CN108849532A - A kind of pueraria lobata Potato microtuber in-vitro inducing method - Google Patents
A kind of pueraria lobata Potato microtuber in-vitro inducing method Download PDFInfo
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- CN108849532A CN108849532A CN201811042118.5A CN201811042118A CN108849532A CN 108849532 A CN108849532 A CN 108849532A CN 201811042118 A CN201811042118 A CN 201811042118A CN 108849532 A CN108849532 A CN 108849532A
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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 present invention provides a kind of pueraria lobata Potato microtuber in-vitro inducing methods, are related to pueraria lobata Cultivating techniques field.The method of the invention includes the following steps：1) pueraria lobata tissue culture plant inoculation is subjected to culture of rootage into root media, obtains rooted seedling；2) rooted seedling is inoculated in Stem covered by vermiculite culture medium and carries out Fiber differentiation, obtain pueraria lobata Potato microtuber.Stem covered by vermiculite rate can be significantly improved using the method for the present invention, shortens the Stem covered by vermiculite time, in the induction time of 20~25d, Stem covered by vermiculite efficiency may be up to 95% or more.
Description
Technical field
The invention belongs to the Cultivating techniques fields of pueraria lobata, and in particular to a kind of pueraria lobata Potato microtuber in-vitro inducing method.
Background technique
Pueraria lobata is called kudzu, Pueraria lobota fiber crops rattan, Pachyrhizua angulatus, is pulse family Pueraria lobota category liana, is rich in starch, is that ministry of Health of China is first batch of
The integration of drinking and medicinal herbs dual purpose plant of approval is known as the good reputation of " joining southern Pueraria lobota in north ", " asia ginseng ".Pueraria lobata is to be with underground storage root tuber
Main economic harvests organ, so research pueraria lobata storage root tuber genesis and development is of great significance.Since pueraria lobata root tuber is from seedling stage
9~10 months are needed to the maturity period, and root tuber growth is in the soil, time-consuming and is inconvenient to draw materials for research observation.Currently, related
The induction of pueraria lobata Potato microtuber has not been reported both at home and abroad.
Summary of the invention
In view of this, induction duration is short the purpose of the present invention is to provide a kind of in-vitro inducing method of pueraria lobata Potato microtuber,
Induced efficiency is high.
In order to achieve the above-mentioned object of the invention, the present invention provides following technical scheme：
The present invention provides a kind of pueraria lobata Potato microtuber in-vitro inducing methods, include the following steps：
1) pueraria lobata tissue culture plant inoculation is subjected to culture of rootage into root media, obtains rooted seedling；The root media
Using 1/2MS culture medium as minimal medium, further include：The NAA of 0.02~0.05mgL, the sucrose of 25~36g/L and 4~8g/L
Agar；
2) rooted seedling for obtaining step 1), which is inoculated in Stem covered by vermiculite culture medium, carries out Fiber differentiation, obtains pueraria lobata
Potato microtuber；The induced medium further includes using MS culture medium as minimal medium：The 6-BA of 1~3.5mg/L, 0.01~
JA, the sucrose of 70~90g/L and the agar of 4~8g/L of the NAA of 0.05mg/L, 1~10 μm of ol/L.
Preferably, the preparation method of step 1) the pueraria lobata tissue-cultured seedling, including：
A) it using pueraria lobata tender tissue as explant, is inoculated in after disinfection in initial culture base and carries out Initial culture, obtained sterile
Seedling；The initial culture base further includes using MS culture medium as minimal medium：The 6-BA of 0.1~0.3mg/L, 0.02~
The agar of the NAA of 0.1mg/L, 25~38g/L sucrose and 4~8g/L；
B) aseptic seedling for obtaining step a), which is inoculated in proliferated culture medium, carries out Multiplying culture, obtains tufted seedling；Institute
Proliferated culture medium is stated using MS culture medium as minimal medium, further includes：6-BA, the 0.02~0.1mg/L of 0.01~0.05mg/L
NAA, the sucrose of 26~32g/L and the agar of 4~8g/L；
C) pueraria lobata tissue-cultured seedling is obtained after the tufted seedling simple bud separation obtained step b).
Preferably, the step a) tender tissue includes that young tender stem is climing.
