CN106035083B - A kind of tissue culture method of purple-colored potato rapid detoxification - Google Patents
A kind of tissue culture method of purple-colored potato rapid detoxification Download PDFInfo
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- CN106035083B CN106035083B CN201610381811.XA CN201610381811A CN106035083B CN 106035083 B CN106035083 B CN 106035083B CN 201610381811 A CN201610381811 A CN 201610381811A CN 106035083 B CN106035083 B CN 106035083B
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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
- A01H4/001—Culture apparatus for tissue culture
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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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention provides a kind of tissue culture method of purple-colored potato rapid detoxification, comprise the following steps：1) leaf stem section of purple-colored potato is cut；2) by the leaf stem section cut carry out successively sterile water wash, alcohol instantaneously sterilize, medicining liquid dipping sterilization and aseptic water washing, the leaf stem section after being sterilized；3) leaf stem section after the sterilization is placed in superclean bench, peels off the stem apex on leaf stem section top under the microscope, stem apex inoculation is then subjected to primary structure culture in the medium, obtains a test tube seedling；4) stem apex of a test tube seedling is peeled off, secondary structure culture is carried out, obtains secondary test tube seedling；5) repeating said steps 4) stripping and tissue cultures, the number of the repetition be 3~5 times, obtain the test tube seedling of detoxification.The virus elimination rate of the purple-colored potato of the method for the invention culture is up to 93%, and the breeding cycle foreshortens to 2~4 months, and the batch that purple-colored potato can be achieved quickly is bred, and reduces toxigenic capacity.
Description
Technical field
The present invention relates to the technical field of tissue culture of plant, more particularly to a kind of tissue culture of purple-colored potato rapid detoxification
Method.
Background technology
Purple-colored potato, potato shape are in oblong, and eye is smaller, and pulp is darkviolet, rich in natural pigments such as anthocyanidin.
Purple-colored potato is goodlooking, and color temptation is strong, and any pigment and toner need not be added in product processing, extremely strong
Health.
Purple-colored potato original producton location is the states such as South America Peru, at present the domestic existing unit in China, personal introduction purple Ma Ling
Some kinds of potato.It is different after plantation but because different purple-colored potatos are in planting process, that introduces a fine variety is regional different
The kind in area unanimously carries out planting experiment in same plot, area, quantity, monomer weight, and very big difference occurs in each kind.
First, plant strain growth fails, compound leaf atrophy；Second, plant type becomes short, blade face shrinkage；Third, there is yellowish green alternate spot, leaf in blade
Arteries and veins necrosis, compound leaf come off, and also part introduced variety yield when planting first time is high, second kind of plant yield after reservation kind
Low, stem tuber is small to degenerate seriously；Purple-colored potato can not be steady in a long-term after introducing a fine variety growth, the offspring especially to reserve seed for planting can not keep
The original performance of plant and yield.Trace it to its cause be after introducing a fine variety planting site plant during, invaded by germ, virus etc.
Dye, but unripe effective purple-colored potato virus-free culture method in the prior art.
The content of the invention
In view of this, it is an object of the invention to provide a kind of tissue culture method of purple-colored potato rapid detoxification.
In order to realize foregoing invention purpose, the present invention provides following technical scheme：
The invention provides a kind of tissue culture method of purple-colored potato rapid detoxification, comprise the following steps：
1) bud of purple-colored potato is cut；
2) by the tender shoots cut carry out successively sterile water wash, alcohol instantaneously sterilize, medicining liquid dipping sterilization and nothing
Bacterium water rinses, the leaf stem section after being sterilized；
3) leaf stem section after the sterilization is placed in superclean bench, peels off the stem apex on leaf stem section top under the microscope,
Then stem apex inoculation is subjected to primary structure culture in the medium, obtains a test tube seedling；
4) stem apex of a test tube seedling is peeled off, secondary structure culture is carried out, obtains secondary test tube seedling；
5) repeating said steps 4) stripping and tissue cultures, the number of the repetition be 3~5 times, obtain the examination of detoxification
Guan Miao.
Preferably, the stem apex after the stripping described in step 1) carries 1 phyllopodium.
Preferably, the time of the sterile water wash described in step 2) is 10~20min.
Preferably, the thimerosal described in step 2) be aqueous sodium hypochlorite solution that mass fraction is 5.5%~6.5% or
Mass fraction is 1.2~2.5% mercury solution.
Preferably, the time that the alcohol described in step 2) instantaneously sterilizes is 2~4s, and the volumetric concentration of the alcohol is 70
~80%.
