CN106613977A - Sterilization and inoculation method of pteridophyte spores - Google Patents
Sterilization and inoculation method of pteridophyte spores Download PDFInfo
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- CN106613977A CN106613977A CN201611200707.2A CN201611200707A CN106613977A CN 106613977 A CN106613977 A CN 106613977A CN 201611200707 A CN201611200707 A CN 201611200707A CN 106613977 A CN106613977 A CN 106613977A
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- spore
- adsorption column
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- 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
Abstract
The invention discloses a sterilization and inoculation method of pteridophyte spores. One set of sterilization and inoculation method applicable to trace pteridophyte spores is designed by combining a sterilization method of a tissue culture technology with tools including a DNA (Deoxyribonucleic Acid) purification column and the like, and is applicable to an in-vitro preservation process. According to the technology disclosed by the invention, the problems of insufficient sterilization and interbreeding and disinfection liquid residues, which easily occur in a disinfection process, are solved; the pollution rate can be effectively reduced and the inoculation efficiency is improved. The sterilization and inoculation method disclosed by the invention has the advantages that the trace pteridophyte spores can be efficiently disinfected and inoculated; experimental apparatuses are easy to obtain; the cost is low, the operation is convenient, the inoculation efficiency is high and the like.
Description
Technical field
The invention belongs to field of plant tissue culture technique, and in particular to one kind is suitable to fern seed during in vitro conservation
Sterilizing methods and inoculation,
Background technology
The fern resource very abundant of China, there are about 2600 kinds, account for global 1/5, wherein being much that China is peculiar.Herba pteridii latiusculi
Class plant not only plays an important role in the research that biological evolution and botanical system are developed, also with human lives' close relation,
Many pteridophyta it is medicinal, numerous aspects such as view and admire there is important value.It can be seen that, the merchandized handling and cultivation with regard to fern
The exploitation of breeding has bright prospects.In pteridophyta tissue culture procedures, tissue culture's tool is carried out as explant using spore
Have the advantages that not damage maternal plant, breeding coefficient big, be the upper most popular Propagation Methods of pteridophyta production.Fern seed is non-
Chang Wei little, in dust-like, when aseptic seeding is carried out, conventional method has filter paper to wrap up or be placed in the vessel such as centrifuge tube, then
Sterilized with sodium hypochlorite or mercuric chloride, sterilized water is inoculated with after repeatedly rinsing.Due to fern seed it is excessively small, it is same in multiple species
When being inoculated with, spore is easily spilt and causes miscegenation by pack, and the filter paper of multilamellar parcel may cause to sterilize it is insufficient；And
In the operating process of centrifugation sterilization, its waste liquid removes difficulty, easily siphons away in spore, or the potential prolongation because of disinfectant solution residual
Sterilization time, and then cause spore to inactivate.
The content of the invention
The purpose of the present invention is the defect existed for prior art, (is included by current commonly available DNA purification columns
Adsorption column and collecting pipe) a kind of easy to operate, efficiency high is provided, fern seed sterilizing and the inoculation being suitable to during in vitro conservation
Method.
