CN106035083A - Tissue culture method for rapid detoxification of purple potatoes - Google Patents
Tissue culture method for rapid detoxification of purple potatoes Download PDFInfo
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- CN106035083A CN106035083A CN201610381811.XA CN201610381811A CN106035083A CN 106035083 A CN106035083 A CN 106035083A CN 201610381811 A CN201610381811 A CN 201610381811A CN 106035083 A CN106035083 A CN 106035083A
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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
Abstract
The invention provides a tissue culture method for rapid detoxification of purple potatoes. The method includes the following steps that 1, bud stems of the purple potatoes are cut; 2, the cut bud stems are subjected to sterile water cleaning, instantaneous alcohol disinfection, disinfectant soaking disinfection and sterile water washing in sequence, and disinfected bud stems are obtained; 3, the disinfected bud stems are placed on an ultra-clean bench, stem tips at the top ends of the bud stems are stripped under a microscope and then inoculated in a culture medium for primary tissue culture, and primary test-tube plantlets are obtained; 4, stem tips of the primary test-tube plantlets are stripped, secondary tissue culture is performed, and secondary test-tube plantlets are obtained; 5, stripping and tissue culture in the step 4 are repeatedly preformed 3-5 times, and detoxified test-tube plantlets are obtained. The detoxification rate of the purple potatoes cultured with the method reaches up to 93%, the reproduction cycle is shortened by 2-4 months, batched and rapid reproduction of the purple potatoes can be achieved, and the culture cost can be lowered.
Description
Technical field
The present invention relates to the technical field of tissue culture of plant, particularly relate to the group training of a kind of purple-colored potato rapid detoxification
Method.
Background technology
Purple-colored potato, potato shape is oblong, and eye is less, and sarcocarp is darkviolet, rich in natural pigment such as anthocyanidin.
Purple-colored potato is goodlooking, and color allure is strong, without adding any pigment and toner in product processing, the most strong
Health.
Purple-colored potato original producton location is the states such as South America Peru, and the domestic existing unit of current China, individual introduce purple Ma Ling
Some kinds of potato.But owing to different purple-colored potatos are in planting process, the area introduced a fine variety is different, different after plantation
The kind in area unanimously carries out planting experiment at same plot, area, quantity, monomer weight, and the biggest difference occurs in each kind.
One is plant strain growth decline, compound leaf atrophy；Two is that plant type becomes short, blade face shrinkage；Three is that blade yellowish green alternate speckle, leaf occurs
Arteries and veins is downright bad, and compound leaf comes off, and also part introduced variety yield when plantation for the first time is high, second time plantation yield after reservation kind
Low, the little degeneration of tuber is serious；Purple-colored potato can not be steady in a long-term after introducing a fine variety growth, the offspring especially reserved seed for planting can not keep
The original performance of plant and yield.Trace it to its cause be introduce a fine variety after planting site plant during, suffer invading of pathogenic bacteria, virus etc.
Dye, but the most ripe effective purple-colored potato virus-free culture method in prior art.
Summary of the invention
In view of this, it is an object of the invention to provide the tissue culture method of a kind of purple-colored potato rapid detoxification.
In order to realize foregoing invention purpose, the present invention provides techniques below scheme:
The invention provides the tissue culture method of a kind of purple-colored potato rapid detoxification, comprise the following steps:
1) bud of purple-colored potato is cut；
2) the described tender shoots cut is carried out successively sterile water wash, the instantaneous sterilization of ethanol, medicining liquid dipping sterilization and nothing
Bacterium water rinses, the leaf stem section after being sterilized；
3) leaf stem section after described sterilization is placed in superclean bench, peels off the stem apex on leaf stem section top under the microscope,
Then stem apex inoculation is carried out primary structure cultivation in the medium, obtain a test tube Seedling；
4) peel off the stem apex of a described test tube Seedling, carry out secondary structure cultivation, obtain secondary test tube Seedling；
5) repeating said steps 4) stripping and tissue culture, the number of times of described repetition is 3～5 times, obtains the examination of detoxification
Guan Miao.
Preferably, step 1) described in stripping after stem apex with 1 phyllopodium.
Preferably, step 2) described in time of sterile water wash be 10～20min.
Preferably, step 2) described in disinfectant solution be mass fraction be 5.5%～6.5% aqueous sodium hypochlorite solution or
Mass fraction is the mercury solution of 1.2～2.5%.
