CN104531606B - The cambial plant stem cell of archangel storage root and its preparation and cultural method - Google Patents
The cambial plant stem cell of archangel storage root and its preparation and cultural method Download PDFInfo
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Abstract
The invention discloses a kind of cambial plant stem cell of archangel storage root and its preparation and cultural method, its preparation method is：Storage root will cut into slices after sterilizing, be placed in after being handled on screening and culturing medium promote root cut into slices in non-formation confluent monolayer cells Mortality or damage or stop growing；Section is put into isolation medium again and cultivated, promotes the division of cambial cell and does not bring it about dedifferentiation, initial gross separation obtains cambial cell；Move it to after being cultivated in subculture medium, then move in isolation medium and repeat alternately to cultivate, the cell containing a large amount of small vacuoles until obtaining a kind of stabilization.The method that the forming layer stem cell culture method that the present invention is provided has suspend culture and fermentation tank culture.The present invention realizes the separation to angelica forming layer stem cell, and expand culture to it there is provided new method, the substantial amounts of industrial production of archangel storage root forming layer stem cell is laid a good foundation, also new cell derived is provided for archangel storage root forming layer stem cell.
Description
Technical field
The present invention relates to the cambial plant stem cell of archangel storage root and its preparation and cultural method.
Background technology
Current Plant Biotechnology is all entered with evoked callus, the culture method such as plant organ or separated protoplast
Row research, and cambial isolated culture method just occurs in the recent period.
Callus is the parenchymal tissue that is formed after plant dedifferentiation, but after dedifferentiation, its secondary metabolite
Amount and species can all reduce, simultaneously containing central big vacuole, expanding culture（Such as fermentation tank culture）The big vacuoles of Shi Zhongyang
Easy fragmentation causes cell death, and culture environment is harsh.
The organ culture of plant generally comprises the culture of plant roots or bud, maintains some shapes of the body cell of plant
State, for callus, the content of secondary metabolite is more, but because its easily differentiation turns into plant individual, causes
It can not preferably be used for industrial production.
The protoplast of plant does not contain cell membrane, and cell is more fragile, to osmotic pressure, and pH etc. requires particularly severe, and one
As be used for transgenosis mediation, be used further to plant regeneration, be difficult in industrial production.
Because forming layer separate living tissue possesses the feature of infinite multiplication, thus it is referred to as " plant stem cell ".Forming layer is thin
The separation and expansion culture of born of the same parents system just occur in the recent period, in November, 2010, and one has been published in Nature Biotechnology
It is entitled《Cultured cambial meristematic cells as a source of plant natural
products》Article, successfully separation expands the forming layer stem cell line of Chinese yew.The cell of this non-dedifferentiation cell
Activity and secondary metabolite content are more than 100 times more than callus, while find that it has a large amount of small Vacuoles Structures,
There is bigger advantage in industrial production.
In the plant containing storage root, the absorption inorganic salts that act as of storage root store nutrient, contain substantial amounts of starch
And more secondary metabolite, and the forming layer of plant roots be by cell horizontal split and break up as root marrow with
And bast, therefore storage root forming layer be these plant root thickenings Main Tissues.But preserved in growth course
Contain substantial amounts of endophyte and fungal component in root.So needing to carry out depth sterilizing when as explant cultured tissue to reduce
Pollution in tissue culture procedures.
International monopoly WO2010/137878 KO and Chinese patent CN102459572A disclose the forming layer of Ginkgoaceae
The plant stem cell and its separation method in source provide the method that the separation of the forming layer stem cell line of xylophyta expands.
International monopoly WO2009/038416 EN and Chinese patent CN101939415A are disclosed from quiescent center
Plant stem cell system and its separation method.
International monopoly WO2009/038417 EN and Chinese patent CN101939414A are disclosed from storage
The cambial plant stem cell system of herbaceous plant of root and its separation method, by doing for some storage root plants such as ginseng, carrot
Cell carries out separation expansion.This method is cut into slices the root containing storage root plant, is handled by osmotic pressure, is made non-in section
Forming layer Mortality or do not divide, then forming layer stem cell line is separated and expanded by suitable culture medium.
Whether detect its cell at present is that the method for stem cell has by the way that whether microexamination is containing a large amount of small vacuoles
State and sensitiveness to radiation or radiomimetic drug determine whether for plant stem cell system.
There is following aspect in the shortcoming of prior art, by practice, imitate on the separation of storage root cambial cell
The experiment of the patent of method, finds in some archangels such as Radix Angelicae Sinensis（Angelica sinensis(Oliv.)