To realize the purpose of the present invention, the invention provides following technical scheme：
Spore is collected, fern seed leaf is fitted in sulphuric acid paper bag, be placed in after labelling code name at aeration-drying, treat spore certainly
By coming off；DNA purification column (specifications：Adsorption column 1ml, collecting pipe 2ml), 1ml pipette tips, pure water is in 121 DEG C of high temperature sterilize 20-
30min is standby；The spore in paper bag that comes off is collected with the template to being folded over, broken leaf, annulus, palea etc. is simply removed miscellaneous
Pour in sterilized adsorption column along folding line after matter, cover lid, in being put into collecting pipe, and carry out labelling, be positioned over centrifugation
On pipe support；Aseptically, the ethanol of 600 μ l 75%, handstand purification column 1-2 time, afterwards 200-1200rpm centrifugations are added
20s.From the beginning of ethanol is added, until completing centrifugation, whole operation process control is in 30s or so；Waste liquid in collecting pipe is abandoned, in nothing
600 μ l sterilized water are added under the conditions of bacterium, are inhaled repeatedly and is beaten solution and makes spore suspended, fully rinse spore, then 200-1200rpm from
Heart 20s, repeats 1-2 time, eluting ethanol；Waste liquid in collecting pipe is abandoned, 600 μ l 0.1-0.2% chlorinations are aseptically added
Hydrargyrum sterilizing 2-4min or 2% hypochlorite disinfectant 6-8min, repeatedly solution is beaten in suction makes the suspended abundant sterilizing of spore, then 200-
1200rpm is centrifuged 20s；Waste liquid in collecting pipe is abandoned, 600 μ l sterilized water are aseptically added, inhaling to beat repeatedly hangs spore
It is turbid, spore is fully rinsed, then 200-1200rpm centrifugations 20s, this operation repeats 3-4 time, makes the thorough eluting of disinfectant solution；Again to
Add 600 μ l sterilized water in adsorption column after centrifugation, suction repeatedly is beaten and is allowed to, in spore suspension, draw 100 μ l spores suspended
Liquid is inoculated in culture medium, realizes inoculation.
A kind of sterilization of fern seed and inoculation method, the method comprises the steps：
Step 1：Spore is collected, fern seed blade is fitted in sulphuric acid paper bag, be placed in after labelling code name at aeration-drying,
Deng spore free dropping；
Step 2：DNA purification columns, specification：Adsorption column 1ml, collecting pipe 2ml, 1ml pipette tips, pure water are in 121 DEG C of high temperature
20-30min is standby for sterilizing；
Step 3：In one layer of the sulphuric acid scraps of paper upper berth lens paper to being folded over, spore of the free dropping in sulphuric acid paper bag is fallen
On lens paper, slight jitter lens paper is simply filtered, spore can by the hole of lens paper, and broken leaf, annulus,
The impurity such as palea can stay in the purpose that simple filtration is reached on lens paper, then the spore after filtration is poured into along folding line and is gone out
In the adsorption column of bacterium, lid is covered, in being put into collecting pipe, carry out labelling, be positioned on centrifuge tube shelf；
Step 4：On aseptic operating platform, adsorption column lid is opened, with 1ml liquid-transfering guns the ethanol of 600 μ l 75% is added, covered
Lid sterilization 30s, period handstand purification column 1-2 time while spore is sterilized, is also sterilized, then inside adsorption column
200-1200rpm is centrifuged 20s, plus ethanol, and handstand purification column, centrifugation completes to control as far as possible in 30s；
Step 5：Adsorption column is taken out, waste liquid in collecting pipe is abandoned.Adsorption column is put back in collecting pipe, is positioned over centrifuge tube shelf
On.In aseptic operating platform, adsorption column lid is opened, with 1ml liquid-transfering guns 600 μ l sterilized water are added, suction repeatedly is beaten solution and fully floated
Spore is washed, then 200-1200rpm centrifugations 20s, repeats 1-2 time；
Step 6：Adsorption column is taken out, waste liquid in collecting pipe is abandoned, adsorption column is put back in collecting pipe, is positioned over centrifuge tube shelf,
In aseptic operating platform, open adsorption column lid, with 1ml liquid-transfering guns add 600 μ l 0.1-0.2% mercuric chloride sterilizing 2-4min or
2% hypochlorite disinfectant 6-8min, repeatedly solution is beaten in suction makes the suspended abundant sterilizing of spore, then 200-1200rpm centrifugations 20s；
Step 7：Adsorption column is taken out, waste liquid in collecting pipe is abandoned, adsorption column is put back in collecting pipe, is positioned over centrifuge tube shelf
On, in aseptic operating platform, adsorption column lid is opened, now adsorption column and spore disappear through ethanol, mercuric chloride or sodium hypochlorite
Poison is in aseptic condition, and with 1ml liquid-transfering guns 600 μ l sterilized water are added, and inhaling to beat repeatedly makes spore suspended, fully rinses spore, so
Afterwards 200-1200rpm is centrifuged 20s；
Step 8：Repeat step 7,3-4 time；
Step 9：600 μ l sterilized water are taken with 1ml liquid-transfering guns to add in adsorption column, suction repeatedly is beaten and is allowed in spore suspension,
Draw 100 μ l spore suspensions to be inoculated in culture medium；
Step 10：Culture is moved on to 25 ± 2 DEG C of temperature light culture 3-5 days, spore after illumination condition culture 2 weeks is then moved to
Sprout.
