CN106973790A - A kind of method of sweet potato stem tip cultured in vitro and tissue-cultured seedling Viral diagnosis - Google Patents
A kind of method of sweet potato stem tip cultured in vitro and tissue-cultured seedling Viral diagnosis Download PDFInfo
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- CN106973790A CN106973790A CN201710213822.1A CN201710213822A CN106973790A CN 106973790 A CN106973790 A CN 106973790A CN 201710213822 A CN201710213822 A CN 201710213822A CN 106973790 A CN106973790 A CN 106973790A
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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
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- 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
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Abstract
The present invention is a kind of sweet potato stem tip cultured in vitro and the method for tissue-cultured seedling Viral diagnosis, is concretely comprised the following steps：(1) it is acquired for materials with sterilizing：In the vigorous sweet potato seedling terminal bud of clip field growing in June, cut blade, running water is rinsed, and uses aseptic filter paper suck dry moisture, be placed on superclean bench sterilize it is standby；(2) stem apex is peeled off and culture：Under binocular anatomical lens, the sweet potato terminal bud after sterilization is peelled off into outer blade with syringe needle point, stem apex is seeded in Initial culture base and cultivated, tissue-cultured seedling is obtained；(3) tissue-cultured seedling Viral diagnosis：Connection is stained with using indicator plant acupuncture juice and carries out Viral diagnosis, the malicious seedling of band is eliminated according to testing result, retains detoxic seedling.The invention provides a kind of sweet potato stem tip cultured in vitro and the method for tissue-cultured seedling Viral diagnosis, Sweetpotato Viruses Elimination success rate is improved, and the Viral diagnosis degree of accuracy is high, and a kind of effective detection means is provided to preventing and treating Virus Diseases of Sweet Potato.
Description
Technical field
The present invention relates to sweet potato tissue-cultured seedling field of virus detection, more particularly to a kind of sweet potato stem tip cultured in vitro and tissue-cultured seedling
The method of Viral diagnosis.
Background technology
Sweet potato is asexually propagated crop, and field planting is easily by pinniform mottle virus (SPFMV), Cauliflower Mosaic shape virus
(SPCLV), cryptovirus (SPLV) and tobacco mosaic virus (TMV) (TMV) infect, and cause kind of a sexual involution, resistance reduction, yield and
Quality decline.China majority growing areas field plant viral infection rate be 60%-70%, have even up to 90%, lead every year
Sweet potato underproduction amplitude is caused up to 29%.The kind and thoroughly immune method to viral complete resistance are there is no at present, also without specific drug
Agent, is still the basic side for preventing and treating Virus Diseases of Sweet Potato, recovering good strains of seeds, improving the yield and quality using Shoot-tip Grafting In Vitro
Method.Therefore cultured in vitro and the Viral diagnosis to tissue-cultured seedling are carried out to sweet potato stem tip turns into the research weight of preventing and treating sweet potato viruses
Point.
The content of the invention
Present invention seek to address that the deficiencies in the prior art, and a kind of sweet potato stem tip cultured in vitro and tissue-cultured seedling virus inspection are provided
The method of survey.
The present invention to achieve the above object, using following technical scheme：
A kind of method of sweet potato stem tip cultured in vitro and tissue-cultured seedling Viral diagnosis, is concretely comprised the following steps：
(1) it is acquired for materials with sterilizing
In the vigorous sweet potato seedling terminal bud of clip field growing in June, blade is cut, running water rinses 40-50min, with nothing
Bacterium filter paper suck dry moisture, is placed on superclean bench and embathes 25s with 75% alcohol, then sterilizes 5min with 0.1% mercuric chloride, finally uses
Aseptic water washing 7-8 times, it is standby；
(2) stem apex is peeled off and culture
Under binocular anatomical lens, the sweet potato terminal bud after sterilization is peelled off into outer blade with syringe needle point, 1-2 leaf is only stayed
Former base, 5-6 size is seeded in Initial culture base for 0.1-0.2mm stem apex and cultivated, sucrose is in culture medium
30g/L, agar is 6.0g/L, and pH is 5.8, and cultivation temperature is 25 DEG C, intensity of illumination 2000-3000lx, light application time 12h/d,
Culture 40 days, obtains tissue-cultured seedling；
(3) tissue-cultured seedling Viral diagnosis
Connection is stained with using indicator plant acupuncture juice and carries out Viral diagnosis：On the nascent healthy leaf of Brazilian morning-glory, with sterilizing
Syringe acanthopore, is then bundled together extrusion juice by Sweet Potato Leaf to be measured, petiole and young stem, is stained with and is connected on pinprick,
And be equally inoculated with using aqua sterilisa and known band Solanum carolinense liquid as negative control and positive control, it is placed in shade in 30 DEG C of insect-proof net chambers
Moisturizing culture；20 days Continuous Observations record symptom after inoculation, according to indicator plant symptom, in all indicator plants be stained with and connect only
There is one plant of performance classical symptom, the sample is the positive；The malicious seedling of band is eliminated according to testing result, retains detoxic seedling.