Preferably, the time of the medicining liquid dipping sterilization described in step 2) is 15~25min.
Preferably, the time of aseptic water washing described in step 2) is 3~5min.
Preferably, the culture medium of step 3) and tissue cultures described in step 4) is the MS culture mediums of improvement, described
The MS culture mediums of improvement are the components that following final concentration is with the addition of in MS culture mediums：BA, 0.04~0.06mg/L, NAA, 0.18
~0.22mg/L, 24-D, 0.4~0.6mg/L, sucrose 30wt% and potato milling liquid 20wt%.
Preferably, step 3) and the intensity of illumination of tissue cultures described in step 4) are 2800~3000Lx.
Preferably, step 3) and the temperature of tissue cultures described in step 4) are 22~25 DEG C
Beneficial effects of the present invention：The method of the invention by peeling off newest on purple-colored potato stem apex grow repeatedly
Phyllopodium, reduce original seed and take viruliferous probability, then by rapid tissue culture, so as to improve purple-colored potato test tube seedling
Virus elimination rate, the further stability for improving purple-colored potato test tube seedling；The purple-colored potato of the method for the invention culture
Virus elimination rate be up to 93%, the breeding cycle foreshortens to 2~4 months, can be achieved purple-colored potato batch quickly breed, reduce training
This is formed, compared with traditional purple-colored potato cultural method, yield increases the purple-colored potato of method culture of the present invention
Add 20%.
Embodiment
The invention provides a kind of tissue culture method of purple-colored potato rapid detoxification, comprise the following steps：
1) bud of purple-colored potato is cut；2) that the tender shoots cut is carried out into sterile water wash, alcohol successively is instantaneous
Sterilization, medicining liquid dipping sterilization and aseptic water washing, the leaf stem section after being sterilized；3) leaf stem section after the sterilization is placed in
Superclean bench, the stem apex on leaf stem section top is peeled off under the microscope, stem apex inoculation is then subjected to once group in the medium
Culture is knitted, obtains a test tube seedling；4) stem apex of a test tube seedling is peeled off, secondary structure culture is carried out, obtains secondary examination
Guan Miao；5) repeating said steps 4) stripping and tissue cultures, the number of the repetition be 3~5 times, obtain the test tube of detoxification
Seedling.
Heretofore described purple-colored potato is preferably the purple-colored potato introduced from foreign countries or domestic seed selection
Purple-colored potato, specific kind are preferably：Yun Site (seven color potato of Yunnan), black beauty, NCC, ruby, purple
Cloud 1 and red potato series S03-2744.
Before stem apex of the present invention on the potato tender shoots to being adopted is peeled off, preferably potato tender shoots is carried out
Pre-sterilization processing, the processing of described pre-sterilization are preferably：By the potato potato wedge with bud as volume fraction be 70~80%
Alcohol in soak 4~6min；Volume fraction is used to be wiped for 70~80% alcohol potato potato wedge surface after drying
Wipe sterilization.The present invention is in order to obtain more preferable great-hearted stem apex material, using above-mentioned more gentle pre-sterilization processing means.
The stem apex that heretofore described stem apex uses in peeling off be preferably from the length of top cutting for 0.2~
0.3mm stem apex, the stem apex preferably carry 1~2 phyllopodium；It is currently preferred that stem apex stripping is carried out to described bud
From so that the stem apex after stripping carries 1 phyllopodium.In the present invention, the concrete operations of described stripping are preferably：By purple
Color potato tender shoots is positioned under 40 times of anatomical lens, is shelled 1~2 phyllopodium position of stem apex point with dissecting needle or scalpel
Cut-out retains 1 phyllopodium, the stem apex after being peeled off.
The purple-colored potato tender shoots that the present invention is cut, the purple-colored potato tender shoots cut is carried out successively sterile
Water cleaning, alcohol instantaneously sterilize, medicining liquid dipping sterilizes and aseptic water washing, the tender shoots after being sterilized.Because tender shoots is smaller
And quantity is more, present invention preferably uses the purple-colored potato tender shoots that gauze wrapped is cut, and then the tender shoots of parcel is carried out
Sterile water wash；With gauze wrapped cleaning of getting up tender shoots will not be caused to be broken up, avoid the waste of material.In the present invention, institute
The time for the sterile water wash stated is preferably 10~20min, more preferably 15min.