A kind of application of the sterilization and inoculation method of described fern seed during fern in vitro conservation.
Compared with prior art, the excellent benefit that possesses of the present invention is：
1. play an important role in research of the present invention to pteridophyta biological evolution and botanical system development, also with the mankind
Life close relation, many pteridophyta it is medicinal, numerous aspects such as view and admire there is important value.Commodity of the present invention to fern
Metaplasia is produced with the exploitation of breeding and cultivation there is provided scientific strong exercisable sterilization and inoculation method.
2. in pteridophyta tissue culture procedures, using spore as explant carry out tissue culture have do not damage maternal plant,
The advantages of breeding coefficient is big, is the upper most popular Propagation Methods of pteridophyta production.Fern seed is very small, in dust
Shape, when aseptic seeding is carried out, conventional method has filter paper to wrap up or be placed in the vessel such as centrifuge tube, then with sodium hypochlorite or chlorine
Change hydrargyrum sterilization, sterilized water is inoculated with after repeatedly rinsing.Because fern seed is excessively small, when multiple species are inoculated with simultaneously, wrap up
Spore is easily spilt and causes miscegenation by method, and the filter paper of multilamellar parcel may cause sterilizing insufficient；And sterilization is centrifuged
In operating process, its waste liquid removes difficulty, easily siphons away in spore, or the potential prolongation sterilization time because of disinfectant solution residual, enters
And cause spore to inactivate.Instant invention overcomes the deficiency that pteridophyta pack and centrifugation sterilization are present.
3. instant invention overcomes the sterilizing that easily occurs in disinfecting process of pteridophyta spore is insufficient, miscegenation and disinfectant solution
The problem of residual, can effectively reduce pollution rate, improve inoculation efficiency.
4. the present invention can efficiently carry out the sterilization and inoculation of micro fern seed, experiment equipment is easy to get, it is with low cost,
Easy to operate, inoculation efficiency is high.
Specific embodiment
Further illustrate the essentiality content of the present invention with embodiments of the invention below, but do not limit with this this
Invention.
Embodiment 1：
1st, material：Cibotium barometz (L.) J. Sm. Cibotiumbarometz (Linnaeus) J.Smith.
2nd, experiment equipment：Super-clean bench, centrifuge, centrifuge tube shelf, DNA purification columns, liquid-transfering gun, 1ml pipette tips, autoclave
3rd, operating procedure：
Step 1：Spore is collected, the blade that Cibotium barometz (L.) J. Sm. length has spore is fitted in sulphuric acid paper bag, be placed in after labelling code name logical
Dry place is air-dried, spore free dropping is waited.
Step 2：DNA purification column (specifications：Adsorption column 1ml, collecting pipe 2ml), 1ml pipette tips, pure water is in 121 DEG C of high temperature
20-30min is standby for sterilizing.
Step 3：In one layer of the sulphuric acid scraps of paper upper berth lens paper to being folded over, spore of the free dropping in sulphuric acid paper bag is fallen
On lens paper, slight jitter lens paper is simply filtered, spore can by the hole of lens paper, and broken leaf, annulus,
The impurity such as palea can stay in the purpose that simple filtration is reached on lens paper.Then the spore after filtration is poured into along folding line and is gone out
In the adsorption column of bacterium, lid is covered, in being put into collecting pipe, carry out labelling, be positioned on centrifuge tube shelf.
Step 4：On aseptic operating platform, adsorption column lid is opened, with 1ml liquid-transfering guns the ethanol of 600 μ l 75% is added, covered
Lid sterilization 30s, period handstand purification column 1-2 time while spore is sterilized, is also sterilized, then inside adsorption column
200-1200rpm is centrifuged 20s.Plus ethanol, handstand purification column, it is centrifuged and completes to control as far as possible in 30s.