Diels）, the root of Dahurain angelica（Angelica dahurica（Fisch. ex Hoffm.）Benth. et Hook. f. ex Franch.
et Sav）, RADIX PEUCEDANI（Angelica decursiva(Miq.) Franch. et Savat）Deng can not be separated in plant
Go out forming layer stem cell line.Illustrate existing storage root cambial cell isolation technics method in as above plant do not apply to, it is necessary to
New method handles this kind of plant, to isolate forming layer stem cell line.
The content of the invention
It is an object of the invention to provide a kind of preparation method of the cambial plant stem cell of archangel storage root.
Another object of the present invention is to provide a kind of culture of the cambial plant stem cell of archangel storage root
Method.
The technical solution used in the present invention is：
The preparation method of the cambial plant stem cell of archangel storage root, comprises the following steps：
1）The selection and sterilizing of explant：Take archangel storage root as explant, after cleaning up, then gone out
Bacterium is handled, and the surface and inside of explant is existed without thalline；
2）The section of explant：Explant after above-mentioned sterilizing is aseptically cut into slices, explant section is obtained；
3）Screening and culturing：Section is attached on screening and culturing medium；16 ~ 72h is handled under the conditions of 4 DEG C ~ 30 DEG C, makes section
In the cell of non-formation layer segment do not divide；
The screening and culturing medium is the MS culture mediums containing 7.5 ~ 8.5g/L of sucrose 1~6% and agar, and the medium pH is 2
~12；
4）It is separately cultured：Explant section taking-up after above-mentioned screening and culturing is put into section liquid and recovered, then
Section is put into isolation medium again and cultivated, promotes the division of cambial cell and does not bring it about dedifferentiation, and its
Hetero-organization cell death or do not divide；Culture obtains the cambial cell that initial gross separation comes out after 6 ~ 16 days；
The isolation medium is the MS cultures containing 0.1~2mg/L of auxin, sucrose 1~6%, 7.5 ~ 8.5g/L of agar
Base, the medium pH is 5.0~5.9；
The auxin is selected from dichlorphenoxyacetic acid（2,4-D）, indolebutyric acid（IBA）, methyl α-naphthyl acetate（NAA）And heteroauxin
（IBA）At least one of；
5）Squamous subculture：The above-mentioned cambial cell isolated is moved in subculture medium and cultivated 5~16 days, Ran Houzhuan
Move in isolation medium and cultivate 5~16 days, transfer to subculture medium and carry out repeating alternately to cultivate, obtained after alternate culture
Still containing a large amount of small Vacuoles Structures in a large amount of cells obtained, these cells are the cambial plant of archangel storage root
Stem cell；
The subculture medium be containing sucrose 1~6%, 0.2 ~ 1.2mg/L 2,4-D, 0.01 ~ 0.5mg/L kinetins KT,
0.5 ~ 2mg/L NAA, 7.5 ~ 8.5g/L agar, the B5 medium of 0.08 ~ 0.12g/L activated carbons, the medium pH be 5.0~
5.9。
Further, above-mentioned steps 1）Described in the detailed process of sterilization treatment be：Surface sterilizing is first carried out, i.e., to cleaning
Explant successively through ethanol solution, liquor natrii hypochloritis, mercuric chloride solution carry out surface sterilizing, after cleaning up；Depth is carried out again
Layer sterilizing, i.e., shake 20~40min by the explant after surface sterilizing in anti-browning deep layer sterilized solution.
Further, above-mentioned anti-browning deep layer sterilized solution is to contain sucrose 1~6%, 100 ~ 400mg/L of penicillin, streptomysin
100 ~ 400mg/L, 100 ~ 400mg/L of cephalosporin, 0.5 ~ 2g/L of carbendazim, 100 ~ 400mg/L of L-AA, citric acid
100 ~ 400mg/L, 0.008 ‰ ~ 0.020 ‰ Tween-20s 1/2MS culture mediums, the medium pH are 5.0~5.9.
Further, above-mentioned steps 2）Described in explant 0.5~2mm of slice thick, contain forming layer.
Further, above-mentioned steps 3）Described in screening and culturing medium pH be 2~5 or 8 ~ 12.
Further, above-mentioned steps 4）Described in section liquid to contain sucrose 1~6%, 100 ~ 400mg/L of penicillin, strepto-
100 ~ 400mg/L of element, 100 ~ 400mg/L of cephalosporin, 100 ~ 400mg/L of L-AA, the 1/ of 100 ~ 400mg/L of citric acid
2MS culture mediums, the medium pH is 5.0~5.9.
Further, above-mentioned steps 4）The condition of culture and step 5 being separately cultured）Described in squamous subculture condition of culture
It is：Temperature is 24 ~ 26 DEG C, humidity is 50 ~ 70%.
