CN105432473A - Orchid seed surface sterilization method - Google Patents
Orchid seed surface sterilization method Download PDFInfo
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- CN105432473A CN105432473A CN201511007094.6A CN201511007094A CN105432473A CN 105432473 A CN105432473 A CN 105432473A CN 201511007094 A CN201511007094 A CN 201511007094A CN 105432473 A CN105432473 A CN 105432473A
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- 238000000034 method Methods 0.000 title claims abstract description 91
- 241000233855 Orchidaceae Species 0.000 title claims abstract description 54
- 238000004659 sterilization and disinfection Methods 0.000 title claims abstract description 50
- 230000001954 sterilising effect Effects 0.000 title claims abstract description 49
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 53
- 239000008223 sterile water Substances 0.000 claims abstract description 40
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 18
- 239000002775 capsule Substances 0.000 claims abstract description 4
- 239000000243 solution Substances 0.000 claims description 29
- 230000000249 desinfective effect Effects 0.000 claims description 27
- 235000013399 edible fruits Nutrition 0.000 claims description 23
- 229960002523 mercuric chloride Drugs 0.000 claims description 20
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 20
- 238000004140 cleaning Methods 0.000 claims description 19
- 238000005119 centrifugation Methods 0.000 claims description 17
- 238000001035 drying Methods 0.000 claims description 13
- 239000007788 liquid Substances 0.000 claims description 10
- 235000013575 mashed potatoes Nutrition 0.000 claims description 9
- 239000011435 rock Substances 0.000 claims description 8
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 6
- 244000061456 Solanum tuberosum Species 0.000 claims description 6
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 230000001476 alcoholic effect Effects 0.000 claims description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 4
- 229930006000 Sucrose Natural products 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 238000005520 cutting process Methods 0.000 claims description 3
- 239000002504 physiological saline solution Substances 0.000 claims description 2
- 241000196324 Embryophyta Species 0.000 abstract description 29
- 241000894006 Bacteria Species 0.000 abstract description 5
- 241000233866 Fungi Species 0.000 abstract description 5
- 230000035784 germination Effects 0.000 abstract description 5
- 244000005700 microbiome Species 0.000 abstract description 5
- 238000005406 washing Methods 0.000 abstract description 5
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 abstract description 4
- 238000002791 soaking Methods 0.000 abstract 2
- 241001313857 Bletilla striata Species 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 230000000694 effects Effects 0.000 description 8
- 230000004083 survival effect Effects 0.000 description 7
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 7
- 229940033663 thimerosal Drugs 0.000 description 7
- 240000004638 Dendrobium nobile Species 0.000 description 5
- 238000002474 experimental method Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 238000011169 microbiological contamination Methods 0.000 description 4
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 230000003311 flocculating effect Effects 0.000 description 3
- 230000007226 seed germination Effects 0.000 description 3
- ZKQDCIXGCQPQNV-UHFFFAOYSA-N Calcium hypochlorite Chemical compound [Ca+2].Cl[O-].Cl[O-] ZKQDCIXGCQPQNV-UHFFFAOYSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 238000013475 authorization Methods 0.000 description 2
- 159000000007 calcium salts Chemical class 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 239000006185 dispersion Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 238000007667 floating Methods 0.000 description 2
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 2
- 239000012452 mother liquor Substances 0.000 description 2
- 238000004806 packaging method and process Methods 0.000 description 2
- 239000012286 potassium permanganate Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000012090 tissue culture technique Methods 0.000 description 2
- 241001517312 Calanthe Species 0.000 description 1
- 241000238557 Decapoda Species 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 241000594394 Hedyotis Species 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000209510 Liliopsida Species 0.000 description 1
- 241000218922 Magnoliophyta Species 0.000 description 1
- 241000415261 Murdannia Species 0.000 description 1
- 208000004880 Polyuria Diseases 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000003356 anti-rheumatic effect Effects 0.000 description 1
- 239000003435 antirheumatic agent Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 230000035619 diuresis Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- 238000012376 hot air sterilization Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000001114 myogenic effect Effects 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000014101 wine Nutrition 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)
- Pretreatment Of Seeds And Plants (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses an orchid seed surface sterilization method. The method comprises steps as follows: S1, shell breaking for seed taking; S2, alcohol soaking; S3, first-time washing with sterile water; S4, sterilization treatment with a mercury chloride solution; S5, second-time treatment with sterile water; S6, follow-up treatment, and seeds are collected centrifugally in S1 to S5. Seeds taken out of capsules are covered with filter paper and gauze, then repeated sterilization is performed sequentially, and direct surface sterilization is performed on ultra-fine plant seeds; besides, according to the method, alcohol soaking in Step 2, sterilization treatment with the mercury chloride solution in Step 4 and second-time treatment with sterile water in Step 5 are accelerated through shaking or vortex and centrifugal treatment. Less equipment used in the method and steps is used, the cost is lower, microorganisms such as fungi, bacteria and the like on surfaces of the plant seeds are eliminated quickly and efficiently, and a better growth foundation is laid for germination and later growth of the seeds.