Step 5：Adsorption column is taken out, waste liquid in collecting pipe is abandoned.Adsorption column is put back in collecting pipe, is positioned over centrifuge tube shelf
On.In aseptic operating platform, adsorption column lid is opened, with 1ml liquid-transfering guns 600 μ l sterilized water are added, suction repeatedly is beaten solution and fully floated
Spore is washed, then 200-1200rpm centrifugations 20s.Repeat 1-2 time.
Step 6：Adsorption column is taken out, waste liquid in collecting pipe is abandoned, adsorption column is put back in collecting pipe, is positioned over centrifuge tube shelf
On.In aseptic operating platform, adsorption column lid is opened, with 1ml liquid-transfering guns 600 μ l 0.1-0.2% mercuric chloride sterilizing 2- is added
4min, repeatedly solution is beaten in suction makes the suspended abundant sterilizing of spore, then 200-1200rpm centrifugations 20s.
Step 7：Adsorption column is taken out, waste liquid in collecting pipe is abandoned, adsorption column is put back in collecting pipe, is positioned over centrifuge tube shelf
On.In aseptic operating platform, adsorption column lid is opened, now adsorption column and spore disappear through ethanol, mercuric chloride or sodium hypochlorite
Poison is in aseptic condition, and with 1ml liquid-transfering guns 600 μ l sterilized water are added, and inhaling to beat repeatedly makes spore suspended, fully rinses spore, so
Afterwards 200-1200rpm is centrifuged 20s.
Step 8：Repeat step 7,3-4 time.
Step 9：600 μ l sterilized water are taken with 1ml liquid-transfering guns to add in adsorption column, suction repeatedly is beaten and is allowed in spore suspension,
Draw 100 μ l spore suspensions to be inoculated in culture medium.
Step 10：Culture is moved on to 25 ± 2 DEG C of temperature light culture 3-5 days, spore after illumination condition culture one week is then moved to
Son is sprouted.
Embodiment 2：
1st, material：Trichroism Herba Pteridis creticae Pterisaspericaulis var.tricolor (Linden) T.Moore ex
E.J.Lowe。
2nd, experiment equipment：Super-clean bench, centrifuge, centrifuge tube shelf, DNA purification columns, liquid-transfering gun, 1ml pipette tips, autoclave
3rd, operating procedure：
Step 1：Spore is collected, the sporophyll piece of trichroism Herba Pteridis creticae is fitted in template bag, be placed in after labelling code name logical
Dry place is air-dried, spore free dropping is waited.
Step 2：DNA purification column (specifications：Adsorption column 1ml, collecting pipe 2ml), 1ml pipette tips, pure water is in 121 DEG C of high temperature
20-30min is standby for sterilizing.
Step 3：In one layer of the sulphuric acid scraps of paper upper berth lens paper to being folded over, spore of the free dropping in sulphuric acid paper bag is fallen
On lens paper, slight jitter lens paper is simply filtered, spore can by the hole of lens paper, and broken leaf, annulus,
The impurity such as palea can stay in the purpose that simple filtration is reached on lens paper.Then the spore after filtration is poured into along folding line and is gone out
In the adsorption column of bacterium, lid is covered, in being put into collecting pipe, carry out labelling, be positioned on centrifuge tube shelf.
Step 4：On aseptic operating platform, adsorption column lid is opened, with 1ml liquid-transfering guns the ethanol of 600 μ l 75% is added, covered
Lid sterilization 30s, period handstand purification column 1-2 time while spore is sterilized, is also sterilized, then inside adsorption column
200-1200rpm is centrifuged 20s.Plus ethanol, handstand purification column, it is centrifuged and completes to control as far as possible in 30s.
Step 5：Adsorption column is taken out, waste liquid in collecting pipe is abandoned.Adsorption column is put back in collecting pipe, is positioned over centrifuge tube shelf
On.In aseptic operating platform, adsorption column lid is opened, with 1ml liquid-transfering guns 600 μ l sterilized water are added, suction repeatedly is beaten solution and fully floated
Spore is washed, then 200-1200rpm centrifugations 20s.Repeat 1-2 time.