Further, above-mentioned archangel includes Radix Angelicae Sinensis Angelica sinensis (Oliv.) Diels, the root of Dahurain angelica
Angelica dahurica（Fisch. ex Hoffm.）Benth. et Hook. f. ex Franch. et Sav, pale reddish brown
Root of purple-flowered peucedanum Angelica decursiva (Miq.) Franch. et Savat, thin leaf Radix Angelicae Sinensis Angelica laxifoliata
Diels, turn celery Angelica polymorpha Maxim, weight tooth Radix Angelicae Sinensis Angelica biserrata (Shan et Yuan)
Yuan et Shan。
The method of the cambial plant stem cell suspension culture of archangel storage root, comprises the following steps：Will be above-mentioned
Plant stem cell prepared by method is inoculated in the suspension medium of liquid with 40 ~ 120g/L of weight in wet base, is 23 DEG C ~ 27 in temperature
DEG C, rotating speed is cultivated for 100 ~ 140rpm condition, cultivates 10 ~ 25 days, you can；
The suspension medium be containing 1~6% sucrose, 0.2 ~ 2mg/L 2,4-D, 0.01 ~ 0.5mg/L kinetins KT, 0.5 ~
2mg/L NAA B5 medium, the medium pH is 5.0 ~ 5.9.
The fermentation tank culture method of the cambial plant stem cell of archangel storage root, comprises the following steps：Will be upper
The plant stem cell for stating method preparation is inoculated in liquid subculture medium, and inoculum concentration is 28 ~ 32g containing weight in wet base in every liter of culture medium
Plant stem cell, temperature be 23 DEG C ~ 27 DEG C, dissolved oxygen rate be 0.20 ~ 0.40, rotating speed be 100 ~ 140rpm CMC model 10
~ 25 days；
The liquid subculture medium be containing 1~6% sucrose, 0.2 ~ 2mg/L 2,4-D, 0.01 ~ 0.5mg/L kinetins KT,
0.5 ~ 2mg/L NAA, 0.08 ~ 0.12g/L activated carbons B5 medium, the medium pH are 5.0 ~ 5.9.
The beneficial effects of the invention are as follows：
The invention provides a kind of preparation method of new plant forming layer stem cell, realize and archangel is preserved
The separation of the cambial plant stem cell of root, and it is expanded the method for culture carried out research there is provided adapt to its expand training
Foster suspension culture method and fermentation tank culture method, this is provided newly in terms of medicinal plant tissue cultures archangel
Prepare and training method, the substantial amounts of industrial production of archangel storage root forming layer stem cell is laid a good foundation, also for work as
Belong to plant storage root forming layer stem cell and provide new cell derived.
Brief description of the drawings
Fig. 1 is the growing state of Radix Angelicae Sinensis storage root explant section during being separately cultured, and wherein a is to cut into slices from screening training
Support the photo of the 1st day that takes out and be separately cultured in base, b is to be separately cultured the state of the 2nd day, c is is separately cultured the 4th day
State, d is to be separately cultured the state of the 6th day；
After Fig. 2 is squamous subculture stabilization, the forming layer stem cell line of acquisition；
Forming layer stem cell and the microstructure of callus cell that Fig. 3 is prepared for the present invention；A is forming layer stem cell
Microstructure, b be neutral red staining after forming layer stem cell, c be callus cell microstructure, d is dimethyl diaminophenazine chloride
Callus cell after dyeing；
The neutral red staining figure for the forming layer stem cell that Fig. 4 is prepared for the present invention；
Fig. 5 callus cells handled with the forming layer stem cell for preparing of the present invention through bleomycin after cell death
Rate；
The state of forming layer stem cell after Fig. 6 is cultivated 15 days to suspend；
Fig. 7 is the picture after callus suspension is cultivated 15 days.
Embodiment
The preparation method of the cambial plant stem cell of archangel storage root, comprises the following steps：
1）The selection and sterilizing of explant：Take archangel storage root as explant, after cleaning up, then gone out
Bacterium is handled, and the surface and inside of explant is existed without thalline；
2）The section of explant：Explant after above-mentioned sterilizing is aseptically cut into slices, explant section is obtained；
3）Screening and culturing：Section is attached on screening and culturing medium；16 ~ 72h is handled under the conditions of 4 DEG C ~ 30 DEG C, makes section
In the cell division of non-formation layer segment do not divide；
4）It is separately cultured：Explant section taking-up after above-mentioned screening and culturing is put into section liquid and recovered, then
Section is put into isolation medium again and cultivated, promotes the division of cambial cell and does not bring it about dedifferentiation, and its
Hetero-organization cell death or do not divide；Culture obtains the cambial cell that initial gross separation comes out after 6 ~ 16 days；
5）Squamous subculture：The above-mentioned cambial cell isolated is moved in subculture medium and cultivated 5~16 days, Ran Houzhuan
Move in isolation medium and cultivate 5~16 days, transfer to subculture medium and carry out repeating alternately to cultivate, obtained after alternate culture
Still containing a large amount of small Vacuoles Structures in a large amount of cells obtained, these cells are the cambial plant of archangel storage root
Stem cell.