After completing sterile water wash, it is instantaneous that the present invention carries out alcohol to the purple-colored potato tender shoots after the sterile water wash
Sterilization.In the present invention, the time that the alcohol instantaneously sterilizes is preferably 2~4s, more preferably 3s；The body of the alcohol
Product concentration is preferably 85~98%, more preferably 88~97%, most preferably 95%.
After the alcohol for completing purple-colored potato tender shoots instantaneously sterilizes, the liquid that carried out disinfection to tender shoots immersion disappears the present invention
Poison.It is 5.5%~6.5% aqueous sodium hypochlorite solution or mass fraction that heretofore described thimerosal, which is preferably mass fraction,
For 1.2~2.5% mercury solutions；More preferably mass fraction is 5.8~6.2% aqueous sodium hypochlorite solutions or mass fraction is
1.5~2.2% mercury solution, most preferably mass fraction are 6.0% aqueous sodium hypochlorite solution or mass fraction is 2.0%
Mercury solution.In the present invention, the time of the medicining liquid dipping sterilization is preferably 15~25min, more preferably 17~
23min, more preferably 20min.
The present invention carries out aseptic water washing after soak soaking disinfection stem apex, to tender shoots.In the present invention, it is described sterile
The time that water rinses is preferably 3~5min, more preferably 4min.
After tender shoots completes aseptic water washing, tender shoots is preferably put on the blotting paper after sterilization and absorbed water by the present invention.
In the present invention, the time of the water suction is preferably 0.5~1.5min, more preferably 1min.The present invention is to described sterile
The source of water can be specifically homemade aqua sterilisa or commercially available using the conventional sterilized water in this area without particular/special requirement
Sterilized water.
The present invention be sterilized after tender shoots, the tender shoots after sterilization is placed in superclean bench, then carried out again under microscope
Stem apex is peeled off, and stem apex is inoculated in progress primary structure culture in culture medium, obtains a test tube seedling.In the present invention, institute
The intensity of illumination for stating primary structure culture is preferably 2800~3000Lx；More preferably 2850~2950Lx, most preferably
For 2900Lx.In the present invention, the temperature of the primary structure culture is preferably 22~25 DEG C, more preferably 23~24 DEG C；
The time of the primary structure culture is preferably 10~25 days, more preferably 15~22 days, most preferably 20 days.
The MS culture mediums that culture medium used in heretofore described primary structure culture is preferably improved, described improvement
MS culture mediums be the component that following final concentration is with the addition of in MS culture mediums：BA 0.04~0.06mg/L, NAA 0.18~
0.4~0.6mg/L of 0.22mg/L, 24-D, sucrose 30wt% and potato milling liquid 20wt%.
The present invention peels off the stem apex of a test tube seedling after a test tube seedling is obtained, and carries out secondary structure culture, obtains
To secondary test tube seedling；The method that the stem apex of a test tube seedling is peeled off in the present invention is identical with the method that above-mentioned stem apex is peeled off,
The method of secondary structure culture is identical with the method for primary structure culture, and the parameter of concrete operations can be in the range of above-mentioned restriction
Independent selection, does not repeat to repeat herein.
The present invention repeats above-mentioned stripping and tissue cultures operation, the number of the repetition after secondary test tube seedling is obtained
Preferably 3~5 times, more preferably 4 times, the stripping repeated each time and the concrete operations parameter of tissue cultures can be upper
State and independently selected in the range of limiting, repeat to peel off and obtain the test tube seedling of detoxification after tissue cultures.
The tissue culture method of purple-colored potato rapid detoxification provided by the invention is carried out specifically with reference to embodiment
It is bright, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
The black beauty potato potato wedge with stem apex of field plots is picked up from, the potato potato wedge with bud is placed in volume fraction
To soak 5min in 75% alcohol, taking-up is placed in ventilation and dried, and it is 75% alcohol to Ma Ling then to use volume fraction
Potato potato wedge surface carries out cleaning disinfection.2~3cm purple-colored potato tender shoots is cut, the tender shoots cut using gauze wrapped is existed successively
Filling and tender shoots 15min is cleaned in the pond of sterilized water, being placed in volume fraction is to sterilize 3s in 95% alcohol, be placed in mass fraction to be
20min is soaked in 6.0% aqueous sodium hypochlorite solution, with the blotting paper after aseptic water washing stem apex 4min, being put in after sterilizing
Absorb water 1min.2~3cm tender shoots is positioned under 40 times of anatomical lens, divides 1 phyllopodium position of stem apex to stripping with dissecting needle
It is disconnected to retain 1 phyllopodium, the stem apex after being peeled off.Then stem apex is connected to progress primary structure training in the MS culture mediums of improvement
Support, the composition of the MS culture mediums of described improvement is：MS culture mediums, add BA0.05mg/L, NAA0.2mg/L, 24-D
0.5mg/L, 30wt% sucrose, 20wt% potatos milling liquid.The intensity of illumination of primary structure culture is 2900Lx, temperature 24
℃；Time is 20 days.