It is MS+6-BA 1.0mg/L+NAA 0.1mg/L that stem apex, which is peeled off with Initial culture base in incubation,.
The classical symptom of the indicator plant performance connect is stained with tissue-cultured seedling virus detection procedure bright for Newborn Leaves appearance systematicness
Arteries and veins, Huang Mai, develop into floral leaf or shrinkage later.
The beneficial effects of the invention are as follows：The invention provides a kind of sweet potato stem tip cultured in vitro and tissue-cultured seedling Viral diagnosis
Method, Sweetpotato Viruses Elimination success rate is improved, and the Viral diagnosis degree of accuracy is high, and a kind of effective detection is provided to preventing and treating Virus Diseases of Sweet Potato
Means.
Embodiment
With reference to specific embodiment, the invention will be further described：
Embodiment 1：
A kind of method of sweet potato stem tip cultured in vitro and tissue-cultured seedling Viral diagnosis, is concretely comprised the following steps：
(1) it is acquired for materials with sterilizing
In the vigorous sweet potato seedling terminal bud of clip field growing in June, blade is cut, running water rinses 40min, with sterile filter
Paper suck dry moisture, is placed on superclean bench and embathes 25s with 75% alcohol, then sterilizes 5min with 0.1% mercuric chloride, finally with sterile
Water is rinsed 7 times, standby；
(2) stem apex is peeled off and culture
Under binocular anatomical lens, the sweet potato terminal bud after sterilization is peelled off into outer blade with syringe needle point, 1 phyllogen is only stayed
Base, 5 sizes are seeded in Initial culture base for 0.1mm stem apex and cultivated, and sucrose is 30g/L, agar in culture medium
For 6.0g/L, pH is 5.8, and cultivation temperature is 25 DEG C, and intensity of illumination 2000lx, light application time 12h/d cultivate 40 days, obtain group
Train seedling；
(3) tissue-cultured seedling Viral diagnosis
Connection is stained with using indicator plant acupuncture juice and carries out Viral diagnosis：On the nascent healthy leaf of Brazilian morning-glory, with sterilizing
Syringe acanthopore, is then bundled together extrusion juice by Sweet Potato Leaf to be measured, petiole and young stem, is stained with and is connected on pinprick,
And be equally inoculated with using aqua sterilisa and known band Solanum carolinense liquid as negative control and positive control, it is placed in shade in 30 DEG C of insect-proof net chambers
Moisturizing culture；20 days Continuous Observations record symptom after inoculation, according to indicator plant symptom, in all indicator plants be stained with and connect only
There is one plant of performance classical symptom, the sample is the positive；The malicious seedling of band is eliminated according to testing result, retains detoxic seedling.
It is MS+6-BA 1.0mg/L+NAA 0.1mg/L that stem apex, which is peeled off with Initial culture base in incubation,.
The classical symptom of the indicator plant performance connect is stained with tissue-cultured seedling virus detection procedure bright for Newborn Leaves appearance systematicness
Arteries and veins, Huang Mai, develop into floral leaf later.