It is preferred that, above-mentioned explant contains cambial storage for a diameter of 0.5~1cm of the annual seedling of archangel
Hide root.
It is preferred that, above-mentioned steps 1）Described in the detailed process of sterilization treatment be：First carry out surface sterilizing, i.e., to cleaning
Explant carries out surface sterilizing through ethanol solution, liquor natrii hypochloritis, mercuric chloride solution successively, after cleaning up；Deep layer is carried out again
Sterilizing, i.e., shake 20~40min by the explant after surface sterilizing in anti-browning deep layer sterilized solution.
It is preferred that, above-mentioned anti-browning deep layer sterilized solution is to contain sucrose 1~6%, 100 ~ 400mg/L of penicillin, streptomysin
100 ~ 400mg/L, 100 ~ 400mg/L of cephalosporin, 0.5 ~ 2g/L of carbendazim, 100 ~ 400mg/L of L-AA, citric acid
100 ~ 400mg/L, 0.008 ‰ ~ 0.020 ‰ Tween-20s 1/2MS culture mediums, the medium pH are 5.0~5.9.
It is preferred that, above-mentioned steps 2）Described in explant 0.5~2mm of slice thick, contain forming layer.
It is preferred that, the formula of above-mentioned section liquid be containing sucrose 1~6%, 100 ~ 400mg/L of penicillin, streptomysin 100 ~
400mg/L, 100 ~ 400mg/L of cephalosporin, 100 ~ 400mg/L of L-AA, 100 ~ 400mg/L of citric acid 1/2MS trainings
Base is supported, the medium pH is 5.0~5.9.
It is preferred that, above-mentioned screening and culturing medium is the MS culture mediums containing 7.5 ~ 8.5g/L of sucrose 1~6% and agar, the culture
Base pH is 2~12.
It is preferred that, the pH value of above-mentioned screening and culturing medium is 2~5 or 8~12.
It is preferred that, above-mentioned isolation medium is to contain 0.1~2mg/L of auxin, sucrose 1~6%, 7.5 ~ 8.5g/L of agar
MS culture mediums, the medium pH be 5.0~5.9.
It is furthermore preferred that the concentration of auxin is 0.2 ~ 1.3 mg/L in above-mentioned isolation medium.
It is preferred that, above-mentioned auxin is selected from dichlorphenoxyacetic acid（2,4-D）, indolebutyric acid（IBA）, methyl α-naphthyl acetate（NAA）With
Heteroauxin（IBA）At least one of.
It is preferred that, above-mentioned steps 4）The condition that the section of middle explant is cultivated in isolation medium is：Temperature 24 ~ 26
DEG C, humidity 50 ~ 70%.
It is preferred that, above-mentioned steps 5）Described in the condition cultivated be：Temperature is 24 ~ 26 DEG C, humidity is 50 ~ 70%.
It is preferred that, above-mentioned subculture medium is containing sucrose 1~6%, 0.2 ~ 2mg/L 2,4-D, 0.01 ~ 0.5mg/L kinetins
KT, 0.5 ~ 2mg/L NAA, 7.5 ~ 8.5g/L agar, the B5 medium of 0.08 ~ 0.12g/L activated carbons, the medium pH are 5.0-
5.9。
It is preferred that, above-mentioned archangel includes Radix Angelicae Sinensis Angelica sinensis (Oliv.) Diels, the root of Dahurain angelica
Angelica dahurica（Fisch. ex Hoffm.）Benth. et Hook. f. ex Franch. et Sav, pale reddish brown
Root of purple-flowered peucedanum Angelica decursiva (Miq.) Franch. et Savat, thin leaf Radix Angelicae Sinensis Angelica laxifoliata
Diels, turn celery Angelica polymorpha Maxim, weight tooth Radix Angelicae Sinensis Angelica biserrata (Shan et Yuan)
Yuan et Shan。
The method of the cambial plant stem cell suspension culture of archangel storage root, comprises the following steps：Will be above-mentioned
Plant stem cell prepared by method is inoculated in the suspension medium of liquid with 40 ~ 120g/L of weight in wet base, is 23 DEG C ~ 27 in temperature
DEG C, rotating speed carries out culture 10 ~ 25 days for 100 ~ 140rpm condition；
The suspension medium be containing 1~6% sucrose, 0.2 ~ 2mg/L 2,4-D, 0.01 ~ 0.5mg/L kinetins KT, 0.5 ~
2mg/L NAA B5 medium, the medium pH is 5.0 ~ 5.9.