Description
Technical field
The present invention relates to a kind of processes for disinfecting surfaces of Orchid Seeds, particularly relate to a kind of Orchid Seeds processes for disinfecting surfaces.
Background technology
The orchid family is the Ge great section being only second to composite family, is the first section in monocotyledon.There are long cultivation history and numerous kinds.Occurring in nature still has manyly has the wild orchid of ornamental value to have to be developed, conservation and utilization.Orchid is precious ornamental plants.According to incompletely statistics, orchid about has 775 genus, 19500 kinds, is widely distributed in the torrid zone, subtropics and temperate zone, mainly originates in South America and Tropical Asian.There is 166 genus kind more than 1000 in China, is mainly distributed in south of the River each province, in the majority with Guangdong, Hainan, Yunnan and Taiwan.Orchid is a special plant group, is one of section evolved the most in angiosperm.Simultaneously because orchid kind is extremely many, and its function of the plant of different cultivars also difference to some extent.As purpleback murdannia herb, herb all can be used as medicine, and has clearing heat and detoxicating, effect of wines used as antirheumatic and anti-inflammatory diuresis; The and for example bletilla striata, is used as medicine with its pseudobulb, can subside a swelling, stop blooding, tonifying lung, myogenic pain relieving.The capable power of profit rib; Also as tasselled calanthe, can be clearing heat and detoxicating, strengthen muscles and bones.In addition, as the orchid of most of kinds such as the lean orchid of Rhizoma Sparganii shrimp, green colored spoon orchid, ribbed hedyotis herb, the stem of noble dendrobium, be all first-class Chinese herbal medicine, all have certain curative effect to some disease.
More than 1200 are had to plant the endangered plants being put into focused protection China orchid; mainly due to its under field conditions (factors); seed germination is more difficult; germination rate is lower etc. causes it cannot meet the demand in market; so people start the artificial cultivation carrying out orchid in a large number now, tissue cultures is the main path of most orchid biological control.
Utilizing tissue culture technique to cultivate in the technical method of orchid seedling, the surface sterilization of its seed is exactly indispensable step.But the seed of orchid is very little, just have several ten thousand in a capsule to up to a million seeds, in superfine fine granularity or cotton-shaped.In tissue culture technique, generally take fruit pod of first sterilizing, then cut the way that pericarp gets seed open.But when fruit pod full maturity cracking, seed is exposed to outside fruit pod, carries out surface sterilization just become very difficult to it; Orchid Seeds is little and light, causes conventional sterilant to operate more difficult.Effective sterilization processing can not be carried out to seed, often cause tissue cultures failure because of microbiological contamination.
First, Orchid Seeds is tiny of powder, and meanwhile, quality is light, and directly immersion or collected by centrifugation are the top (Fig. 3) substantially all floating on alcohol or water; Could soak or centrifugal (Fig. 4) after needing packaging.
In addition, Authorization Notice No. is CN101352120B, name is called a kind of sterilizing surface of plant seed, it discloses dust-like plant seed, especially after Orchid Seeds strip off fruit pod, seed in fruit pod is carried out to the method for surface sterilization, it comprises the steps: to use medicining liquid dipping seed, filters and removes described thimerosal; Use seed described in sterile water wash again, filter and remove residual described thimerosal and described sterile water, obtain sterile seed; And above-mentioned steps is carried out in centrifuge tube, adopt agar solution or water by described sterile seed mixing, obtain seed suspension liquid, then with pipettor, described seed suspension liquid is shifted out from described centrifuge tube.