Embodiment 2：
A kind of method of sweet potato stem tip cultured in vitro and tissue-cultured seedling Viral diagnosis, is concretely comprised the following steps：
(1) it is acquired for materials with sterilizing
In the vigorous sweet potato seedling terminal bud of clip field growing in June, blade is cut, running water rinses 50min, with sterile filter
Paper suck dry moisture, is placed on superclean bench and embathes 25s with 75% alcohol, then sterilizes 5min with 0.1% mercuric chloride, finally with sterile
Water is rinsed 8 times, standby；
(2) stem apex is peeled off and culture
Under binocular anatomical lens, the sweet potato terminal bud after sterilization is peelled off into outer blade with syringe needle point, 2 phyllogens are only stayed
Base, 6 sizes are seeded in Initial culture base for 0.2mm stem apex and cultivated, and sucrose is 30g/L, agar in culture medium
For 6.0g/L, pH is 5.8, and cultivation temperature is 25 DEG C, and intensity of illumination 3000lx, light application time 12h/d cultivate 40 days, obtain group
Train seedling；
(3) tissue-cultured seedling Viral diagnosis
Connection is stained with using indicator plant acupuncture juice and carries out Viral diagnosis：On the nascent healthy leaf of Brazilian morning-glory, with sterilizing
Syringe acanthopore, is then bundled together extrusion juice by Sweet Potato Leaf to be measured, petiole and young stem, is stained with and is connected on pinprick,
And be equally inoculated with using aqua sterilisa and known band Solanum carolinense liquid as negative control and positive control, it is placed in shade in 30 DEG C of insect-proof net chambers
Moisturizing culture；20 days Continuous Observations record symptom after inoculation, according to indicator plant symptom, in all indicator plants be stained with and connect only
There is one plant of performance classical symptom, the sample is the positive；The malicious seedling of band is eliminated according to testing result, retains detoxic seedling.
It is MS+6-BA 1.0mg/L+NAA 0.1mg/L that stem apex, which is peeled off with Initial culture base in incubation,.
The classical symptom of the indicator plant performance connect is stained with tissue-cultured seedling virus detection procedure bright for Newborn Leaves appearance systematicness
Arteries and veins, Huang Mai, develop into shrinkage later.
Embodiment 3：
A kind of method of sweet potato stem tip cultured in vitro and tissue-cultured seedling Viral diagnosis, is concretely comprised the following steps：
(1) it is acquired for materials with sterilizing
In the vigorous sweet potato seedling terminal bud of clip field growing in June, blade is cut, running water rinses 45min, with sterile filter
Paper suck dry moisture, is placed on superclean bench and embathes 25s with 75% alcohol, then sterilizes 5min with 0.1% mercuric chloride, finally with sterile
Water is rinsed 8 times, standby；
(2) stem apex is peeled off and culture
Under binocular anatomical lens, the sweet potato terminal bud after sterilization is peelled off into outer blade with syringe needle point, 1 phyllogen is only stayed
Base, 6 sizes are seeded in Initial culture base for 0.2mm stem apex and cultivated, and sucrose is 30g/L, agar in culture medium
For 6.0g/L, pH is 5.8, and cultivation temperature is 25 DEG C, and intensity of illumination 2500lx, light application time 12h/d cultivate 40 days, obtain group
Train seedling；
(3) tissue-cultured seedling Viral diagnosis
Connection is stained with using indicator plant acupuncture juice and carries out Viral diagnosis：On the nascent healthy leaf of Brazilian morning-glory, with sterilizing
Syringe acanthopore, is then bundled together extrusion juice by Sweet Potato Leaf to be measured, petiole and young stem, is stained with and is connected on pinprick,
And be equally inoculated with using aqua sterilisa and known band Solanum carolinense liquid as negative control and positive control, it is placed in shade in 30 DEG C of insect-proof net chambers
Moisturizing culture；20 days Continuous Observations record symptom after inoculation, according to indicator plant symptom, in all indicator plants be stained with and connect only
There is one plant of performance classical symptom, the sample is the positive；The malicious seedling of band is eliminated according to testing result, retains detoxic seedling.
It is MS+6-BA 1.0mg/L+NAA 0.1mg/L that stem apex, which is peeled off with Initial culture base in incubation,.
The classical symptom of the indicator plant performance connect is stained with tissue-cultured seedling virus detection procedure bright for Newborn Leaves appearance systematicness
Arteries and veins, Huang Mai, develop into floral leaf later.
The invention provides a kind of sweet potato stem tip cultured in vitro and the method for tissue-cultured seedling Viral diagnosis, Sweetpotato Viruses Elimination success rate
Improve, the Viral diagnosis degree of accuracy is high, a kind of effective detection means is provided to preventing and treating Virus Diseases of Sweet Potato.