The fermentation tank culture method of the cambial plant stem cell of archangel storage root, comprises the following steps：Will be upper
The plant stem cell for stating method preparation is inoculated in liquid subculture medium, and inoculum concentration is 28 ~ 32g containing weight in wet base in every liter of culture medium
Plant stem cell, temperature be 23 DEG C ~ 27 DEG C, dissolved oxygen rate be 0.2 ~ 0.4, rotating speed for 100 ~ 140rpm CMC model 10 ~
25 days；
It is described contain 1~6% sucrose, 0.2 ~ 2mg/L 2,4-D, 0.01 ~ 0.5mg/L kinetins KT, 0.5 ~ 2mg/L NAA,
The B5 medium of 0.08 ~ 0.12g/L activated carbons, the medium pH is 5.0 ~ 5.9.
With reference to specific embodiment, the present invention is further illustrated, but is not limited thereto.
Embodiment 1：The preparation method of the cambial plant stem cell of archangel storage root
（1）The selection and sterilizing of explant
Annual Radix Angelicae Sinensis seedling diameter 0.8cm or so complete root is chosen, surface soil is removed, 30 points are rinsed with running water
Clock, obtains the explant of cleaning.Explant is soaked 30 seconds with 75% ethanol in aseptic operating platform, with sterile water wash 2 times.
Then soaked and shaken with 1 ~ 2% sodium hypochlorite and sterile water wash is used after 4 minutes 2 times, finally with 4 points of 0.1% mercuric chloride immersion shaking
Clock, and with sterile water wash 4 ~ 5 times, obtain the explant of surface complete sterilizing.
The explant that surface sterilizing is finished is put into anti-browning deep layer sterilized solution and shaken 30 minutes.The anti-browning deep layer
Sterilized solution contain sucrose 3%, penicillin 200mg/L, streptomysin 300mg/L, cephalosporin 300mg/L, carbendazim 1g/L, L- resist
Bad hematic acid 100mg/L, citric acid 100mg/L, the 1/2MS culture mediums of 0.01 ‰ Tween-20s（As shown in table 1）, pH is 5.0-
5.9。
The anti-browning deep layer of table 1 sterilizing formula of liquid
Wherein in process for preparation, by penicillin, streptomysin, cephalosporin, L-AA and citric acid sterilized water
PH is adjusted after mixed dissolution to add in the culture medium cooled down after sterilizing to 5.8, then by 0.22 micron membrane filter filtration sterilization.Sterilizing
The culture medium cooled down afterwards is culture medium of the other compositions mixing after 121 DEG C of high-temperature sterilizations, 20 minutes gained in table 1.
Wherein, MS formulas are：NH4NO31650mg/L, KNO3 1900mg/L, CaCl2·2H2O 440mg/L, MgSO4·
7H2O 370mg/L, KH2PO4 170mg/L, FeSO4 27.8mg/L, Na2-EDTA·2H2The mg/ of O 37.3mg/L, KI 0.83
L, H3BO46.2 mg/L, MnSO4·4H2O 2.23 mg/L, ZnSO4·7H2O 8.6mg/L, NaMoO4·2H2O 0.25mg/
L, CuSO4·5H2O 0.025mg/L, CoCl2·6H2O 0.025mg/L, L- glycine 20mg/L, puridoxine hydrochloride（Dimension life
Plain B6）5mg/L, nicotinic acid 5mg/L, thiamine hydrochloride (vitamin B1) 1mg/L, inositol 100mg/L, surplus is water（Following institute
It is this to have MS formulas）.
1/2MS is：A great number of elements CaCl2, KNO3, NH4NO3, MgSO4, KH2PO4 consumption is the half of MS culture mediums,
Other compositions are identical with MS culture mediums.
（2）The section of explant
Explant is taken out to be placed in culture dish and cut into slices, slice thickness is 0.5~2mm, containing forming layer, and as far as possible
Keep the complete of section.
The formula of the section liquid is to contain sucrose 3%, penicillin 100mg/L, streptomysin 150mg/L, cephalosporin
150mg/L, L-AA 100mg/L, citric acid 200mg/L 1/2MS culture mediums（Formula is as shown in table 2）, the culture medium
PH is 5.0 ~ 5.9.