This Plants the surface of the seed sterilization method, which overcomes the difficulty of plant seed surface sterilization difficulty because volume is little, achieves and carry out direct surface sterilization to dust-like plant seed, has broken away from prior art the constraint of fruit pod of must sterilizing.Experimental result shows, this invention sterilization method effective, and the seed after sterilization sprouts failed ratio because of pollution extremely low.But use the plant seed that said method obtains, although its survival rate arrives, as long as the seed not soaked through thimerosal cleaning and sterile water just can reach the survival rate very in later stage, still have Some seeds infected.In addition, the survival rate in the orchid later stage survived neither be very high, easily causes disease worm.
In addition, existing Authorization Notice No. CN101569256B, name is called a kind of patent of sterilization method of rush mat seeds, discloses (1) by the potassium permanganate solution process 70 ~ 75 hours of rush mat seeds 0.6 ~ 0.65g/L, clean with aseptic water washing; (2) by weight percent concentration be 70 ~ 75% ethanol postincubation 55 ~ 65 minutes, clean with aseptic water washing; (3) with the aqueous sodium hypochlorite solution process 3 ~ 5 minutes of the calcium hypochlorite tablet aqueous solution of 19 ~ 21g/L or weight percent concentration 1 ~ 2%, clean with aseptic water washing; (4) by weight percent concentration be 0.1 ~ 0.15% mercuric chloride aqueous solution process 7 ~ 9 minutes, clean with aseptic water washing.This invention sterilization method is simple to operate, sterilization is thorough, and detergency reaches as high as 90%, and the seed germination rate after sterilization is up to more than 80%, effectively ensure that the success rate of follow-up tissue cultures, and cost is low, is suitable for suitability for industrialized production.
Rush mat seeds is also small volume, and the seed of lighter weight, without the seed of cleaning, survival rate is lower, can be used for cleaning Orchid Seeds, but its concrete thimerosal and can not directly be used for sterilization, even if directly also clean Orchid Seeds with potassium permanganate solution, follow-up process directly can not be cleaned with calcium hypochlorite tablet.And for the seed of orchid, seed germination rate reaches 80% and still seems lower.
Above method does not all embody the collection problem in minute seeds sterilization cleaning process, and make minute seeds be difficult to gather in cleaning process, it directly soaks or collected by centrifugation is the top (Fig. 3) substantially all floating on alcohol or water; Could soak or centrifugal (Fig. 4) after needing packaging, cause it to disinfect in process at entire surface of the seed, cause unnecessary loss owing to being difficult to collect, decrease the weight of disinfecting seed, cause economic loss and the wasting of resources.
So the present invention is directed to above-mentioned deficiency, carry out exploring and improving, define minute seeds sterilization method fast and effectively, be proven, it has important application prospect.
Summary of the invention
In order to overcome above-mentioned the deficiencies in the prior art, the invention provides a kind of Orchid Seeds processes for disinfecting surfaces, the particularly processes for disinfecting surfaces of the Orchid Seeds of cotton-shaped or particulate, its object is to surface sterilization and the cleaning effect of the plant seed promoting cotton-shaped or particulate, and strengthen its flocculating result in the process of disinfecting, avoid the loss that it causes when surface sterilization process, and strengthen the germination rate of the plant seed of cotton-shaped or particulate simultaneously, and its sprout after growth survival rate.