Preferably, the step a) Initial culture is illumination cultivation, and the light application time of the Initial culture is 11~20h/
D, intensity of illumination are 1500~2000lx；The temperature of the Initial culture is 22~28 DEG C, and the time of the Initial culture is 28
~35d.
Preferably, the step b) Multiplying culture is illumination cultivation, and the light application time of the Multiplying culture is 11~20h/
D, intensity of illumination are 1500~2000lx；The temperature of the Multiplying culture is 22~28 DEG C, and the time of the Multiplying culture is 25
~30d.
Preferably, the step 1) culture of rootage is dark culture, and the cultivation temperature of the culture of rootage is 22~28 DEG C, training
Supporting the time is 5~7d.
Preferably, the root long of the step 1) rooted seedling is 0.5~1cm.
Preferably, the step 2) Fiber differentiation is illumination cultivation, and the light application time of the Fiber differentiation is 11~20h/
D, intensity of illumination are 1500~2000lx；The temperature of the Fiber differentiation is 22~28 DEG C, and the time of the Fiber differentiation is 20
~25d.
The present invention provides a kind of pueraria lobata Potato microtuber in-vitro inducing methods, carry out pueraria lobata potato by material of pueraria lobata tissue-cultured seedling
In-vitro inducing can significantly improve Stem covered by vermiculite rate, shorten the Stem covered by vermiculite time, cultivate pueraria lobata tissue-cultured seedling described in 25~30d,
Stem covered by vermiculite efficiency may be up to 95% or more, and inductive condition is not limited by season, it can be achieved that the anniversary ties potato.
Detailed description of the invention
The pueraria lobata Potato microtuber figure that Fig. 1 is induced by the embodiment of the present invention 1.
Specific embodiment
The present invention provides a kind of pueraria lobata Potato microtuber in-vitro inducing methods, include the following steps：
1) pueraria lobata tissue culture plant inoculation is subjected to culture of rootage into root media, obtains rooted seedling；The root media
Using 1/2MS culture medium as minimal medium, further include：The NAA of 0.02~0.05mgL, the sucrose of 25~36g/L and 4~8g/L
Agar；
2) rooted seedling for obtaining step 1), which is inoculated in Stem covered by vermiculite culture medium, carries out Fiber differentiation, obtains pueraria lobata
Potato microtuber；The induced medium further includes using MS culture medium as minimal medium：The 6-BA of 1~3.5mg/L, 0.01~
JA, the sucrose of 70~90g/L and the agar of 4~8g/L of the NAA of 0.05mg/L, 1~10 μm of ol/L.
In pueraria lobata Potato microtuber in-vitro inducing method of the present invention, by pueraria lobata tissue culture plant inoculation into root media into
Row culture of rootage, obtains rooted seedling；The root media further includes using 1/2MS culture medium as minimal medium：0.02~
The agar of the NAA of 0.05mg/L, the sucrose of 25~36g/L and 4~8g/L.The preparation method of pueraria lobata tissue-cultured seedling of the present invention,
It preferably includes：
A) it using pueraria lobata tender tissue as explant, is inoculated in after disinfection in initial culture base and carries out Initial culture, obtained sterile
Seedling；The initial culture base further includes using MS culture medium as minimal medium：The 6-BA of 0.1~0.3mg/L, 0.02~
The agar of the NAA of 0.1mg/L, 25~38g/L sucrose and 4~8g/L；
B) aseptic seedling for obtaining step a), which is inoculated in proliferated culture medium, carries out Multiplying culture, obtains tufted seedling；Institute
Proliferated culture medium is stated using MS culture medium as minimal medium, further includes：6-BA, the 0.02~0.1mg/L of 0.01~0.05mg/L
NAA, the sucrose of 26~32g/L and the agar of 4~8g/L；
C) pueraria lobata tissue-cultured seedling is obtained after the tufted seedling simple bud separation obtained step b).