Preferably, step 2) described in time of the instantaneous sterilization of ethanol be 2～4s, the volumetric concentration of described ethanol is 70
～80%.
Preferably, step 2) described in medicining liquid dipping sterilization time be 15～25min.
Preferably, step 2) described in time of aseptic water washing be 3～5min.
Preferably, step 3) and step 4) described in the MS culture medium that culture medium is improvement of tissue culture, described
The MS culture medium of improvement is the component that with the addition of following final concentration in MS culture medium: BA, 0.04～0.06mg/L, NAA, 0.18
～0.22mg/L, 24-D, 0.4～0.6mg/L, sucrose 30wt% and Rhizoma Solani tuber osi pulverizing liquid 20wt%.
Preferably, step 3) and step 4) described in the intensity of illumination of tissue culture be 2800～3000Lx.
Preferably, step 3) and step 4) described in the temperature of tissue culture be 22～25 DEG C
Beneficial effects of the present invention: the method for the invention is by repeatedly peeling off up-to-date on purple-colored potato stem apex growing
Phyllopodium, is reduced original seed and takes viruliferous probability, then cultivated by rapid tissue, thus improves purple-colored potato test tube Seedling
Virus elimination rate, further improve the stability of purple-colored potato test tube Seedling；The purple-colored potato that the method for the invention is cultivated
Virus elimination rate be up to 93%, the breeding cycle foreshortens to 2～4 months, can realize the batch Fast-propagation of purple-colored potato, reduces training
Forming this, the purple-colored potato that method of the present invention is cultivated is compared with traditional purple-colored potato cultural method, and yield increases
Add 20%.
Detailed description of the invention
The invention provides the tissue culture method of a kind of purple-colored potato rapid detoxification, comprise the following steps:
1) bud of purple-colored potato is cut；2) the described tender shoots cut is carried out successively sterile water wash, ethanol instantaneous
Sterilization, medicining liquid dipping sterilization and aseptic water washing, the leaf stem section after being sterilized；3) leaf stem section after described sterilization is placed in
Superclean bench, peels off the stem apex on leaf stem section top under the microscope, then stem apex inoculation is carried out once group in the medium
Knit cultivation, obtain a test tube Seedling；4) peel off the stem apex of a described test tube Seedling, carry out secondary structure cultivation, obtain secondary examination
Guan Miao；5) repeating said steps 4) stripping and tissue culture, the number of times of described repetition is 3～5 times, obtains the test tube of detoxification
Seedling.
Heretofore described purple-colored potato is preferably from the external purple-colored potato introduced or domestic selection-breeding
Purple-colored potato, concrete kind is preferably: Yun Site (the color Rhizoma Solani tuber osi in Yunnan seven), black beauty, NCC, ruby, purple
Cloud 1 and red potato series S03-2744.
Rhizoma Solani tuber osi tender shoots, before peeling off the stem apex on the Rhizoma Solani tuber osi tender shoots adopted, is preferably carried out by the present invention
Pre-sterilization processes, and described pre-sterilization processes preferably: be 70～80% by the potato block of band bud as volume fraction
Ethanol in soak 4～6min；After drying use volume fraction be 70～80% ethanol potato block surface is wiped
Wipe sterilization.The present invention, in order to obtain more preferable great-hearted stem apex material, uses above-mentioned more gentle pre-sterilization to process means.
The stem apex that heretofore described stem apex uses in peeling off preferably from a length of the 0.2 of top cutting～
The stem apex of 0.3mm, described stem apex is preferably with 1～2 phyllopodium；Currently preferred described bud is carried out stem apex stripping
From so that the stem apex after stripping is with 1 phyllopodium.In the present invention, the concrete operations of described stripping are preferably: by purple
Color Rhizoma Solani tuber osi tender shoots is positioned under the anatomical lens of 40 times, divides stripping with dissecting needle or dissecting knife by stem apex 1～2 phyllopodium positions
Cut off and retain 1 phyllopodium, the stem apex after being peeled off.