The technical solution adopted in the present invention is: a kind of Orchid Seeds processes for disinfecting surfaces, comprises the steps:
S1: kind is got in broken shell, orchid fruit pod is opened in cutting, adopts at least 1 layer of qualitative filter paper and at least 2 layers of hospital gauze wrapping Orchid Seeds; Then seed is put into centrifuge tube, within least 1 minute, collected with the centrifugation process of 350-550 rpm;
S2: alcohol-pickled, puts into 70-85% alcoholic solution and rocks or vortex process 2-3 minute by the seed wrapped, then with the centrifugation process at least 1 minute of 350-550 rpm, remove alcohol;
S3: sterile water wash for the first time, the seed after step S2 process, puts it into and is equipped with 3 times with eccentric cleaning in the centrifuge tube of upper volume sterile water once;
S4: mercuric chloride solution is disinfected, immerses the seed after cleaning in 0.05-0.3% mercuric chloride solution and disinfects 3-5 minute or vortex process 2-3 minute, then with the centrifugation process at least 1 minute of 350-550 rpm, removes mercuric chloride solution;
S5: second time sterile water process, after step S4 process, pours 3 times into the sterile water of upper volume, then with the centrifugation process at least 1 minute of 350-550 rpm; Then adopt blotting paper to absorb top moisture, carry out sterilizing-drying, repeat drying at least 2 times;
S6: subsequent treatment, finally sucks residual liquid with the blotting paper of sterilizing-drying, obtains aseptic seed.The processes for disinfecting surfaces of superfine fine granularity plant seed of the present invention, first plant seed is difficult to carry out surface sterilization difficulty because volume is little is effectively overcomed, filter paper and gauze is adopted to be wrapped up the seed taken out from fruit pod, then bring filter paper and gauze that it is carried out alcohol disinfecting successively, first time sterile water sterilization, mercury chloride and the sterilization of second time sterile water, achieve and direct surface sterilization is carried out to superfine shape plant seed, break away from prior art the constraint of fruit pod of must sterilizing, and the method adopts directly rocks or vortex, and centrifugal treating method is alcohol-pickled to step S2, step S4 mercuric chloride solution is disinfected and is accelerated with the process of step S5 second time sterile water, help the surface of its rapidly and efficiently disinfection seed, to eliminate in fruit pod the microorganism such as the multiple fungi of carrying or bacterium to the not expected impact of plant seed follow-up cultivations generation.Most importantly, the equipment that method of the present invention and step use is less, cost is lower, eliminate the microorganisms such as the surperficial fungi of plant seed and bacterium quickly and efficiently, grows basis preferably for the rudiment of seed and later stage grow up to provide.
Further improvement of the present invention is, when step S1 broken shell is got kind, by filter paper pads in bottom, after fruit pod is cut open, seed is directly fallen into above filter paper; And seed is fine powdered.
Further improvement of the present invention is, the fruit pod in step S1 is ripe orchid capsule.
Further improvement of the present invention is, the centrifugal treating in step S2, step S4 and step S5 is the centrifugation process 2 minutes of 500 rpms.
Further improvement of the present invention is, the alcoholic solution concentration in step S2 is 50% ~ 85%, preferably 70 ~ 80%.
Further improvement of the present invention is, the sterile water in step S3 and step S5 is pure water after HTHP moist heat sterilization or physiological saline.
Further improvement of the present invention is, the sterile water in step S3 and step S5, be 121 ~ 125 DEG C and pressure is in the hygrothermal environment of 0.1 ~ 0.15Mpa through excess temperature, and sterilization time 10 ~ 15min obtains.
Further improvement of the present invention is, is immersed in 0.1% mercuric chloride solution by the seed after cleaning and disinfect 3-5 minute in described step S2.The blotting paper of the sterilizing-drying in S5 is through 160 ° of C hot air sterilizations filter paper of 1 hour or blotting paper.
Further improvement of the present invention is, the aseptic seed obtained in step S6, puts into and is equipped with in the blake bottle of medium, then in blake bottle, injects sterile water with liquid-transfering gun, is 21-27 DEG C, cultivates 8-12 days under dark condition in temperature; Being moved into temperature is again 25 DEG C, and light intensity is 1500 ~ 2500lx, and light application time is cultivate under the condition of 10h/d.
Further improvement of the present invention is, the formula of medium is as follows: 1/2MS+NAA0.5mg/L+20g/L mashed potatoes+30g/L sucrose+5.5g/L agar, pH5.8; Wherein, the preparation method of mashed potatoes is as follows: by potato clean after peel, be cut into fritter by knife, then put into juice extractor add water squeeze broken, the homogenate obtained is mashed potatoes.