Step 6：Adsorption column is taken out, waste liquid in collecting pipe is abandoned, adsorption column is put back in collecting pipe, is positioned over centrifuge tube shelf.
In aseptic operating platform, open adsorption column lid, with 1ml liquid-transfering guns add 600 μ l 0.1-0.2% mercuric chloride sterilizing 2-4min or
2% hypochlorite disinfectant 6-8min, repeatedly solution is beaten in suction makes the suspended abundant sterilizing of spore, then 200-1200rpm centrifugations 20s.
Step 7：Adsorption column is taken out, waste liquid in collecting pipe is abandoned, adsorption column is put back in collecting pipe, is positioned over centrifuge tube shelf
On.In aseptic operating platform, adsorption column lid is opened, now adsorption column and spore disappear through ethanol, mercuric chloride or sodium hypochlorite
Poison is in aseptic condition, and with 1ml liquid-transfering guns 600 μ l sterilized water are added, and inhaling to beat repeatedly makes spore suspended, fully rinses spore, so
Afterwards 200-1200rpm is centrifuged 20s.
Step 8：Repeat step 7,3-4 time.
Step 9：600 μ l sterilized water are taken with 1ml liquid-transfering guns to add in adsorption column, suction repeatedly is beaten and is allowed in spore suspension,
Draw 100 μ l spore suspensions to be inoculated in culture medium.
Step 10：Culture is moved on to 25 ± 2 DEG C of temperature light culture 3-5 days, spore after illumination condition culture 2 weeks is then moved to
Sprout.
Claims (3)
1. a kind of sterilization of fern seed and inoculation method, it is characterised in that the method first collects fern seed, fern seed
Leaf is fitted in sulphuric acid paper bag, is placed in after labelling code name at aeration-drying, treats spore free dropping；DNA purification columns, specification：Inhale
Attached column 1ml, collecting pipe 2ml, 1ml pipette tips, pure water are standby in 121 DEG C of high temperature sterilize 20-30min；With the template to being folded over
The spore in paper bag that comes off is collected, simply goes after the broken leaf of the removal of impurity, annulus, palea to pour sterilized absorption into along folding line
In post, lid is covered, in being put into collecting pipe, and carry out labelling, be positioned on centrifuge tube shelf；Aseptically, 600 μ l are added
75% ethanol, handstand purification column 1-2 time, the 20s of 200-1200rpm centrifugations afterwards；It is whole until completing centrifugation from the beginning of ethanol is added
Individual operating process is controlled in 25-35s；Waste liquid in collecting pipe is abandoned, 600 μ l sterilized water are aseptically added, is inhaled repeatedly
Beating solution makes spore suspended, fully rinses spore, and then 200-1200rpm centrifugations 20s, repeats 1-2 time, eluting ethanol；Abandon
Waste liquid in collecting pipe, aseptically adds 600 μ l 0.1-0.2% mercuric chloride sterilizing 2-4min or 2% hypochlorite disinfectant
6-8min, repeatedly solution is beaten in suction makes the suspended abundant sterilizing of spore, then 200-1200rpm centrifugations 20s；Abandon in collecting pipe and give up
Liquid, aseptically adds 600 μ l sterilized water, and inhaling to beat repeatedly makes spore suspended, fully rinses spore, then 200-
1200rpm is centrifuged 20s, and this operation repeats 3-4 time, makes the thorough eluting of disinfectant solution；Add 600 in the adsorption column after centrifugation again
μ l sterilized water, repeatedly suction is beaten and is allowed in spore suspension, is drawn 100 μ l spore suspensions and is inoculated in culture medium, realizes inoculation.