In the preparation method of pueraria lobata test tube seedling of the present invention, using pueraria lobata tender tissue as explant, it is inoculated with after disinfection
Initial culture is carried out in initial culture base, obtains aseptic seedling；The initial culture base is also wrapped using MS culture medium as minimal medium
It includes：The 6-BA of 0.1~0.3mg/L, the NAA of 0.02~0.1mg/L, 25~38g/L sucrose and 4~8g/L agar.The present invention
The tender tissue preferably includes that young tender stem is climing, and there is no particular determinations in source of the present invention to the tender tissue, preferably select
It takes robust growth, the young tender stem of no disease and pests harm plant climing, and cuts its blade.Disinfection of the present invention preferably includes will be described outer
Implant is placed in 15~20min of immersion in the aqueous solution containing dish washing liquid, cleans up after taking-up；Being placed in mass concentration again is 0.1%
Mercuric chloride solution in impregnate 6~10min, take out, rinse 4~5 times.The present invention to the concentration of the aqueous solution containing dish washing liquid simultaneously
It is not particularly limited, utilizes the routine aqueous solution containing dish washing liquid of this field.The present invention is in the aqueous solution containing dish washing liquid
When middle immersion, preferably include with banister brush clean the surface dirt.Mercuric chloride solution of the present invention impregnates preferably aseptically
It carries out, more preferably carries out in superclean bench.The soaking time of mercuric chloride solution of the present invention is preferably 7~9min, more excellent
It is selected as 8min.It include 6-BA in initial culture base of the present invention, concentration of the 6-BA in the initial culture base is preferred
For 0.12~0.28mg/L, more preferably 0.18~0.25mg/L, most preferably 0.2mg/L.
It include NAA in initial culture base of the present invention, concentration of the NAA in the initial culture base is preferably
0.03~0.08mg/L, more preferably 0.04~0.06mg/L, most preferably 0.05mg/L.In initial culture base of the present invention
Comprising sucrose, concentration of the sucrose in the initial culture base is preferably 27~35g/L, more preferably 28~32g/L, most
Preferably 30g/L.It include agar in Initial culture of the present invention, concentration of the agar in the initial culture base is preferred
For 4~8g/L, more preferably 5~7g/L, most preferably 6g/L.The present invention to each raw material sources in the initial culture base simultaneously
It is not particularly limited, utilizes the conventional reagent of this field.Initial culture of the present invention is preferably illumination cultivation, the light
Light application time according to culture is preferably 11~20h/d, more preferably 13~18h/d, most preferably 16h/d.It is of the present invention first
Feeding intensity of illumination of being commissioned to train is preferably 1500~2000lx, more preferably 1600~1850lx, most preferably 1800lx.The present invention
The temperature of the Initial culture is preferably 22~28 DEG C, more preferably 24~26 DEG C, most preferably 25 DEG C.It is of the present invention primary
The time of culture is preferably 28~35d, more preferably 29~32d, most preferably 30d.Initial culture of the present invention can incite somebody to action
Explant is induced into aseptic seedling.
After obtaining aseptic seedling, the aseptic seedling is inoculated in proliferated culture medium and carries out Multiplying culture by the present invention, obtains tufted seedling；
The proliferated culture medium further includes using MS culture medium as minimal medium：6-BA, the 0.02~0.1mg/ of 0.01~0.05mg/L
The agar of the NAA of L, the sucrose of 26~32g/L and 4~8g/L.Proliferated culture medium of the present invention includes 6-BA, and the 6-BA exists
Concentration in the proliferated culture medium is preferably 0.02~0.04mg/L, more preferably 0.03mg/L.Proliferation training of the present invention
Supporting in base includes NAA, and the concentration in the proliferated culture medium of the NAA is preferably 0.03~0.08mg/L, more preferably
0.04~0.06mg/L, most preferably 0.05mg/L.It include sucrose in proliferated culture medium of the present invention, the sucrose is described
Concentration in proliferated culture medium is preferably 27~31.5g/L, more preferably 28~31g/L, most preferably 30g/L.Institute of the present invention
It states comprising agar in Multiplying culture, concentration of the agar in the proliferated culture medium is preferably 4~8g/L, and more preferably 5
~7g/L, most preferably 6g/L.There is no particular determinations to each raw material sources in the proliferated culture medium by the present invention, utilize this
The conventional reagent in field.Multiplying culture of the present invention is preferably illumination cultivation, and the time of the illumination is preferably 11~
20h/d, more preferably 13~18h/d.Most preferably 16h/d.The intensity of illumination of Multiplying culture of the present invention is preferably 1500
~2000lx, more preferably 1600~1850lx, most preferably 1800lx.The temperature of Multiplying culture of the present invention is preferably 22
~28 DEG C, more preferably 24~26 DEG C, most preferably 25 DEG C.The period of Multiplying culture of the present invention is preferably 25~30d, more
Preferably 26~29d, most preferably 28d.The proliferation rate of Multiplying culture of the present invention is 11.3.
