CN108812305A - Obtain the method and its application of sterile arrowhead explant - Google Patents
Obtain the method and its application of sterile arrowhead explant Download PDFInfo
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- CN108812305A CN108812305A CN201810297303.2A CN201810297303A CN108812305A CN 108812305 A CN108812305 A CN 108812305A CN 201810297303 A CN201810297303 A CN 201810297303A CN 108812305 A CN108812305 A CN 108812305A
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- arrowhead
- stem
- explant
- stolon
- bulb
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Environmental Sciences (AREA)
- Botany (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention discloses a kind of method and its application for obtaining sterile arrowhead explant, and this approach includes the following steps：(1) the arrowhead bulb by disinfection is put into after being cultivated 40-50 days in river sand, takes the stolon of sprouting；(2) after rinsing the stolon of above-mentioned sprouting well, the stem apex of stolon is acquired, clip goes out the first stem section on the stem apex, and first stem section is placed in desinfection chamber 5-10min；And (3) intercept out the second stem section in first stem section on the super-clean bench, second stem section, 75% alcohol is submerged, and then use aseptic water washing, it is impregnated with 0.1% mercury chloride disinfection or 2% sodium hypochlorite, is then sealed in sterilized culture bottle for inoculation with draining after aseptic water washing again, arrowhead explant pollution rate accessed by this method is low, proliferation rate is high, and saves kind of a ball material, has preferable economic benefit and social benefit.
Description
Technical field
The present invention relates to field of plant tissue culture technique, in particular to a kind of method for obtaining sterile arrowhead explant and
It is applied.
Background technique
Arrowhead (Sagittaria trifoliaL.) belongs to the perennial aquatic herbaceous plant of Alismataceae Sagittaria, with its bulb
It is edible, it is one of the main aquatic vegetable in Winter-Spring vegetable supply dull season.Arrowhead is with higher medicinal and health value, nature and flavor
It is bitter sweet, slightly cold, nontoxic, there is the effect of cool blood, hemostasis, removing toxic substances, detumescence.80% products export is sold to Southeast Asia, the U.S. plus is taken
Country of great Deng state, is important export-oriented commodity.
Arrowhead is water plant, entire growth cycle be all in water, bulb be also sprout, expand in water, so
It is more vulnerable to infecting for microorganism compared with ground crop, the formal structure of arrowhead bulb is more special in addition, belong to half package status,
Thus sterile stem apex is just more difficult, and it is to pollute most important reason that explant itself, which carries disease germs, especially endophyte, pole is not
It is easy to remove, is unfavorable for the quick breeding of arrowhead.
The information disclosed in the background technology section is intended only to increase the understanding to general background of the invention, without answering
When being considered as recognizing or imply that the information constitutes the prior art already known to those of ordinary skill in the art in any form.
Summary of the invention
The purpose of the present invention is to provide a kind of method and its application for obtaining sterile arrowhead explant, acquired in this method
The arrowhead explant pollution rate arrived is low, and proliferation rate is high, and saves kind of a ball material, has preferable economic benefit and social benefit.
To achieve the above object, the present invention provides a kind of method for obtaining sterile arrowhead explant, include the following steps：
(1) the arrowhead bulb by disinfection is put into after being cultivated 40-50 days in river sand, takes the stolon of sprouting；
(2) after rinsing the stolon of above-mentioned sprouting well, the stem apex (that is, stolon stem apex) of stolon is acquired, in institute
It states clip on stem apex and goes out the first stem section, first stem section is placed in desinfection chamber 5-10min；And
(3) intercept out the second stem section in first stem section on the super-clean bench, by second stem section with 75% wine
Essence submergence, and then use aseptic water washing, then with 0.1% mercury chloride sterilize or 2% sodium hypochlorite immersion, then rushed with sterile water
After washing, drains and be sealed in sterilized culture bottle for inoculation.
Preferably, in above-mentioned technical proposal, the arrowhead bulb is with by 800 times of diluted 50% wettables of carbendazim
What pulvis carried out disinfection, wherein 50% wettable powder of the carbendazim is purchased from two Co., Ltd., Factory of Jiangyin pesticide.