Compared with prior art, the invention has the beneficial effects as follows: the processes for disinfecting surfaces of superfine fine granularity plant seed of the present invention, first plant seed is difficult to carry out surface sterilization difficulty because volume is little is effectively overcomed, filter paper and gauze is adopted to be wrapped up the seed taken out from fruit pod, then bring filter paper and gauze that it is carried out alcohol disinfecting successively, first time sterile water sterilization, mercury chloride and the sterilization of second time sterile water, achieve and direct surface sterilization is carried out to superfine shape plant seed, break away from prior art the constraint of fruit pod of must sterilizing, and the method adopts directly rocks or vortex, and centrifugal treating method is alcohol-pickled to step S2, step S4 mercuric chloride solution is disinfected and is accelerated with the process of step S5 second time sterile water, help the surface of its rapidly and efficiently disinfection seed, to eliminate in fruit pod the microorganism such as the multiple fungi of carrying or bacterium to the not expected impact of plant seed follow-up cultivations generation.Most importantly, the equipment that method of the present invention and step use is less, cost is lower, eliminate the microorganisms such as the surperficial fungi of plant seed and bacterium quickly and efficiently, grows basis preferably for the rudiment of seed and later stage grow up to provide.
The inventive method also has the features such as simple to operate, cost is low, save time, and is thus particularly suitable for the front process to Orchid Seeds of tissue cultures.Improve surface sterilization and the cleaning effect of the plant seed of cotton-shaped or particulate, and strengthen its flocculating result in the process of disinfecting, avoid the loss that it causes when surface sterilization process, and enhance the germination rate of the plant seed of cotton-shaped or particulate simultaneously, and its sprout after growth survival rate.
Accompanying drawing explanation
Fig. 1 is a kind of structural representation of an embodiment of Orchid Seeds processes for disinfecting surfaces;
Fig. 2 is a kind of structural representation of another embodiment of Orchid Seeds processes for disinfecting surfaces;
Fig. 3 is view when Orchid Seeds is used or alcohol directly soaks;
View when Fig. 4 is Orchid Seeds collected by centrifugation.
Embodiment
In order to deepen the understanding of the present invention, below in conjunction with drawings and Examples, the present invention is further described, and this embodiment, only for explaining the present invention, not forming protection scope of the present invention and limiting.
As depicted in figs. 1 and 2, a kind of Orchid Seeds processes for disinfecting surfaces, comprises the steps:
S1: kind is got in broken shell, orchid fruit pod is opened in cutting, adopts at least 1 layer of qualitative filter paper and at least 2 layers of hospital gauze wrapping Orchid Seeds; Then seed is put into centrifuge tube, within least 1 minute, collected with the centrifugation process of 350-550 rpm;
S2: alcohol-pickled, puts into 70-85% alcoholic solution and rocks or vortex process 2-3 minute by the seed wrapped, then with the centrifugation process at least 1 minute of 350-550 rpm, remove alcohol;
S3: sterile water wash for the first time, the seed after step S2 process, puts it into and is equipped with 3 times with eccentric cleaning in the centrifuge tube of upper volume sterile water once;
S4: mercuric chloride solution is disinfected, immerses the seed after cleaning in 0.05-0.3% mercuric chloride solution and disinfects 3-5 minute or vortex process 2-3 minute, then with the centrifugation process at least 1 minute of 350-550 rpm, removes mercuric chloride solution;
S5: second time sterile water process, after step S4 process, pours 3 times into the sterile water of upper volume, then with the centrifugation process at least 1 minute of 350-550 rpm; Then adopt blotting paper to absorb top moisture, carry out sterilizing-drying, repeat drying at least 2 times;
S6: subsequent treatment, finally sucks residual liquid with the blotting paper of sterilizing-drying, obtains aseptic seed.
To be described in detail according to following embodiment below.
Embodiment 1
For the fruit pod of the bletilla striata (Bletillastriata) in the present embodiment, the inventive method is described.
One. disinfect
The experimental ware used in this experiment (as first-class in gauze, blotting paper, tweezers, rifle) is all through autoclaving.
This sterilization method all carries out on superclean bench.
Thimerosal process: get full ripe bletilla striata fruit pod, cut pericarp open and take out seed, seed is placed in 1 sterilized metafiltration paper and 2 gauzes bandage, the gauze wrapping seed is put in the centrifuge tube filling 75% alcohol, rock to evenly, or centrifugal 2 minutes of vortex process 2-3 minute, 500rpm, remove alcohol.The seed wrapped is put into 3 times to be cleaned once with upper volume sterile water; Seed after cleaning immerses in 0.06% mercuric chloride solution disinfects 3-5 minute or centrifugal 2 minutes of vortex process 2-3 minute, 500rpm, removes mercuric chloride solution.Pour 3 times in centrifuge tube into the sterile water wash of upper volume, centrifugal 2 minutes of 500rpm, absorb top moisture, repeat 3 times.Finally suck residual liquid with the blotting paper of sterilizing-drying, obtain aseptic seed.