After obtaining tufted seedling, the present invention will obtain pueraria lobata tissue-cultured seedling after tufted seedling simple bud separation.The present invention is to the simple bud
There is no particular determinations for isolated method, utilize the conventional paradigm method of this field.
After obtaining pueraria lobata tissue-cultured seedling, the pueraria lobata tissue culture plant inoculation is carried out culture of rootage into root media by the present invention,
Obtain rooted seedling；The root media further includes using 1/2MS culture medium as minimal medium：The NAA of 0.02~0.05mg/L,
The sucrose of 25~36g/L and the agar of 4~8g/L.It include NAA in root media of the present invention, the NAA is in the life
Concentration in root culture medium is preferably 0.025~0.046mg/L, more preferably 0.03~0.04mg/L, most preferably
0.035mg/L.It include sucrose in root media of the present invention, concentration of the sucrose in the root media is preferred
For 26~34g/L, more preferably 29~32g/L, most preferably 30g/L.It include agar, institute in root media of the present invention
Stating concentration of the agar in the root media is preferably 4.5~7.2g/L, more preferably 5.6~6.5g/L, most preferably
6g/L.Root media of the present invention is preferably semisolid culturemedium.The present invention is to each raw material of the root media
There is no particular determinations in source, utilize the conventional reagent of this field.Culture of rootage of the present invention is preferably dark culture, institute
The temperature for stating dark culture is preferably 22~28 DEG C, more preferably 24~26 DEG C, most preferably 25 DEG C.Culture of rootage of the present invention
Time be preferably 5~7d.The rooting rate of culture of rootage of the present invention is 97%, in the 5~7d of culture of rootage, is taken root
The root long of seedling can reach 0.5~1cm.
After obtaining rooted seedling, the rooted seedling is inoculated in induced medium and carries out Fiber differentiation by the present invention, obtains pueraria lobata examination
Pipe potato；The induced medium further includes using MS culture medium as minimal medium：The 6-BA of 1~3.5mg/L, 0.01~
JA, the sucrose of 70~90g/L and the agar of 4~8g/L of the NAA of 0.05mg/L, 1~10 μm of ol/L.Induction training of the present invention
Supporting in base includes 6-BA, and concentration of the 6-BA in the induced medium is preferably 1.2~3mg/L, and more preferably 1.8
~2.5mg/L, most preferably 2mg/L.It include NAA in induced medium of the present invention, the NAA is in the Fiber differentiation
Concentration in base is preferably 0.012~0.04mg/L, more preferably 0.016~0.03mg/L, most preferably 0.02mg/L.This hair
It include JA in the bright induced medium, concentration of the JA in the induced medium is preferably 2~8 μm of ol/L, more excellent
It is selected as 3~6 μm of ol/L, most preferably 5 μm of ol/L.It include sucrose in induced medium of the present invention, the sucrose is described
Concentration in induced medium is preferably 75~88g/L, more preferably 78~83g/L, most preferably 80g/L.It is of the present invention
It include agar in induced medium, concentration of the agar in the induced medium is preferably 4.5~7.2g/L, more preferably
For 5.6~6.5g/L, most preferably 6g/L.There is no special limits in source of the present invention to each raw material in the induced medium
It is fixed, utilize the conventional reagent of this field.Fiber differentiation of the present invention is preferably illumination cultivation, the light of the illumination cultivation
It is preferably 11~20h/d according to the time, more preferably 14~18h/d, most preferably 16h/d.The light of Fiber differentiation of the present invention
It is preferably 1500~2000lx according to intensity, more preferably 1700~1900lx, most preferably 1800lx.Induction training of the present invention
Feeding temperature is preferably 22~28 DEG C, more preferably 24~26 DEG C, most preferably 25 DEG C.The time of Fiber differentiation of the present invention
Preferably 20~25d, more preferably 22~24d, most preferably 23d.The inductivity of pueraria lobata potato of the present invention is 95% or more.
Pueraria lobata Potato microtuber in-vitro inducing method provided by the invention is described in detail below with reference to embodiment, still
They cannot be interpreted as limiting the scope of the present invention.