The section formula of liquid of table 2
Wherein in process for preparation, penicillin, streptomysin, cephalosporin, ascorbic acid and citric acid are mixed with sterilized water
Regulation pH after dissolving is closed to add in the culture medium cooled down after sterilizing to 5.8, then by 0.22 micron membrane filter filtration sterilization.After sterilizing
The culture medium of cooling is culture medium of the other compositions mixing after 121 DEG C of high-temperature sterilizations, 20 minutes gained in table 2.
（3）Screening and culturing
Above-mentioned section is attached on the solid screening and culturing medium that pH is 8, handled 16 ~ 72 hours under the conditions of 4 ~ 30 DEG C,
The histocyte beyond forming layer is set not divide.When temperature is relatively low, the long period can be handled, and effect is preferable.The screening training
It is the MS culture mediums containing sucrose 3% and agar 8g/L to support base, and the medium pH is 8（As shown in table 3）, the pH of the screening and culturing
Value can also be the arbitrary value between 2 ~ 5 or 8 ~ 12.
Explant section, which is attached in above-mentioned screening and culturing, handles a period of time so that the tissue beyond storage root forming layer
（Including bast, marrow etc.）Dedifferentiation is lost into the ability of callus, and crust（Phellem layer）In surface sterilizing
Generation is dead.
The screening and culturing based formulas of table 3
（4）Isolation medium grows the forming layer stem cell line of explant：
The separated process of forming layer in storage root as shown in Figure 1；Explant section after above-mentioned screening and culturing is put
Soak and recovered in section liquid for 30 seconds, cutting temperature is reverted to 24 ~ 26 DEG C, pH is reverted to 5.0 ~ 5.9；Then again will
It is put into isolation medium（As shown in Figure 1a）, cultivated under conditions of 25 DEG C, culture to such as Fig. 1 b institutes of state at the 2nd day
Show, cultivate to state at the 4th day as illustrated in figure 1 c, it is observed that cambial cell horizontal split makes section arch upward in bowl
To state at the 6th day as shown in Figure 1 d, the bowl-type state become apparent can be observed, cambial cell is further in shape, culture
Increase；Terminate to be separately cultured after cultivating 6 ~ 16 days, the cambial cell that initial gross separation comes out can be obtained；
During being separately cultured, cut into slices and divide rapidly in the isolation medium for suitably forming confluent monolayer cells growth and do not send out
Raw dedifferentiation, obtains a number of cambial cell, and other histocytes are dead after being handled in screening and culturing medium
Or do not divide, it is ignored and disregards；So as to isolated cambial cell.
During above-mentioned be separately cultured, the present invention has also carried out substantial amounts of screening to the formula of isolation medium, especially
The screening to hormone in formula so that in section cambial cell will not dedifferentiation into callus, so that it is guaranteed that final point
The forming layer stem cell separated out is pure natural, rather than formed by dedifferentiation.
The isolation medium is containing each 0.6~0.8mg/L of auxin 2,4-D and IAA, sucrose 3%, agar 8g/L
MS culture mediums, the medium pH is 5.0 ~ 5.9（As shown in table 4）.
The total concentration of auxin can also be the arbitrary value in 0.1~2mg/L scopes in above-mentioned isolation medium, wherein raw
Long element can select dichlorphenoxyacetic acid（2,4-D）, indolebutyric acid（IBA）, methyl α-naphthyl acetate（NAA）And heteroauxin（IBA）In extremely
Few one kind.
The formula of the isolation medium of table 4
（5）Squamous subculture
Although cambial cell is separated and has certain amount, but now the speed of growth of cell is slow, separation
Culture medium is not suitable for continuing subculture.It is advantageous to the suitable culture medium that it is mushroomed out as subculture medium（It is formulated such as table 5
It is shown）.
The concrete operations of squamous subculture are：By the above-mentioned cambial cell isolated move in subculture medium culture 5~
16 days, be then transferred in isolation medium cultivate 5~16 days, transfer in subculture medium, with isolation medium and after
Squamous subculture alternately and repeatedly is carried out for culture medium, by microscopic examination of cell after multiple cycles are repeated, it is determined that still protecting
Hold a large amount of small Vacuoles Structures, then these cells are the cambial plant stem cell system of archangel storage root.
The subculture medium is containing sucrose 3%, 2,4-D 1mg/L, kinetin（KT）0.01mg/L, NAA 1mg/L, fine jade
Fat 8g/L, activated carbon 0.1g/L B5 medium, the medium pH are 5.0~5.9（As shown in table 5）.