Preferably, it in above-mentioned technical proposal, in step (1), is trained being put into river sand by the arrowhead bulb of disinfection
It is feeding the specific steps are：It will be put on the river sand with a thickness of 8-10 centimetres by the arrowhead bulb of disinfection, further described kind
Bedding takes the stolon of sprouting, wherein the mesh of the river sand after culture 40-50 days with a thickness of the river sand of 5-8cm on aunt's bulb
Number is 20-40 mesh.
Preferably, in above-mentioned technical proposal, in step (1), on the arrowhead bulb after bedding river sand, it is also necessary to paving
The river sand of lid carries out disinfection.
Preferably, in above-mentioned technical proposal, the river sand of bedding is with by 800 times of diluted 50% wettable powders of carbendazim
The processing that carries out disinfection is sprayed in agent.
Preferably, in above-mentioned technical proposal, in step (2), after the stem apex for acquiring stolon, it is also necessary to by the stolon
Stem apex be put into washing powder water and carry out immersion 25-35min, rinse 2-3 after with tap water, and then the clip on the stem apex
First stem section out, it is preferred that the mass percentage concentration of the washing powder water is 0.2%-0.5%, and the washing powder water is for killing
Bacterium.
Preferably, in above-mentioned technical proposal, in step (2), the length of the first stem section is 2-3cm.
Preferably, in above-mentioned technical proposal, in step (3), the diameter of second stem section is 3-5mm, length 5-
10mm。
Preferably, in above-mentioned technical proposal, in step (3), after second stem section, 75% alcohol submergence, gently
10-30s is rocked, pours out alcohol, and then with aseptic water washing 3-5 times, then sterilizes 10-15min or 2% with 0.1% mercury chloride
Sodium hypochlorite impregnates 8-10min, then uses aseptic water washing 3-5 times.
The present invention also provides application of the method for the sterile arrowhead explant of above-mentioned acquisition in Plant Tissue Breeding.
Compared with prior art, the present invention has the advantages that：
(1) the application selected materials are the stolon stem apex (general each bulb has 2-5 item) of arrowhead bulb, instead of biography
Unite bulb terminal bud (bulb only one terminal bud), therefore, more horn of plenty of drawing materials；
(2) the application draws materials again after arrowhead bulb is first passed through sand culture, and uses the time specifically sterilized and concentration,
After sterilization processing, so that the thallus infection rate of arrowhead explant is effectively controlled within 10%, substantially less than traditional 30
~50%；
(3) the application is that arrowhead tissue-culturing rapid propagation, preserving seed, biotechnology operation etc. based on tissue culture technique provide
Suitable aseptic explant material, this method is easy to operate, and acquired arrowhead explant pollution rate is low, it is kind to be significantly increased
Aunt's tissue culture success rate.
Specific embodiment
Specific embodiments of the present invention will be described in detail below, it is to be understood that protection scope of the present invention is not
It is restricted by specific implementation.
Unless otherwise explicitly stated, otherwise in entire disclosure and claims, term " includes " or its change
Changing such as "comprising" or " including " etc. will be understood to comprise stated element or component, and not exclude other members
Part or other component parts.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples, it is unless otherwise specified, commercially commercially available.
50% wettable powder of carbendazim used in the present embodiment is purchased from the limited public affairs of two factory of Jiangyin pesticide
Department.
The method that embodiment 1 obtains sterile arrowhead explant
Previous materials prepare：The arrowhead bulb of robust growth, no disease and pests harm is taken, 800 times of diluted carbendazim 50% are wettable
Property pulvis to bulb submergence 20min disinfection treatment after, be put on the thin river sand (20-40 mesh) of 8-10 cm thick one by one, thereon again
Cover the thin river sand of 5-8 cm thick, 800 times of diluted 50% wettable powders of carbendazim sprinklings carry out disinfection processing to fine sand, pass through
It often keeps fine sand gentle, after 45d, takes the stolon of sprouting, rinse well.
Pretreatment：The stem apex for acquiring stolon, stem apex is put into and impregnates 30min added in 0.4% washing powder warm water, with certainly
Water is rinsed 2-3 times, and clip goes out the first stem section 3cm, is placed in desinfection chamber the 7min that dries in the air.