Embodiment 1
It is climing to choose robust growth, the young tender stem of no disease and pests harm plant, is put into the water added with dish washing liquid after cutting its blade
It impregnates 15min and rinses 20min in flowing water with banister brush clean the surface dirt.
On superclean bench, 0.1%HgCl is used2Immersion treatment 8min, then with aseptic water washing 5 times, use aseptic filter paper
Blot explant surface moisture.
Aseptically, stem section is cut into the stem section of one axillary bud of 1.2cm long band, biology lower end is vertically erected and is inserted in
It is trained in initial culture base MS+0.2mg/L 6-BA+0.05mg/L NAA+30.0g/L sucrose+6.0g/L agar (pH, 5.8)
It supports.Condition of culture：Cultivation temperature (25 ± 1) DEG C, intensity of illumination 1800lx, light application time 16h/d.
After cultivating 4 weeks, the aseptic seedling stem-segment with single bud of Initial culture is transferred to subculture multiplication medium MS+0.03mg/L 6-
BA+0.05mg/L NAA+30.0g/L sucrose+6.0g/L agar (pH, 5.8).Condition of culture：Cultivation temperature (25 ± 1) DEG C, light
According to intensity 1800lx, light application time 16h/d.Every 25 days are a circulation subculture cycle, i.e. acquisition pueraria lobata tissue-cultured seedling.
Sterile pueraria lobata tissue-cultured seedling is transferred to root media 1/2MS+NAA0.02mg/L+ sucrose 30g/L+ agar powder 6g/
Culture of rootage 5d is carried out in the semisolid culturemedium of L, condition of culture is dark culture, cultivation temperature (25 ± 1) DEG C.It is to root long
When 0.5cm, it is transferred to 1 μm of ol/L+ sucrose 80g/L+ of Stem covered by vermiculite culture medium MS+6-BA 2mg/L+NAA 0.02mg/L+JA
Agar powder 6g/L, condition of culture are：Cultivation temperature (25 ± 1) DEG C, illumination 1500lx, light application time 16h/d, Fiber differentiation week
Phase is 20d.After Fiber differentiation 20d, as shown in Figure 1, wherein horizontal line is bar, bar=1cm.Through detecting, pueraria lobata Stem covered by vermiculite
Rate is 95.6%.
Comparative example
For comparative example in addition to different from the culture of rootage of embodiment 1 and Fiber differentiation, remaining condition is all the same, i.e., will be obtained
Pueraria lobata tissue-cultured seedling be directly transferred to Stem covered by vermiculite culture medium MS+6-BA 1 μm of ol/L+ sucrose of 2mg/L+NAA0.02mg/L+JA
In 80g/L+ agar powder 6g/L culture medium, the inductivity of pueraria lobata Potato microtuber is 25.1% after 25 days.
Embodiment 2
It is climing to choose robust growth, the young tender stem of no disease and pests harm plant, is put into the water added with dish washing liquid after cutting its blade
It impregnates 20min and rinses 20min in flowing water with banister brush clean the surface dirt.
On superclean bench, 0.1%HgCl is used2Immersion treatment 6min, then with aseptic water washing 4 times, use aseptic filter paper
Blot explant surface moisture.
Aseptically, stem section is cut into the stem section of one axillary bud of 1cm long band, biology lower end is vertically erected and is inserted in just
For being trained in culture medium MS+0.1mg/L 6-BA+0.05mg/L NAA+30.0g/L sucrose+6.0g/L agar (pH, 5.8)
It supports.Condition of culture：Cultivation temperature (25 ± 1) DEG C, intensity of illumination 1500lx, light application time 16h/d.
After cultivating 5 weeks, the aseptic seedling stem-segment with single bud of Initial culture is transferred to subculture multiplication medium MS+0.01mg/L 6-
BA+0.05mg/L NAA+30.0g/L sucrose+6.0g/L agar (pH, 5.8).Condition of culture：Cultivation temperature (25 ± 1) DEG C, light
According to intensity 1500lx, light application time 16h/d.Every 28 days are a circulation subculture cycle, i.e. acquisition pueraria lobata tissue-cultured seedling.