Two, the checking of Disinfection Effect
Open gauze and filter paper, put in blake bottle with tweezers by seed, in blake bottle, then inject the sterile water of 0.5mL with liquid-transfering gun, object makes seeded dispersion on blake bottle surface.Be 22-25 DEG C in temperature, cultivate 10 days under dark condition, then to be moved into temperature be 25 DEG C, light intensity is 1500 ~ 2500lx, and light application time is cultivate under the condition of 10h/d.
Formula and the composition of described medium are as follows:
Formula: 1/2MS+NAA0.5mg/L+20g/L mashed potatoes+30g/L sucrose+5.5g/L agar, pH5.8
Wherein, the preparation method of mashed potatoes is as follows: by potato clean after peel, be cut into fritter by knife, then put into juice extractor add water squeeze broken, the homogenate obtained is mashed potatoes.
Three repetitions are established in experiment.Result shows, all seeds are all normally sprouted, and does not all have microbiological contamination phenomenon.
Note: the main component of MS mother liquor, refers to table 1, table 2, table 3, table 4, table 5 and table 6:
Table 1: macroelement 20*
| Composition | Concentration (mg/L) |
| NH 4NO 3 | 33000 |
| KNO 3 | 38000 |
| MgSO 4.7H 2O | 7400 |
| KH 2PO 4 | 3400 |
Table 2: organic 200*
| Composition | Concentration (mg/L) |
| Inositol | 20000 |
| Nicotinic acid | 100 |
| V B1 | 100 |
| V B6 | 100 |
Table 3: micro-200*
| Composition | Concentration (mg/L) |
| KI | 166 |
| H 3BO 3 | 1240 |
| MnSO 4.4H 2O or MnSO 4 | 4460 or 3380 |
| ZnSO 4.7H 2O | 1720 |
| Na Mo.2H 2O | 50 |
| CuSO 4.5H 2O | 5 |
| CoCl 2.6H 2O | 5 |
Table 4: calcium salt 200*
| Composition | Concentration (mg/L) |
| CaCl 2.H 2O | 88000 |
Table 5: molysite 200*
| Composition | Concentration (mg/L) 5--> |
| FeSO 4.7H 2O | 5560 |
| Na EDTA.2H 2O | 7640 |
The formula of table 6:MS minimal medium
| Composition | Mother liquor content (mL/L) |
| Macroelement 20* | 50 |
| Organic 200* | 5 |
| Trace element 200* | 5 |
| Calcium salt 200* | 5 |
| Molysite 200* | 5 |
Embodiment 2
For the seed of the bletilla striata (Bletillastriata) in the present embodiment, the inventive method is described.
One, disinfect
The experimental ware used in this experiment (as first-class in gauze, filter paper, blotting paper, tweezers, rifle) is all through autoclaving.
This sterilization method all carries out on superclean bench.
Thimerosal process: bletilla striata seeds is placed in sterilized gauze and bandages, the gauze wrapping seed is put in the centrifuge tube filling 80% alcohol, rock, or vortex process 2-3 minute, centrifugal 5 minutes of 500rpm, remove alcohol, the seed wrapped is put into 3 times and cleans once with upper volume sterile water; Seed after cleaning immerses in 0.1% mercuric chloride solution disinfects 3-5 minute or centrifugal 5 minutes of vortex process 2-3 minute, 500rpm, removes mercuric chloride solution; Pour 3 times in centrifuge tube into the sterile water wash of upper volume, centrifugal 5 minutes of 500rpm, absorb top moisture, repeat 4 times.Finally suck residual liquid with the blotting paper of sterilizing-drying, obtain aseptic seed.
Two, the checking of Disinfection Effect
Open gauze, with tweezers seed put in liquid nutrient medium and carry out Shaking culture.Be 22-26 DEG C in temperature, cultivate 9 days under dark condition, then to be moved into temperature be 25 DEG C, light intensity is 1500 ~ 2500lx, and light application time is cultivate under the condition of 10h/d.