After obtaining a test tube seedling, the stem apex of a test tube seedling is peeled off, under conditions of above-mentioned tissue cultures successively
Secondary structure culture is carried out, obtains secondary test tube seedling；Then peel off secondary test tube seedling stem apex carry out tissue cultures three times again after
The test tube seedling of detoxification is obtained, the time that the tissue cultures three times finally give detoxification test tube plantlet is 2 months.
In the present embodiment, the growth of the detoxification test tube plantlet survived and detoxification situation are shown in Table 1.
The growth for the detoxification test tube plantlet survived that the embodiment of the present invention 1 of table 1 obtains and detoxification situation
As can be seen from Table 1, the detoxicating cuvette shoot survival percent obtained in the present embodiment is 60%, growth is fast, virus elimination rate is high
Up to 93%.
Embodiment 2
No. 1 potato potato wedge of purple cloud with stem apex of field plots is picked up from, the potato potato wedge with bud is placed in volume integral
Number is then taken out and is placed in ventilation and dries to soak 5min in 75% alcohol, then use volume fraction for 75% alcohol
Cleaning disinfection is carried out to potato potato wedge surface.Cut 2~3cm purple-colored potato tender shoots, the tender shoots cut using gauze wrapped
Tender shoots 15min is cleaned in the pond for fill sterilized water successively, is placed in volume fraction to sterilize 3s in 95% alcohol, being placed in quality
Fraction be 6.0% aqueous sodium hypochlorite solution in soak 20min, with after aseptic water washing stem apex 4min, be put in sterilization after suction
Absorb water 1min on water paper.2~3cm tender shoots is positioned under 40 times of anatomical lens, with scalpel by 2 phyllopodium positions of stem apex
Divide stripping is disconnected to retain 1 phyllopodium, the stem apex after being peeled off.Then stem apex is connected in the MS culture mediums of improvement and carried out once
Tissue cultures, the composition of the MS culture mediums of described improvement are：MS culture mediums, add BA0.05mg/L, NAA0.2mg/L, 24-D
0.5mg/L, 30wt% sucrose, 20wt% potatos milling liquid.The intensity of illumination of primary structure culture is 2950Lx, temperature 22
℃；Time is 23 days.
After obtaining a test tube seedling, the stem apex of a test tube seedling is peeled off, secondary structure culture is carried out, obtains secondary examination
Guan Miao；Then the stem apex for peeling off secondary test tube seedling carries out tissue cultures three times again, repeat point peel off and tissue cultures 4 times after obtain
The test tube seedling of detoxification, the time that 4 tissue cultures finally give detoxification test tube plantlet are 3 months.In the present embodiment, survive
The growth of detoxification test tube plantlet and detoxification situation are shown in Table 2.
Table 2
| Stem apex number | 4~6 leaf test tube seedling numbers | The speed of growth | Survival rate (%) | Virus elimination rate (%) |
| 20 | 10 | Quickly | 50 | 92 |
As can be seen from Table 2, the detoxicating cuvette shoot survival percent obtained in the present embodiment is 50%, growth is fast, virus elimination rate is high
Up to 92%.
Embodiment 3
The NCC potato potato wedge with stem apex of field plots is picked up from, by the potato potato wedge with bud as volume fraction
To soak 3min in 75% alcohol, then take out and be placed in ventilation and dry, then use volume fraction for 75% alcohol pair
Potato potato wedge surface carries out cleaning disinfection.Cut 2~3cm purple-colored potato tender shoots, using the tender shoots that gauze wrapped is cut according to
It is secondary that tender shoots 15min is cleaned in the pond for fill sterilized water, is placed in volume fraction to sterilize 3s in 95% alcohol, being placed in quality point
Number for 6.0% aqueous sodium hypochlorite solutions in soak 20min, with after aseptic water washing stem apex 4min, be put in sterilization after water suction
Absorb water 1min on paper.2~3cm tender shoots is positioned under 40 times of anatomical lens, divided 2 phyllopodium positions of stem apex with dissecting needle
Stripping is disconnected to retain 1 phyllopodium, the stem apex after being peeled off.Then stem apex is connected in the MS culture mediums of improvement and carries out once group
Culture is knitted, the composition of the MS culture mediums of described improvement is：MS culture mediums, add BA0.05mg/L, NAA0.2mg/L, 24-D
0.5mg/L, 30wt% sucrose, 20wt% potatos milling liquid.The intensity of illumination of primary structure culture is 2850Lx, temperature 24
℃；Time is 18 days.