Sterile pueraria lobata tissue-cultured seedling is transferred to root media 1/2MS+NAA0.02mg/L+ sucrose 30g/L+ agar powder 6g/
Culture of rootage 5d is carried out in the semisolid culturemedium of L, condition of culture is dark culture, cultivation temperature (25 ± 1) DEG C.It is to root long
When 1.0cm, it is transferred to 5 μm of ol/L+ sucrose 80g/L+ fine jades of Stem covered by vermiculite culture medium MS+6-BA2mg/L+NAA0.02mg/L+JA
Cosmetics 6g/L, condition of culture are optical culture, cultivation temperature (25 ± 1) DEG C, illumination 1800lx, light application time 16h/d, induction training
Supporting the period is 22d.Through detecting, pueraria lobata Stem covered by vermiculite rate is 98.4%.
Comparative example
For comparative example in addition to different from the culture of rootage of embodiment 2 and Fiber differentiation, remaining condition is all the same, i.e., will be obtained
Pueraria lobata tissue-cultured seedling be directly transferred to 1 μm of ol/L+ sucrose of Stem covered by vermiculite culture medium MS+6-BA2mg/L+NAA0.02mg/L+JA
In 80g/L+ agar powder 6g/L culture medium, the inductivity that pueraria lobata Potato microtuber is counted after 25 days is 24.0%.
Embodiment 3
It is climing to choose robust growth, the young tender stem of no disease and pests harm plant, is put into the water added with dish washing liquid after cutting its blade
18min is impregnated, with banister brush clean the surface dirt, 25min is rinsed in flowing water.
On superclean bench, 0.1%HgCl is used2Immersion treatment 10min, then with aseptic water washing 5 times, use aseptic filter paper
Blot explant surface moisture.
Aseptically, stem section is cut into the stem section of one axillary bud of 1.5cm long band, biology lower end is vertically erected and is inserted in
It is trained in initial culture base MS+0.3mg/L 6-BA+0.05mg/L NAA+30.0g/L sucrose+6.0g/L agar (pH, 5.8)
It supports.Condition of culture：Cultivation temperature (25 ± 1) DEG C, intensity of illumination 1500lx, light application time 16h/d.
After cultivating 4 weeks, the aseptic seedling stem-segment with single bud of Initial culture is transferred to subculture multiplication medium MS+0.05mg/L 6-
BA+0.05mg/L NAA+30.0g/L sucrose+6.0g/L agar (pH, 5.8).Condition of culture：Cultivation temperature (25 ± 1) DEG C, light
According to intensity 1500lx, light application time 16h/d.Every 30 days are a circulation subculture cycle, i.e. acquisition pueraria lobata tissue-cultured seedling.
Sterile pueraria lobata tissue-cultured seedling is transferred to root media 1/2MS+NAA0.02mg/L+ sucrose 30g/L+ agar powder 6g/
Culture of rootage 5d is carried out in the semisolid culturemedium of L, condition of culture is dark culture, cultivation temperature (25 ± 1) DEG C.It is to root long
When 0.8cm, it is transferred to 10 μm of ol/L+ sucrose 80g/L+ of Stem covered by vermiculite culture medium MS+6-BA 2mg/L+NAA0.02mg/L+JA
Agar powder 6g/L, condition of culture are optical culture, cultivation temperature (25 ± 1) DEG C, illumination 2000lx, light application time 16h/d, induction
Cultivation cycle is 25d.Through detecting, pueraria lobata Stem covered by vermiculite rate is 95.1%.
Comparative example
For comparative example in addition to different from the culture of rootage of embodiment 3 and Fiber differentiation, remaining condition is all the same, i.e., will be obtained
Pueraria lobata tissue-cultured seedling be directly transferred to Stem covered by vermiculite culture medium MS+6-BA 1 μm of ol/L+ sucrose of 2mg/L+NAA0.02mg/L+JA
In 80g/L+ agar powder 6g/L culture medium, the inductivity that pueraria lobata Potato microtuber is counted after 25 days is 24.8%.
Reference examples
The method that control group uses general planting pueraria lobata, i.e. late March to early April, the pueraria lobata transplantation of seedlings that will be cultivated
Into kind of plant hole, kind plant hole spacing is 1.0~1.2 meters, by soil compaction, compacting after transplanting, is watered with root water.It is cultivated in field
After 120~130d, root tuber can be just formed.