The present invention obtains the purple-colored potato tender shoots cut, and carries out aseptic successively by the described purple-colored potato tender shoots cut
Water cleaning, the instantaneous sterilization of ethanol, medicining liquid dipping sterilization and aseptic water washing, the tender shoots after being sterilized.Owing to tender shoots is less
And quantity is more, present invention preferably uses gauze and wrap up the purple-colored potato tender shoots cut, then the tender shoots of parcel is carried out
Sterile water wash；It is not result in that tender shoots is broken up with the bundle cleaning that wraps, it is to avoid the waste of material.In the present invention, institute
The time of 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 ethanol to the purple-colored potato tender shoots after described sterile water wash
Sterilization.In the present invention, the time of the instantaneous sterilization of described ethanol is preferably 2～4s, more preferably 3s；The body of described ethanol
Volume concentrations is preferably 85～98%, more preferably 88～97%, and most preferably 95%.
The present invention is after the instantaneous sterilization of the ethanol completing purple-colored potato tender shoots, and the immersion that carries out disinfection tender shoots bubble disappears
Poison.Heretofore described disinfectant solution preferably mass fraction is 5.5%～6.5% aqueous sodium hypochlorite solution or mass fraction
It it is 1.2～2.5% mercury solution；More preferably mass fraction be 5.8～6.2% aqueous sodium hypochlorite solution or mass fraction be
1.5～the mercury solution of 2.2%, most preferably mass fraction are 6.0% aqueous sodium hypochlorite solution or mass fraction is 2.0%
Mercury solution.In the present invention, described medicining liquid dipping sterilization time be preferably 15～25min, more preferably 17～
23min, more preferably 20min.
The present invention, after soak soaking disinfection stem apex, carries out aseptic water washing to tender shoots.In the present invention, described aseptic
The time that water rinses is preferably 3～5min, more preferably 4min.
After tender shoots completes aseptic water washing, the present invention preferably tender shoots is put in sterilization after absorbent paper on absorb water.?
In the present invention, the time of described water suction is preferably 0.5～1.5min, more preferably 1min.The present invention is to described aseptic
The source of water, without particular/special requirement, uses the sterilized water that this area is conventional, and concrete is homemade aquesterilisa or commercially available
Sterilized water.
The present invention be sterilized after tender shoots, the tender shoots after sterilization is placed in superclean bench, carries out under microscope the most again
Stem apex is peeled off, and stem apex is inoculated in culture medium carries out primary structure cultivation, obtains a test tube Seedling.In the present invention, institute
The intensity of illumination stating primary structure cultivation is preferably 2800～3000Lx；More preferably 2850～2950Lx, most preferably
For 2900Lx.In the present invention, the temperature that described primary structure is cultivated is preferably 22～25 DEG C, more preferably 23～24 DEG C；
The time that described primary structure is cultivated is preferably 10～25 days, more preferably 15～22 days, most preferably 20 days.
The heretofore described culture medium used by primary structure cultivation is preferably the MS culture medium of improvement, described improvement
MS culture medium be the component that with the addition of following final concentration in MS culture medium: BA 0.04～0.06mg/L, NAA 0.18～
0.22mg/L, 24-D 0.4～0.6mg/L, sucrose 30wt% and Rhizoma Solani tuber osi pulverizing liquid 20wt%.
The present invention, after obtaining a test tube Seedling, peels off the stem apex of a described test tube Seedling, carries out secondary structure cultivation,
To secondary test tube Seedling；The method of the stem apex peeling off a described test tube Seedling in the present invention is identical with the method that above-mentioned stem apex is peeled off,
The method that secondary structure is cultivated is identical with the method that primary structure is cultivated, and the parameter of concrete operations can be in the range of above-mentioned restriction
Independently selected, do not repeat at this to repeat.
The present invention repeats above-mentioned stripping and tissue culture's operation, the number of times of described repetition after obtaining secondary test tube Seedling
Preferably 3～5 times, more preferably 4 times, the stripping repeated each time and the concrete operations parameter of tissue culture all can be upper
State in the range of restriction independently selected, repeat to peel off and obtain after tissue culture the test tube Seedling of detoxification.