After obtaining a test tube seedling, the stem apex of a test tube seedling is peeled off, secondary structure culture is carried out, obtains secondary examination
Guan Miao；Then the stem apex for peeling off secondary test tube seedling carries out tissue cultures three times again, repeat stem apex peel off and tissue cultures 5 times after
To the test tube seedling of detoxification, the incubation time of the detoxification test tube plantlet is 3 months.
In the present embodiment, the growth of the detoxification test tube plantlet survived and detoxification situation are shown in Table 3.
Table 3
| Stem apex number | 4~6 leaf test tube seedling numbers | The speed of growth | Survival rate (%) | Virus elimination rate (%) |
| 20 | 9 | Comparatively fast | 45 | 91 |
As can be seen from Table 3, the detoxicating cuvette shoot survival percent obtained in the present embodiment is 45%, growth is fast, virus elimination rate is high
Up to 91%.
Embodiment 4
The black beauty potato potato wedge with stem apex of field plots is picked up from, the potato potato wedge of bud is placed in into volume fraction is
Soak 5min in 75% alcohol, taking-up is placed in ventilation and dried, and it is 75% alcohol to potato then to use volume fraction
Potato wedge surface carries out cleaning disinfection.2~3cm purple-colored potato tender shoots is cut, the tender shoots cut using gauze wrapped is being filled successively
It is to sterilize 3s in 95% alcohol, be placed in mass fraction to be that tender shoots 15min is cleaned in the pond of full sterilized water, is placed in volume fraction
20min is soaked in 6.0% aqueous sodium hypochlorite solution, with the blotting paper after aseptic water washing stem apex 4min, being put in after sterilizing
Absorb water 1min.2~3cm tender shoots is positioned under 40 times of anatomical lens, divides 1 phyllopodium position of stem apex to stripping with dissecting needle
It is disconnected to retain 1 phyllopodium, the stem apex after being peeled off.Then stem apex is connected to progress primary structure training in the MS culture mediums of improvement
Support, the composition of the MS culture mediums of described improvement is：MS culture mediums, add BA0.05mg/L, NAA0.2mg/L, 24-D
0.5mg/L, 30wt% sucrose, 20wt% potatos milling liquid.The intensity of illumination of primary structure culture is 2900Lx, temperature 24
℃；Time is 20 days.
After obtaining a test tube seedling, the stem apex of a test tube seedling is peeled off, the stem apex of a test tube seedling is positioned over 40
Under anatomical lens again, divide stripping is disconnected to retain 1 phyllopodium, the stem after being peeled off at 1 phyllopodium position of stem apex with dissecting needle
Point.Then the stem apex of a described test tube seedling is connected to progress secondary structure culture in the MS culture mediums of improvement, described changes
The composition of good MS culture mediums is：MS culture mediums, add BA0.05mg/L, NAA0.2mg/L, 24-D 0.5mg/L, 30wt% sugarcanes
Sugar, 20wt% potatos milling liquid.The intensity of illumination of secondary structure culture is 2950Lx, and temperature is 22 DEG C；Time is 23 days, is obtained
To secondary test tube seedling；
After obtaining secondary test tube seedling, the stem apex of the secondary test tube seedling is peeled off, the stem apex of secondary test tube seedling is positioned over 40
Under anatomical lens again, divide stripping is disconnected to retain 1 phyllopodium, the stem after being peeled off at 1 phyllopodium position of stem apex with dissecting needle
Point.Then the stem apex of described secondary test tube seedling is connected in the MS culture mediums of improvement and carries out tissue cultures three times, described changes
The composition of good MS culture mediums is：MS culture mediums, add BA0.05mg/L, NAA0.2mg/L, 24-D 0.5mg/L, 30wt% sugarcanes
Sugar, 20wt% potatos milling liquid.The intensity of illumination of tissue cultures is 2950Lx three times, and temperature is 22 DEG C；Time is 19 days, is obtained
To test tube seedling three times；
In the present embodiment, the growth of the detoxification test tube plantlet survived and detoxification situation are shown in Table 4.