The squamous subculture based formulas of table 5
B5 medium is formulated：KNO3 2500mg/L, (NH4)2SO4134 mg/LL, MgSO4·7H2O 121.56mg/L,
CaCl2·2H2O 113.23mg/L, FeSO4 27.8mg/L, Na2-EDTA·2H2O 37.3mg/L, NaH2PO4·H2O
130.44 mg/L, MnSO4·4H2O 10mg/L, ZnSO4·7H2O 2mg/L, NaMoO4·2H2O 0.25mg/L, H3BO4 3
Mg/L, KI 0.75 mg/L, CuSO4·5H2O 0.025mg/L, CoCl2·6H2O 0.025mg/L, inositol 100mg/L, hydrochloric acid
Thiamine (vitamin B1) 20mg/L, nicotinic acid 2mg/L, puridoxine hydrochloride（Vitamin B6）2mg/L, L-AA 50
Mg/L, the mg/L of citric acid 75, the mg/L of L-Aspartic acid 133, L-arginine 175 mg/L, L- glycine 75 mg/L, L-
The mg/L of proline 115.
Forming layer stem cell line such as Fig. 2 to be obtained after above-mentioned squamous subculture；The stem cell line is faint yellow soft group
Knit state.
The identification of correlation properties is carried out to the forming layer stem cell prepared in above-described embodiment below.
First, microstructure is identified
The microstructure of forming layer stem cell prepared by embodiment 1 is observed, and the callus group formed with dedifferentiation
Knit cell to be contrasted, as a result as shown in figure 3, Fig. 3 a are the microphoto of forming layer stem cell prepared by embodiment 1, Fig. 3 c are
The microphoto of callus cell, Fig. 3 b and Fig. 4 are photograph of the forming layer stem cell of the preparation of embodiment 1 after neutral red staining
Piece, Fig. 3 d are the photo after callus cell neutral red staining, pass through the contrast of neutral red staining vacuole picture（Fig. 3 b, Fig. 4
With Fig. 3 d）, it is evident that there is substantial amounts of small Vacuoles Structure, with tip of a root stem apex in the forming layer stem cell that the present invention is prepared
The feature of separate living tissue is similar, and in callus cell is the structure of central big vacuole.
2nd, radiomimetic drug processing experiment
By callus cell and the forming layer stem cell for preparing of the present invention handled under various concentrations bleomycin 24h and
After 48h, cell mortality is counted.
Hanged using the radiomimetic drug bleomycin 0-200 μ g/mL liquid for handling forming layer stem cell line and callus
Floating cell 24 hours and 48 hours, with Evans Blue and FDA dyeing statistics cell mortality（FDA will swash after dyeing in red
Green fluorescence is dissipated under light emitting source, expression cell is living cells；Evans Blue by after cell dyeing under blue excitation light source
Red fluorescence is dissipated, expression cell is dead cell）, find at higher concentrations（More than 80 μ g/mL）, forming layer stem cell it is dead
Die rate higher.Illustrate that forming layer stem cell is more sensitive to bleomycin than callus.I.e. forming layer stem cell radiates to class
Medicine is more sensitive, further illustrates that the forming layer stem cell that the inventive method prepares is natural stem cell, be not by
The callus of dedifferentiation.
Embodiment 2：The cultural method of the cambial plant stem cell of archangel storage root
The method of the cambial plant stem cell suspension culture of archangel storage root
, it is necessary to set up suspension system and contribute to further research in the utilization of plant cell engineering.It is specific outstanding
Floating culture operation is as follows：The forming layer stem cell line obtained in embodiment 1 is inoculated in fluid nutrient medium with weight in wet base 40g/L,
Cultivated under 100 ~ 120rpm, 23 DEG C ~ 27 DEG C environment, wherein suspension medium formula is as shown in table 5.After suspension culture 15 days
Observation finds the cell that forming layer stem cell line is white uniformity（As shown in Figure 6）, and callus cell has the feature of browning,
And tissue agglomerate is of different sizes in callus（As shown in Figure 7）.
The suspension medium composition of table 6
The correlation function of the forming layer stem cell obtained below to Maitland culture described in embodiment 2 is detected.
The detection of forulic acid biosynthesis
L-phenylalanine is in the raw material compared with upstream in the synthesis of forulic acid, the biosynthesis order in plant Radix Angelicae Sinensis
For the synthetic route of L-phenylalanine → cinnamic acid → p-Coumaric Acid → caffeic acid → forulic acid, natural Radix Angelicae Sinensis cell can be utilized
L-phenylalanine in environment synthesizes forulic acid by vivo biodistribution route of synthesis.And forulic acid is the index for detecting Radix Angelicae Sinensis quality
Property composition.
The 50mL suspension mediums for containing 500mg/L L-phenylalanines are added in 250mL conical flask, then will be implemented
Forming layer stem cell line, the callus prepared in example 1 is inoculated into conical flask by 80g/L amount.Suspend after cultivating 10 days, receive
Collect cell, and extracted with methanol, after ethyl acetate extraction, the amount of forulic acid in detection cell extraction liquid.