The present invention is exemplarily described above in conjunction with specific embodiment, it is clear that the present invention is implemented not by upper
The limitation of mode is stated, if the various improvement of inventive concept and technical scheme of the present invention progress are employed, or it is not improved straight
Scoop out for other occasions, within protection scope of the present invention.
Claims (3)
1. a kind of method of sweet potato stem tip cultured in vitro and tissue-cultured seedling Viral diagnosis, it is characterised in that concretely comprise the following steps：
(1) it is acquired for materials with sterilizing
In the vigorous sweet potato seedling terminal bud of clip field growing in June, blade is cut, running water rinses 40-50min, with sterile filter
Paper suck dry moisture, is placed on superclean bench and embathes 25s with 75% alcohol, then sterilizes 5min with 0.1% mercuric chloride, finally with sterile
Water is rinsed 7-8 times, standby；
(2) stem apex is peeled off and culture
Under binocular anatomical lens, the sweet potato terminal bud after sterilization is peelled off into outer blade with syringe needle point, 1-2 phyllogen is only stayed
Base, 5-6 size is seeded in Initial culture base for 0.1-0.2mm stem apex and cultivated, and sucrose is 30g/ in culture medium
L, agar is 6.0g/L, and pH is 5.8, and cultivation temperature is 25 DEG C, intensity of illumination 2000-3000lx, light application time 12h/d, culture
40 days, obtain tissue-cultured seedling；
(3) tissue-cultured seedling Viral diagnosis
Connection is stained with using indicator plant acupuncture juice and carries out Viral diagnosis：On the nascent healthy leaf of Brazilian morning-glory, sterile injection is used
Device acanthopore, is then bundled together extrusion juice by Sweet Potato Leaf to be measured, petiole and young stem, is stained with and is connected on pinprick, and with
Aqua sterilisa and known band Solanum carolinense liquid are equally inoculated with as negative control and positive control, are placed in shade moisturizing in 30 DEG C of insect-proof net chambers
Culture；20 days Continuous Observations record symptom after inoculation, according to indicator plant symptom, as long as having in all indicator plants be stained with and connect
One plant of performance classical symptom, the sample is the positive；The malicious seedling of band is eliminated according to testing result, retains detoxic seedling.
2. the method for a kind of sweet potato stem tip cultured in vitro according to claim 1 and tissue-cultured seedling Viral diagnosis, its feature exists
In it is MS+6-BA 1.0mg/L+NAA0.1mg/L that stem apex, which is peeled off with Initial culture base in incubation,.
3. the method for a kind of sweet potato stem tip cultured in vitro according to claim 2 and tissue-cultured seedling Viral diagnosis, its feature exists
In the classical symptom that the indicator plant performance connect is stained with tissue-cultured seedling virus detection procedure systematicness Ming Mai, Huang occurs for Newborn Leaves
Arteries and veins, develops into floral leaf or shrinkage later.
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| CN201710213822.1A CN106973790A (en) | 2017-03-29 | 2017-03-29 | A kind of method of sweet potato stem tip cultured in vitro and tissue-cultured seedling Viral diagnosis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108552059A (en) * | 2018-06-06 | 2018-09-21 | 武汉叶动力生物科技有限公司 | A kind of method for plant tissue culture promoting potato seedling strengthening root |
| CN115644063A (en) * | 2022-11-11 | 2023-01-31 | 青岛农业大学 | Rapid propagation method of virus-free raw seedling of sweet potato |
| CN116114599A (en) * | 2022-12-01 | 2023-05-16 | 湖南省棉花科学研究所 | Method and device for rapidly cutting sweet potato detoxification tissue culture stem tip |
-
2017
- 2017-03-29 CN CN201710213822.1A patent/CN106973790A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108552059A (en) * | 2018-06-06 | 2018-09-21 | 武汉叶动力生物科技有限公司 | A kind of method for plant tissue culture promoting potato seedling strengthening root |
| CN115644063A (en) * | 2022-11-11 | 2023-01-31 | 青岛农业大学 | Rapid propagation method of virus-free raw seedling of sweet potato |
| CN116114599A (en) * | 2022-12-01 | 2023-05-16 | 湖南省棉花科学研究所 | Method and device for rapidly cutting sweet potato detoxification tissue culture stem tip |
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Application publication date: 20170725 |