The growth for the detoxification test tube plantlet survived that the embodiment of the present invention 4 of table 4 obtains and detoxification situation
| Stem apex number | 4~6 leaf test tube seedling numbers | The speed of growth | Survival rate (%) | Virus elimination rate (%) |
| 20 | 12 | Quickly | 60 | 92 |
As can be seen from Table 4, the detoxicating cuvette shoot survival percent obtained in the present embodiment is 60%, growth is fast, virus elimination rate is high
Up to 92%.
Comparative example 1
Traditional introduces a fine variety method for planting, is directly separated the stem apex stem sharp for cultivating to obtain after pretreatment that carries out disinfection and survives
Situation and growing state are shown in Table 5.The culture medium that stem sharp culture uses is that MS culture mediums add BA0.05mg/L, IAA 1.0mg/
L, 2,4-D 0.5mg/L.
Table 5
| Stem apex number | 4~6 leaf test tube seedling numbers | The speed of growth | Survival rate (%) | Virus elimination rate (%) |
| 30 | 3 | Slowly | 10 | 33 |
As can be seen from Table 5, the detoxicating cuvette shoot survival percent obtained in comparative example is 10%, growth is slow, virus elimination rate is
33%
From above-described embodiment and comparative example, the survival rate of the purple-colored potato of the method for the invention culture is high, takes off
Malicious rate is up to 93%, and the breeding cycle foreshortens to 2~4 months, and survival rate, the speed of growth and virus elimination rate are all significantly larger than traditional draw
Kind method for planting, method of the present invention can realize that the batch of purple-colored potato is quickly bred, and reduce toxigenic capacity.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (1)
1. a kind of tissue culture method of purple-colored potato rapid detoxification, comprises the following steps：
1) tender shoots of purple-colored potato is cut, obtains the tender shoots cut；
2) by the tender shoots cut carry out successively sterile water wash, alcohol instantaneously sterilize, medicining liquid dipping sterilization and sterilized water
Rinse, the leaf stem section after being sterilized；
The time of described sterile water wash is 10~20min；
The time that described alcohol instantaneously sterilizes is 2~4s, and the volumetric concentration of the alcohol is 85~98%；
Described thimerosal is the aqueous sodium hypochlorite solution that mass fraction is 5.5%~6.5%；
The time of described medicining liquid dipping sterilization is 15~25min；
The time of the aseptic water washing is 3~5min；
3) leaf stem section after the sterilization is placed in superclean bench, peels off the stem apex on leaf stem section top under the microscope, obtained
Stem apex after stripping；Stem apex after described stripping carries 1 phyllopodium, then carries out stem apex inoculation once in the medium
Tissue cultures, obtain a test tube seedling；
4) stem apex of a test tube seedling is peeled off, secondary structure culture is carried out, obtains secondary test tube seedling；
5) repeating said steps 4) stripping and tissue cultures, the number of the repetition be 3~5 times, obtain the test tube seedling of detoxification；
The culture medium of tissue cultures described in step 3) and step 4) is the MS culture mediums of improvement, and the MS of described improvement is cultivated
Base is the component that following final concentration is with the addition of in MS culture mediums：BA0.04~0.06mg/L, NAA0.18~0.22mg/L, 2,
4-D0.4~0.6mg/L, sucrose 30wt% and potato milling liquid 20wt%；
The intensity of illumination of tissue cultures is 2800~3000Lx described in step 3) and step 4)；
The temperature of tissue cultures described in step 3) and step 4) is 22~25 DEG C；
The time of the primary structure culture is 10~25 days.
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| CN106718887B (en) * | 2016-11-30 | 2018-12-14 | 云南省农业科学院生物技术与种质资源研究所 | A kind of method of fast eliminating Potyvirus |
| CN107318656B (en) * | 2017-08-21 | 2019-12-03 | 山西省农业科学院作物科学研究所 | A kind of repetition culture poison-removing method of Potato Shoot-tips |
| CN107736244A (en) * | 2017-10-22 | 2018-02-27 | 威海市农业科学院 | Potato Shoot-tips peel off detoxifying fast breeding method |
| CN109042337A (en) * | 2018-09-30 | 2018-12-21 | 天津大学 | A method of utilizing potato leaf bud callus fast seedling growing |
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| CN104026009A (en) * | 2014-05-23 | 2014-09-10 | 甘肃省农业科学院生物技术研究所 | Purple potato test tube mini-tuber induction method |
| CN105191793A (en) * | 2015-09-10 | 2015-12-30 | 宁波市农业科学研究院 | Efficient breeding method of purple potato tissue culture seedlings |
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