Formula and the composition of described medium are as follows:
Formula: 1/2MS+NAA0.5mg/L+50g/L murphy juice+30g/L sucrose, pH5.8
Wherein, the preparation method of murphy juice is as follows: peel after being cleaned by potato, be cut into fritter by knife, then put into sterile water and boil to potato well done, leach potato by gauze, the juice obtained is murphy juice.
Three repetitions are established in experiment.Result shows, all seeds are all normally sprouted, and does not all have microbiological contamination phenomenon.
Embodiment 3
For the fruit pod of a kind of stem of noble dendrobium (Dendrobiumsp.) in the present embodiment, the inventive method is described.
One, disinfect
This disinfection process all carries out on superclean bench.
Thimerosal process: get full ripe stem of noble dendrobium fruit pod, cut pericarp open and take out seed, seed is placed in sterilized gauze to bandage, the gauze wrapping seed is put in the centrifuge tube filling 75% alcohol and rocks, or vortex process 2-3 minute, centrifugal 3 minutes of 500rpm, removes alcohol, the seed wrapped is put into 3 times and cleans once with upper volume sterile water; Seed after cleaning immerses in 0.26% mercuric chloride solution disinfects 3-5 minute or centrifugal 3 minutes of vortex process 2-3 minute, 500rpm, removes mercuric chloride solution; Pour 3 times in centrifuge tube into the sterile water wash of upper volume, centrifugal 3 minutes of 500rpm, absorb top moisture, repeat 3 times.Finally suck residual liquid with the blotting paper of sterilizing-drying, obtain aseptic seed.
Two, the checking of Disinfection Effect
Open gauze, put in water agar blake bottle by seed, in blake bottle, then inject the sterile water of 0.8mL with liquid-transfering gun with tweezers, object makes seeded dispersion on blake bottle surface.Be 24-27 DEG C in temperature, cultivate 11 days under dark condition, then to be moved into temperature be 25 DEG C, light intensity is 1500 ~ 2500lx, and light application time is cultivate under the condition of 10h/d.
Formula and the composition of described medium are as follows:
Formula: add 5g-8g agar in 1L sterile water, pH5.8
Three repetitions are established in experiment.Result shows, all seeds are all normally sprouted, and does not all have microbiological contamination phenomenon.
Experimental result shows: sterilization method of the present invention effective, and the seed after sterilization sprouts failed ratio because of pollution extremely low, and the growth strength after later stage plantation is better.The inventive method also has the features such as simple to operate, cost is low, save time in addition, is thus particularly suitable for the front process to Orchid Seeds of tissue cultures.Improve surface sterilization and the cleaning effect of the plant seed of cotton-shaped or particulate, and strengthen its flocculating result in the process of disinfecting, avoid the loss that it causes when surface sterilization process, and enhance the germination rate of the plant seed of cotton-shaped or particulate simultaneously, and its sprout after growth survival rate.In addition, by the bletilla striata after above-mentioned rudiment, stem of noble dendrobium budlet implanting and cultivating, after cultivation, 3-5 month is observed, the bletilla striata that damage by disease and insect equal proportion obtains much smaller than the rudiment of, the seed adopting original processing method to obtain, the stem of noble dendrobium.
What embodiments of the invention were announced is preferred embodiment, but is not limited thereto, those of ordinary skill in the art; very easily according to above-described embodiment, understand spirit of the present invention, and make different amplifications and change; but only otherwise depart from spirit of the present invention, all in protection scope of the present invention.
