endophytic fungi.
Tests prove that the sterilization effect is better when the concentration of the carbendazim in the combination 1 is 1g/L and 1.5g/L, and the concentration of the carbendazim is finally determined to be 1g/L according to the principle of low-efficiency safe use of the medicament; the combination 2 has better treatment effect when the concentration of the thiophanate methyl is 1.5g/L, but the fungal contamination rate is still higher than that of the combination 1. Therefore, the disinfectant of chlorobromoisocyanuric acid (3g/L) and carbendazim (1g/L) has the best sterilizing effect on the endophytic fungi.
Claims (5)
1. A safe, low-toxicity and high-efficiency sterilization method for plant explants is characterized by comprising the following steps:
step 1: removing unused parts of the collected explants, and then cleaning;
step 2: operating on a clean bench, soaking the cleaned explant in 70-75% alcohol solution for 10-30s, and performing primary sterilization;
and step 3: performing secondary sterilization on the explants after primary sterilization by using a disinfectant consisting of chlorobromoisocyanuric acid and carbendazim;
and 4, step 4: the explants after the secondary sterilization are washed with sterile water.
2. The method for safely and efficiently sterilizing plant explants according to claim 1, wherein the cleaning treatment in step 1 comprises: after being cleaned by liquid detergent, the mixture is washed for 30min by a large amount of tap water.
3. The method for safely and efficiently sterilizing plant explants according to claim 1, wherein the mass concentration of chlorobromoisocyanuric acid in the step 3 is 3 g/L; the mass concentration of the carbendazim is 1 g/L.
4. The method for safely and efficiently sterilizing plant explants according to claim 1, wherein the secondary sterilization in step 3 is performed by shaking to ensure that the plant materials are in full contact with the disinfectant.
5. The method for safely and efficiently sterilizing plant explants according to claim 1, wherein the number of washing in step 4 is 3-5.
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