The tissue culture method of the purple-colored potato rapid detoxification provided the present invention below in conjunction with embodiment is carried out specifically
Bright, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1
Pick up from black beauty's potato block of the band stem apex of field plots, the potato block of band bud is placed in volume fraction
Being immersion 5min in the ethanol of 75%, taking-up is placed in ventilation and dries, and then using volume fraction is that the ethanol of 75% is to Ma Ling
Potato potato block surface carries out cleaning disinfection.Cutting 2～3cm purple-colored potato tender shoots, the tender shoots using gauze parcel to cut exists successively
Fill the pond of sterilized water is cleaned tender shoots 15min, be placed in volume fraction be 95% ethanol is sterilized 3s, be placed in mass fraction and be
After the aqueous sodium hypochlorite solution of 6.0% soaks 20min, use aseptic water washing stem apex 4min, it is put in the absorbent paper after sterilization
Water suction 1min.The tender shoots of 2～3cm is positioned under the anatomical lens of 40 times, divides stripping with dissecting needle by 1 phyllopodium position of stem apex
1 phyllopodium of disconnected reservation, the stem apex after being peeled off.Then it is connected to stem apex in the MS culture medium of improvement carry out primary structure training
Support, consisting of of the MS culture medium of described improvement: MS culture medium, add BA0.05mg/L, NAA0.2mg/L, 24-D
0.5mg/L, 30wt% sucrose, 20wt% Rhizoma Solani tuber osi pulverizing liquid.The intensity of illumination that primary structure is cultivated is 2900Lx, and temperature is 24
℃；Time is 20 days.
After obtaining a test tube Seedling, peel off the stem apex of a described test tube Seedling, under conditions of above-mentioned tissue culture successively
Carry out secondary structure cultivation, obtain secondary test tube Seedling；Then after the stem apex of stripping secondary test tube Seedling carries out three tissue cultures again
Obtaining the test tube Seedling of detoxification, the time that described three tissue cultures finally give detoxification test tube plantlet is 2 months.
In the present embodiment, growth and the detoxification situation of the detoxification test tube plantlet survived are shown in Table 1.
The growth of the detoxification test tube plantlet survived that table 1 embodiment of the present invention 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
Reach 93%.
Embodiment 2
Pick up from No. 1 potato block of purple cloud of the band stem apex of field plots, the potato block of band bud is placed in volume integral
Number be 75% ethanol in soak 5min, then take out and be placed in ventilation and dry, then using volume fraction is the ethanol of 75%
Potato block surface is carried out cleaning disinfection.Cut 2～3cm purple-colored potato tender shoots, use gauze to wrap up the tender shoots cut
Successively in the pond fill sterilized water clean tender shoots 15min, be placed in volume fraction be in 95% ethanol sterilize 3s, be placed in quality
Mark be 6.0% aqueous sodium hypochlorite solution in soak 20min, with after aseptic water washing stem apex 4min, be put in the suction after sterilization
Absorb water on water paper 1min.The tender shoots of 2～3cm is positioned under the anatomical lens of 40 times, with dissecting knife by 2 phyllopodium positions of stem apex
Divide stripping 1 phyllopodium of disconnected reservation, the stem apex after being peeled off.Then it is connected to stem apex in the MS culture medium of improvement carry out once
Tissue culture, consisting of of the MS culture medium of described improvement: MS culture medium, add BA0.05mg/L, NAA0.2mg/L, 24-D
0.5mg/L, 30wt% sucrose, 20wt% Rhizoma Solani tuber osi pulverizing liquid.The intensity of illumination that primary structure is cultivated is 2950Lx, and temperature is 22
℃；Time is 23 days.
After obtaining a test tube Seedling, peel off the stem apex of a described test tube Seedling, carry out secondary structure cultivation, obtain secondary examination
Guan Miao；Then the stem apex peeling off secondary test tube Seedling carries out three tissue cultures again, repeats point and peels off and obtain after tissue culture 4 times
The test tube Seedling of detoxification, the time that described 4 tissue cultures finally give detoxification test tube plantlet is 3 months.In the present embodiment, survive
Growth and the detoxification situation of detoxification test tube plantlet 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
Reach 92%.