2. a kind of sterilization of fern seed and inoculation method, it is characterised in that the method comprises the steps：
Step 1：Spore is collected, fern seed blade is fitted in sulphuric acid paper bag, be placed in after labelling code name at aeration-drying, wait spore
Sub- free dropping；
Step 2：DNA purification columns, specification：Adsorption column 1ml, collecting pipe 2ml, 1ml pipette tips, pure water are in 121 DEG C of high temperature sterilizes
20-30min is standby；
Step 3：In one layer of the sulphuric acid scraps of paper upper berth lens paper to being folded over, spore of the free dropping in sulphuric acid paper bag is poured on wiping
On mirror paper, slight jitter lens paper is simply filtered, and spore can be by the hole of lens paper, and broken leaf, annulus, palea
The purpose that simple filtration is reached on lens paper can be stayed in Deng impurity, then the spore after filtration is poured into along folding line sterilized
In adsorption column, lid is covered, in being put into collecting pipe, carry out labelling, be positioned on centrifuge tube shelf；
Step 4：On aseptic operating platform, adsorption column lid is opened, with 1ml liquid-transfering guns the ethanol of 600 μ l 75% is added, cover lid
Sterilization 30s, period handstand purification column 1-2 time while spore is sterilized, is also sterilized, then 200- inside adsorption column
1200rpm is centrifuged 20s, plus ethanol, and handstand purification column, centrifugation completes to control as far as possible in 30s；
Step 5：Adsorption column is taken out, waste liquid in collecting pipe is abandoned.Adsorption column is put back in collecting pipe, is positioned on centrifuge tube shelf.
In aseptic operating platform, adsorption column lid is opened, with 1ml liquid-transfering guns 600 μ l sterilized water are added, suction repeatedly is beaten solution and fully rinses spore
Son, then 200-1200rpm centrifugations 20s, repeats 1-2 time；
Step 6：Adsorption column is taken out, waste liquid in collecting pipe is abandoned, adsorption column is put back in collecting pipe, is positioned over centrifuge tube shelf, in nothing
In bacterium operating board, adsorption column lid is opened, add 600 μ l 0.1-0.2% mercuric chloride to sterilize 2-4min or 2% time with 1ml liquid-transfering guns
Sodium chlorate sterilization 6-8min, repeatedly solution is beaten in suction makes the suspended abundant sterilizing of spore, then 200-1200rpm centrifugations 20s；
Step 7：Adsorption column is taken out, waste liquid in collecting pipe is abandoned, adsorption column is put back in collecting pipe, is positioned on centrifuge tube shelf,
In aseptic operating platform, adsorption column lid is opened, now adsorption column and spore are in through ethanol, mercuric chloride or hypochlorite disinfectant
Aseptic condition, with 1ml liquid-transfering guns 600 μ l sterilized water are added, and inhaling to beat repeatedly makes spore suspended, fully rinses spore, then 200-
1200rpm is centrifuged 20s；
Step 8：Repeat step 7,3-4 time；
Step 9：600 μ l sterilized water are taken with 1ml liquid-transfering guns to add in adsorption column, suction repeatedly is beaten and is allowed to, in spore suspension, draw
100 μ l spore suspensions are inoculated in culture medium；
Step 10：Culture is moved on to 25 ± 2 DEG C of temperature light culture 3-5 days, spore after illumination condition culture 2 weeks is then moved to and is sprouted
Send out.
3. a kind of sterilization of the fern seed described in claim 1 or 2 and inoculation method during fern in vitro conservation should
With.
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| CN201611200707.2A CN106613977A (en) | 2016-12-22 | 2016-12-22 | Sterilization and inoculation method of pteridophyte spores |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611200707.2A CN106613977A (en) | 2016-12-22 | 2016-12-22 | Sterilization and inoculation method of pteridophyte spores |
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Family
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| CN201611200707.2A Pending CN106613977A (en) | 2016-12-22 | 2016-12-22 | Sterilization and inoculation method of pteridophyte spores |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112293253A (en) * | 2020-10-28 | 2021-02-02 | 中国科学院昆明植物研究所 | Isolated preservation culture medium and isolated preservation method for Adiantum furiosaeanum |
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| CN104663443A (en) * | 2015-02-25 | 2015-06-03 | 杨惠才 | Construction method of in-vitro regeneration system of adiantum reniforme var.sinense |
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Application publication date: 20170510 |