The present invention provides a kind of pueraria lobata Potato microtuber in-vitro inducing methods, can significantly improve Stem covered by vermiculite rate, shorten examination
Pipe potato induction time, in the induction time of 20~25d, Stem covered by vermiculite efficiency may be up to 95% or more.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (8)
1. a kind of pueraria lobata Potato microtuber in-vitro inducing method, includes the following steps：
1) pueraria lobata tissue culture plant inoculation is subjected to culture of rootage into root media, obtains rooted seedling；The root media is with 1/
2MS culture medium is minimal medium, further includes：The fine jade of the NAA of 0.02~0.05mg/L, the sucrose of 25~36g/L and 4~8g/L
Rouge；
2) rooted seedling for obtaining step 1), which is inoculated in Stem covered by vermiculite culture medium, carries out Fiber differentiation, obtains pueraria lobata test tube
Potato；The induced medium further includes using MS culture medium as minimal medium：6-BA, the 0.01~0.05mg/ of 1~3.5mg/L
JA, the sucrose of 70~90g/L and the agar of 4~8g/L of the NAA of L, 1~10 μm of ol/L.
2. in-vitro inducing method according to claim 1, which is characterized in that the preparation side of step 1) the pueraria lobata tissue-cultured seedling
Method, including：
A) it using pueraria lobata tender tissue as explant, is inoculated in after disinfection in initial culture base and carries out Initial culture, obtain aseptic seedling；Institute
Initial culture base is stated using MS culture medium as minimal medium, further includes：The 6-BA of 0.1~0.3mg/L, 0.02~0.1mg/L
The agar of NAA, 25~38g/L sucrose and 4~8g/L；
B) aseptic seedling for obtaining step a), which is inoculated in proliferated culture medium, carries out Multiplying culture, obtains tufted seedling；The increasing
Culture medium is grown using MS culture medium as minimal medium, further includes：The 6-BA of 0.01~0.05mg/L, 0.02~0.1mg/L
The agar of NAA, the sucrose of 26~32g/L and 4~8g/L；
C) pueraria lobata tissue-cultured seedling is obtained after the tufted seedling simple bud separation obtained step b).
3. in-vitro inducing method according to claim 2, which is characterized in that the step a) tender tissue includes young tender stem
It is climing.
4. in-vitro inducing method according to claim 2, which is characterized in that the step a) Initial culture is illumination cultivation,
The light application time of the Initial culture is 11~20h/d, and intensity of illumination is 1500~2000lx；The temperature of the Initial culture is
22~28 DEG C, the time of the Initial culture is 28~35d.
5. in-vitro inducing method according to claim 2, which is characterized in that the step b) Multiplying culture is illumination cultivation,
The light application time of the Multiplying culture is 11~20h/d, and intensity of illumination is 1500~2000lx；The temperature of the Multiplying culture is
22~28 DEG C, the time of the Multiplying culture is 25~30d.
6. in-vitro inducing method according to claim 1, which is characterized in that the step 1) culture of rootage is dark culture, institute
The cultivation temperature for stating culture of rootage is 22~28 DEG C, and incubation time is 5~7d.
7. in-vitro inducing method according to claim 1, which is characterized in that the root long of the step 1) rooted seedling be 0.5~
1cm。
8. in-vitro inducing method according to claim 1, which is characterized in that the step 2) Fiber differentiation is illumination cultivation,
The light application time of the Fiber differentiation is 11~20h/d, and intensity of illumination is 1500~2000lx；The temperature of the Fiber differentiation is
22~28 DEG C, the time of the Fiber differentiation is 20~25d.
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| CN109258477A (en) * | 2018-12-05 | 2019-01-25 | 怀化学院 | Pachyrhizua angulatus method for tissue culture |
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| CN113331051A (en) * | 2020-03-02 | 2021-09-03 | 中国科学院分子植物科学卓越创新中心 | Culture medium and method for inducing plants to form storage roots |
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| WO2021179527A1 (en) * | 2020-03-13 | 2021-09-16 | 广西壮族自治区农业科学院 | Method for culturing pueraria thomsonii by proliferation of single-bud stem segment and one-step seedling formation |
| CN112273226A (en) * | 2020-05-09 | 2021-01-29 | 江西农业大学 | Kudzu tissue culture method |
| CN115005101A (en) * | 2022-06-15 | 2022-09-06 | 广西壮族自治区农业科学院 | Method for promoting stem node elongation of kudzu root tissue culture seedling |
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