Embodiment 3
Pick up from the NCC potato block of the band stem apex of field plots, by the potato block of band bud as volume fraction
Being immersion 3min in the ethanol of 75%, then take out and be placed in ventilation and dry, then using volume fraction is the ethanol pair of 75%
Potato block surface carries out cleaning disinfection.Cutting 2～3cm purple-colored potato tender shoots, the tender shoots using gauze parcel to cut depends on
Secondary clean in the pond fill sterilized water tender shoots 15min, be placed in volume fraction be 95% ethanol is sterilized 3s, be placed in quality and divide
Number be 6.0% aqueous sodium hypochlorite solution in soak 20min, with after aseptic water washing stem apex 4min, be put in the water suction after sterilization
Absorb water on paper 1min.The tender shoots of 2～3cm is positioned under the anatomical lens of 40 times, with dissecting needle, 2 phyllopodium positions of stem apex is divided
Stripping is disconnected retains 1 phyllopodium, the stem apex after being peeled off.Then it is connected to stem apex in the MS culture medium of improvement carry out once group
Knit cultivation, consisting of of the MS culture medium of described improvement: MS culture medium, add BA0.05mg/L, NAA0.2mg/L, 24-D
0.5mg/L, 30wt% sucrose, 20wt% Rhizoma Solani tuber osi pulverizing liquid.The intensity of illumination that primary structure is cultivated is 2850Lx, and temperature is 24
℃；Time is 18 days.
After obtaining a test tube Seedling, peel off the stem apex of a described test tube Seedling, carry out secondary structure cultivation, obtain secondary examination
Guan Miao；Then the stem apex peeling off secondary test tube Seedling carries out three tissue cultures again, repeats stem apex and peels off and after tissue culture 5 times
To the test tube Seedling of detoxification, the incubation time of described detoxification test tube plantlet is 3 months.
In the present embodiment, growth and the detoxification situation of the detoxification test tube plantlet survived 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
Reach 91%.
Embodiment 4
Picking up from black beauty's potato block of the band stem apex of field plots, the potato block of bud is placed in volume fraction is
Soaking 5min in the ethanol of 75%, taking-up is placed in ventilation and dries, and then using volume fraction is that the ethanol of 75% is to Rhizoma Solani tuber osi
Potato block surface carries out cleaning disinfection.Cut 2～3cm purple-colored potato tender shoots, use the tender shoots that gauze parcel cuts successively at dress
The pond of full sterilized water is cleaned tender shoots 15min, be placed in volume fraction be 95% ethanol is sterilized 3s, be placed in mass fraction and be
After the aqueous sodium hypochlorite solution of 6.0% soaks 20min, use aseptic water washing stem apex 4min, it is put in the absorbent paper after sterilization
Water suction 1min.The tender shoots of 2～3cm is positioned under the anatomical lens of 40 times, divides stripping with dissecting needle by 1 phyllopodium position of stem apex
1 phyllopodium of disconnected reservation, the stem apex after being peeled off.Then it is connected to stem apex in the MS culture medium of improvement carry out primary structure training
Support, consisting of of the MS culture medium of described improvement: MS culture medium, add BA0.05mg/L, NAA0.2mg/L, 24-D
0.5mg/L, 30wt% sucrose, 20wt% Rhizoma Solani tuber osi pulverizing liquid.The intensity of illumination that primary structure is cultivated is 2900Lx, and temperature is 24
℃；Time is 20 days.
After obtaining a test tube Seedling, peel off the stem apex of a described test tube Seedling, the stem apex of a test tube Seedling is positioned over 40
Under anatomical lens again, divide stripping to break at 1 phyllopodium position of stem apex with dissecting needle and retain 1 phyllopodium, the stem after being peeled off
Point.Then being connected to the stem apex of a described test tube Seedling in the MS culture medium of improvement carry out secondary structure cultivation, described changes
Consisting of of good MS culture medium: MS culture medium, adds BA0.05mg/L, NAA0.2mg/L, 24-D 0.5mg/L, 30wt% sugarcane
Sugar, 20wt% Rhizoma Solani tuber osi pulverizing liquid.The intensity of illumination that secondary structure is cultivated is 2950Lx, and temperature is 22 DEG C；Time is 23 days,
To secondary test tube Seedling；
After obtaining secondary test tube Seedling, peel off the stem apex of described secondary test tube Seedling, the stem apex of secondary test tube Seedling is positioned over 40
Under anatomical lens again, divide stripping to break at 1 phyllopodium position of stem apex with dissecting needle and retain 1 phyllopodium, the stem after being peeled off
Point.Then being connected to the stem apex of described secondary test tube Seedling in the MS culture medium of improvement carry out three tissue cultures, described changes
Consisting of of good MS culture medium: MS culture medium, adds BA0.05mg/L, NAA0.2mg/L, 24-D 0.5mg/L, 30wt% sugarcane
Sugar, 20wt% Rhizoma Solani tuber osi pulverizing liquid.The intensity of illumination of three tissue cultures is 2950Lx, and temperature is 22 DEG C；Time is 19 days,
To three test tube Seedlings；
In the present embodiment, growth and the detoxification situation of the detoxification test tube plantlet survived are shown in Table 4.