Claims (10)
1. an Orchid Seeds processes for disinfecting surfaces, is characterized in that, comprises the steps:
S1: kind is got in broken shell, orchid fruit pod is opened in cutting, adopts at least 1 layer of qualitative filter paper and at least 2 layers of hospital gauze wrapping Orchid Seeds; Then seed is put into centrifuge tube, within least 1 minute, collected with the centrifugation process of 350-550 rpm;
S2: alcohol-pickled, puts into 70-85% alcoholic solution and rocks or vortex process 2-3 minute by the seed wrapped, then with the centrifugation process at least 1 minute of 350-550 rpm, remove alcohol;
S3: sterile water wash for the first time, the seed after step S2 process, puts it into and is equipped with 3 times with eccentric cleaning in the centrifuge tube of upper volume sterile water once;
S4: mercuric chloride solution is disinfected, immerses the seed after cleaning in 0.05-0.3% mercuric chloride solution and disinfects 3-5 minute or vortex process 2-3 minute, then with the centrifugation process at least 1 minute of 350-550 rpm, removes mercuric chloride solution;
S5: second time sterile water process, after step S4 process, pours 3 times into the sterile water of upper volume, then with the centrifugation process at least 1 minute of 350-550 rpm; Then adopt blotting paper to absorb top moisture, carry out sterilizing-drying, repeat drying at least 2 times;
S6: subsequent treatment, finally sucks residual liquid with the blotting paper of sterilizing-drying, obtains aseptic seed.
2. a kind of Orchid Seeds processes for disinfecting surfaces according to claim 1, is characterized in that: when described step S1 broken shell is got kind, by filter paper pads in bottom, and after fruit pod is cut open, seed is directly fallen into above filter paper; And described seed is fine powdered.
3. a kind of Orchid Seeds processes for disinfecting surfaces according to claim 1 and 2, is characterized in that: the fruit pod in described step S1 is ripe orchid capsule.
4. a kind of Orchid Seeds processes for disinfecting surfaces according to claim 1, is characterized in that: the centrifugal treating in described step S2, step S4 and step S5 is the centrifugation process 2 minutes of 500 rpms.
5. a kind of Orchid Seeds processes for disinfecting surfaces according to claim 1 or 4, is characterized in that: the alcoholic solution concentration in step S2 is 50% ~ 85%, preferably 70 ~ 80%.
6. a kind of Orchid Seeds processes for disinfecting surfaces according to claim 1, is characterized in that: the sterile water in described step S3 and step S5 is pure water after HTHP moist heat sterilization or physiological saline.
7. a kind of Orchid Seeds processes for disinfecting surfaces according to claim 1 or 6, it is characterized in that: the sterile water in described step S3 and step S5, be 121 ~ 125 DEG C and pressure through excess temperature be in the hygrothermal environment of 0.1 ~ 0.15Mpa, sterilization time 10 ~ 15min obtains.
8. a kind of Orchid Seeds processes for disinfecting surfaces according to claim 7, is characterized in that: immersed in 0.1% mercuric chloride solution by the seed after cleaning in described step S2 and disinfect 3-5 minute.
9. a kind of Orchid Seeds processes for disinfecting surfaces according to claim 1, it is characterized in that: the aseptic seed obtained in step S6, put into and be equipped with in the blake bottle of medium, then in blake bottle, sterile water is injected with liquid-transfering gun, be 21-27 DEG C in temperature, under dark condition, cultivate 8-12 days; Being moved into temperature is again 25 DEG C, and light intensity is 1500 ~ 2500lx, and light application time is cultivate under the condition of 10h/d.
10. a kind of Orchid Seeds processes for disinfecting surfaces according to claim 9, is characterized in that: the formula of described medium is as follows: 1/2MS+NAA0.5mg/L+20g/L mashed potatoes+30g/L sucrose+5.5g/L agar, pH5.8; Wherein, the preparation method of mashed potatoes is as follows: by potato clean after peel, be cut into fritter by knife, then put into juice extractor add water squeeze broken, the homogenate obtained is mashed potatoes.
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| CN105850730A (en) * | 2016-04-06 | 2016-08-17 | 南京晓庄学院 | Plant material surface disinfection method |
| CN105917956A (en) * | 2016-07-17 | 2016-09-07 | 湘南学院 | Sowing method of orchid seeds |
| CN106135007A (en) * | 2016-09-27 | 2016-11-23 | 中国科学院昆明植物研究所 | A kind of sterilizing being suitable to trace Orchid Seeds during in vitro conservation and inoculation method |
| CN106717307A (en) * | 2017-02-22 | 2017-05-31 | 山东博华高效生态农业科技有限公司 | A kind of miniature seed disinfection forwarding method |
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| CN105850730A (en) * | 2016-04-06 | 2016-08-17 | 南京晓庄学院 | Plant material surface disinfection method |
| CN105917956A (en) * | 2016-07-17 | 2016-09-07 | 湘南学院 | Sowing method of orchid seeds |
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