Stem apex sterilizing：On the super-clean bench, diameter 4mm, the second stem section of long 7mm, by second are intercepted out in the first stem section
Stem section is put into beaker, is poured into 75% alcohol submergence, is jiggled 30s, pour out alcohol, with aseptic water washing explant 3 times,
10min is sterilized with 0.1% mercury chloride again, after then using aseptic water washing 4 times, drains and is sealed in confession in sterilized culture bottle
Inoculation is used.
2 comparative test of embodiment
(1) sand culture processing and traditional crop field processing comparison
Traditional crop field processing：Arrowhead bulb is adopted back from big Tanaka, bulb terminal bud is acquired from the arrowhead bulb, and then will
The bulb terminal bud is submerged with 75% alcohol, is jiggled 30s, is poured out alcohol, with aseptic water washing explant 3 times, then uses
0.1% mercury chloride sterilizes 10min, after then using aseptic water washing 4 times, drains and is sealed in sterilized culture bottle for inoculation
With.
It is further that above-mentioned sand culture processing (embodiment 1) obtained stolon stem apex (that is, second stem section) is big with tradition
Field handles obtained bulb terminal bud and is cultivated and compared.
The processing of 1 sand culture of table handles contrast table with traditional crop field
| Processing | Pollution rate | The death rate |
| Sand culture | 22 | 12 |
| Crop field | 50 | 23 |
As it can be seen from table 1 culture sand culture processing the obtained tissue-cultured seedling of stolon stem apex pollution rate well below
The effect of earth culture processing, and the tissue-cultured seedling death rate reduces.
(2) sterilization method compares
In embodiment 1, bulb also is acquired from arrowhead bulb while acquiring stolon stem apex (that is, second stem section)
Terminal bud, and identical processing operation is carried out to it, further by the processing time point of 75% alcohol disinfecting involved in embodiment 1
It is not set as 10s, 30s, 60s, 90s, sterile water wash 3-5 sterilizes 5min, 10min with 0.1% mercury chloride after respectively,
15min, with aseptic water washing 3-5 times, each 10 bottles of processing, sterilization processing result is shown in Table 2.
Table 2：The influence of materials and disinfectant to arrowhead stem apex Disinfection Effect
From table 2 it can be seen that 0.1% mercury chloride disinfecting time 5min, though take bulb terminal bud or stolon stem apex (that is,
Second stem section) it is cultivated, pollution rate is above 60%；When 0.1% mercury chloride disinfecting time is 10-15min, pollution rate has
It is reduced, in 75% alcohol disinfecting 10-30s, stolon stem apex pollution rate is lower than 30%, and when being higher than 30s, tissue-cultured seedling occurs dead
Die phenomenon；And 75% alcohol disinfecting of bulb terminal bud be less than 60s when, tissue-cultured seedling does not occur the phenomena of mortality.
The aforementioned description to specific exemplary embodiment of the invention is in order to illustrate and illustration purpose.These descriptions
It is not wishing to limit the invention to disclosed precise forms, and it will be apparent that according to the above instruction, can much be changed
And variation.The purpose of selecting and describing the exemplary embodiment is that explaining specific principle of the invention and its actually answering
With so that those skilled in the art can be realized and utilize a variety of different exemplary implementation schemes of the invention and
Various chooses and changes.The scope of the present invention is intended to be limited by claims and its equivalents.
Claims (10)
1. a kind of method for obtaining sterile arrowhead explant, which is characterized in that include the following steps：
(1) the arrowhead bulb by disinfection is put into after being cultivated 40-50 days in river sand, takes the stolon of sprouting；
(2) after rinsing the stolon of above-mentioned sprouting well, the stem apex of stolon is acquired, clip goes out the first stem on the stem apex
Section, is placed in desinfection chamber 5-10min for first stem section；And
(3) the second stem section is intercepted out in first stem section on the super-clean bench, second stem section, 75% alcohol is soaked
Not yet, and then with aseptic water washing, it then with 0.1% mercury chloride disinfection or 2% sodium hypochlorite impregnates, then uses aseptic water washing
Afterwards, it drains and is sealed in sterilized culture bottle for inoculation.