The growth of the detoxification test tube plantlet survived that table 4 embodiment of the present invention 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
Reach 92%.
Comparative example 1
Traditional introduces a fine variety method for planting, is directly separated stem apex and carries out disinfection and cultivate surviving of the stem sharp that obtains after pretreatment
Situation and growing state are shown in Table 5.It is that MS culture medium adds BA0.05mg/L, IAA 1.0mg/ that stem sharp cultivates the culture medium used
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 that the method for the invention is cultivated is high, de-
Poison 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 drawing
Planting method for planting, method of the present invention can realize the batch Fast-propagation of purple-colored potato, reduces toxigenic capacity.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For Yuan, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (10)
1. a tissue culture method for purple-colored potato rapid detoxification, comprises the following steps:
1) bud of purple-colored potato is cut；
2) the described tender shoots cut is carried out successively sterile water wash, the instantaneous sterilization of ethanol, medicining liquid dipping sterilization and sterilized water
Rinse, the leaf stem section after being sterilized；
3) leaf stem section after described sterilization is placed in superclean bench, peels off the stem apex on leaf stem section top under the microscope, then
Stem apex inoculation is carried out primary structure cultivation in the medium, obtains a test tube Seedling；
4) peel off the stem apex of a described test tube Seedling, carry out secondary structure cultivation, obtain secondary test tube Seedling；
5) repeating said steps 4) stripping and tissue culture, the number of times of described repetition is 3～10 times, obtains the test tube of detoxification
Seedling.
Tissue culture method the most according to claim 1, it is characterised in that step 1) described in stripping after stem apex with 1
Individual phyllopodium.
Tissue culture method the most according to claim 1, it is characterised in that step 2) described in time of sterile water wash be
10～20min.
Tissue culture method the most according to claim 1, it is characterised in that step 2) described in disinfectant solution be that mass fraction is
The aqueous sodium hypochlorite solution of 5.5%～6.5% or the mercury solution that mass fraction is 1.2～2.5%.
Tissue culture method the most according to claim 1, it is characterised in that step 2) described in time of the instantaneous sterilization of ethanol
Being 2～4s, the volumetric concentration of described ethanol is 85～98%.
6. according to the tissue culture method described in claim 1 or 4, it is characterised in that step 2) described in medicining liquid dipping sterilization
Time be 15～25min.
Tissue culture method the most according to claim 1, it is characterised in that step 2) described in time of aseptic water washing be 3
～5min.
Tissue culture method the most according to claim 1, it is characterised in that step 3) and step 4) described in tissue culture
Culture medium is the MS culture medium of improvement, and the MS culture medium of described improvement is the group that with the addition of following final concentration in MS culture medium
Point: BA, 0.04～0.06mg/L, NAA, 0.18～0.22mg/L, 24-D, 0.4～0.6mg/L, sucrose 30wt% and Rhizoma Solani tuber osi
Pulverizing liquid 20wt%.
Tissue culture method the most according to claim 1, it is characterised in that step 3) and step 4) described in the light of tissue culture
It is 2800～3000Lx according to intensity.
10. according to the tissue culture method described in claim 1 or 9, it is characterised in that step 3) and step 4) described in tissue culture
Temperature be 22～25 DEG C.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106718887A (en) * | 2016-11-30 | 2017-05-31 | 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 |
| CN107318656A (en) * | 2017-08-21 | 2017-11-07 | 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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| JP2006141274A (en) * | 2004-11-19 | 2006-06-08 | Oita General Service Kk | Method for culturing shoot apex |
| 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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| JP2006141274A (en) * | 2004-11-19 | 2006-06-08 | Oita General Service Kk | Method for culturing shoot apex |
| 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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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106718887A (en) * | 2016-11-30 | 2017-05-31 | 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 |
| CN107318656A (en) * | 2017-08-21 | 2017-11-07 | 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 |
| 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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|---|---|
| CN106035083B (en) | 2017-12-05 |
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