2. the method according to claim 1 for obtaining sterile arrowhead explant, which is characterized in that the arrowhead bulb is to use
It carries out disinfection by 800 times of diluted 50% wettable powders of carbendazim.
3. the method according to claim 1 for obtaining sterile arrowhead explant, which is characterized in that in step (1), will pass through
The arrowhead bulb of disinfection be put into cultivated in river sand the specific steps are：It will be put by the arrowhead bulb of disinfection with a thickness of 8-
On 10 centimetres of river sand, further bedding takes and sprouts after culture 40-50 days with a thickness of the river sand of 5-8cm on the arrowhead bulb
The stolon of hair.
4. the method according to claim 3 for obtaining sterile arrowhead explant, which is characterized in that in step (1), described
On arrowhead bulb after bedding river sand, it is also necessary to carry out disinfection to the river sand of bedding.
5. the method according to claim 4 for obtaining sterile arrowhead explant, which is characterized in that the river sand of bedding is with warp
800 times of diluted 50% wettable powders of carbendazim are crossed to carry out disinfection processing.
6. the method according to claim 1 for obtaining sterile arrowhead explant, which is characterized in that in step (2), acquisition is crawled
After the stem apex of crawl stem, it is also necessary to the stem apex of the stolon is put into washing powder water and carry out immersion 25-35min, use tap water
2-3 is rinsed after, and then clip goes out the first stem section on the stem apex, it is preferred that the mass percentage concentration of the washing powder water
For 0.2%-0.5%.
7. the method according to claim 1 for obtaining sterile arrowhead explant, which is characterized in that in step (2), the first stem
The length of section is 2-3cm.
8. the method according to claim 1 for obtaining sterile arrowhead explant, which is characterized in that in step (3), described the
The diameter of two stem sections is 3-5mm, length 5-10mm.
9. the method according to claim 1 for obtaining sterile arrowhead explant, which is characterized in that, will be described in step (3)
After second stem section, 75% alcohol submergence, 10s-30s is jiggled, pours out alcohol, and then with aseptic water washing 3-5 times, then
10-15min is sterilized with 0.1% mercury chloride or 2% sodium hypochlorite impregnates 8-10min, is then used aseptic water washing 3-5 times.
10. application of the method according to claim 1 for obtaining sterile arrowhead explant in Plant Tissue Breeding.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201810297303.2A CN108812305A (en) | 2018-04-04 | 2018-04-04 | Obtain the method and its application of sterile arrowhead explant |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201810297303.2A CN108812305A (en) | 2018-04-04 | 2018-04-04 | Obtain the method and its application of sterile arrowhead explant |
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| CN201810297303.2A Pending CN108812305A (en) | 2018-04-04 | 2018-04-04 | Obtain the method and its application of sterile arrowhead explant |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117158321A (en) * | 2023-10-20 | 2023-12-05 | 江苏北环生物科技有限公司 | Tissue culture propagation-expanding explant treatment and disinfection method for fritillaria thunbergii |
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| CN103070073A (en) * | 2013-01-22 | 2013-05-01 | 武汉市蔬菜科学研究所 | Method for fast propagation of test tube arrowhead by tissue culture |
| CN104542277A (en) * | 2014-12-19 | 2015-04-29 | 广西壮族自治区农业科学院生物技术研究所 | Rapid propagation method for sagittaria sagittifolia tissue culture seedling and culture medium |
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2018
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| CN103070073A (en) * | 2013-01-22 | 2013-05-01 | 武汉市蔬菜科学研究所 | Method for fast propagation of test tube arrowhead by tissue culture |
| CN104542277A (en) * | 2014-12-19 | 2015-04-29 | 广西壮族自治区农业科学院生物技术研究所 | Rapid propagation method for sagittaria sagittifolia tissue culture seedling and culture medium |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117158321A (en) * | 2023-10-20 | 2023-12-05 | 江苏北环生物科技有限公司 | Tissue culture propagation-expanding explant treatment and disinfection method for fritillaria thunbergii |
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Application publication date: 20181116 |