Absworption peak under 316nm is detected by HPLC, it is found that the forming layer that Maitland culture described in embodiment 2 is obtained is dry thin
Forulic acid amount in born of the same parents is 9.118mg/ kilograms of wet weight cells, and forulic acid is not detected in callus.
The above results illustrate that forming layer stem cell line produced by the present invention is different from callus, formation produced by the present invention
It is active stronger that the secondary metabolite of layer stem cell line is synthesized.
Embodiment 3：The cultural method of the cambial plant stem cell of archangel storage root
The method of the cambial plant stem cell fermentation tank culture of archangel storage root
Cultivated, by the amount that wet cell weight is 40g/L, respectively prepared embodiment 1 using 5L airlift fermentor
Forming layer plant stem cell and callus be inoculated in liquid subculture medium, and in rotating speed 105-115rpm, temperature 25
DEG C, dissolved oxygen rate（OD values）Culture 21 days is carried out under conditions of 0.3.
As a result the growth rate for finding callus is only 1.67, and browning occurs for cell.Forming layer prepared by the present invention
The growth rate of stem cell line is 2.83.Illustrate that forming layer stem cell prepared by the present invention has soon under conditions of fermentation tank culture
The ability of speed propagation.
In addition, applicant has also carried out the experiment described in above-described embodiment 1 ~ 3 to other kinds in angelica of plant, it is selected
The plant taken has the root of Dahurain angelica（Angelica dahurica（Fisch. ex Hoffm.）Benth. et Hook. f. ex
Franch. et Sav）, RADIX PEUCEDANI（Angelica decursiva(Miq.) Franch. et Savat）, dredge leaf Radix Angelicae Sinensis
（Angelica laxifoliata Diels）, turn celery（Angelica polymorpha Maxim.）, weight tooth Radix Angelicae Sinensis
（Angelica biserrata(Shan et Yuan) Yuan et Shan）Deng the class achieved as described in embodiment 1 ~ 3
As result, further illustrate the inventive method be applied to the cambial plant stem cell of archangel storage root preparation
And its culture.
It is readily appreciated that for those skilled in the art, the foregoing is only the preferred embodiment of patent of the present invention, and
Not to limit any modifications, equivalent substitutions and improvements made within the present invention, all the spirit and principles in the present invention etc., fall
Within the protection domain of application claims.
Claims (9)
1. the preparation method of the cambial plant stem cell of archangel storage root, it is characterised in that：Comprise the following steps：
1）The selection and sterilizing of explant：Archangel storage root is taken as explant, after cleaning up, then is carried out at sterilizing
Reason, makes the surface and inside of explant exist without thalline；
2）The section of explant：Explant after above-mentioned sterilizing is aseptically cut into slices, explant section is obtained；
3）Screening and culturing：Section is attached on screening and culturing medium；16 ~ 72h is handled under the conditions of 4 DEG C ~ 30 DEG C, is made non-in section
The cell for forming layer segment does not divide；
The screening and culturing medium be the MS culture mediums containing 7.5 ~ 8.5g/L of sucrose 1~6% and agar, the medium pH be 8~
12；
4）It is separately cultured：Explant section taking-up after above-mentioned screening and culturing is put into section liquid and recovered, then again will
Section, which is put into isolation medium, is cultivated, and is promoted the division of cambial cell and is not brought it about dedifferentiation, and other groups
Knit cell death or do not divide；Culture obtains the cambial cell that initial gross separation comes out after 6 ~ 16 days；
The isolation medium be containing 0.1~2mg/L of auxin, sucrose 1~6%, 7.5 ~ 8.5g/L of agar MS culture mediums,
The medium pH is 5.0~5.9；
The auxin is 2,4-D and IAA；
5）Squamous subculture：The above-mentioned cambial cell isolated is moved in subculture medium and cultivated 5~16 days, is then transferred to
Cultivated 5~16 days in isolation medium, transfer to subculture medium and carry out repeating alternately to cultivate, obtained after alternate culture
Still containing a large amount of small Vacuoles Structures in a large amount of cells, these cells are that the cambial plant of archangel storage root is dry thin
Born of the same parents；
The subculture medium be containing sucrose 1~6%, 0.2 ~ 1.2mg/L 2,4-D, 0.01 ~ 0.5mg/L kinetins KT, 0.5 ~
2mg/L NAA, 7.5 ~ 8.5g/L agar, the B5 medium of 0.08 ~ 0.12g/L activated carbons, the medium pH are 5.0~5.9.
2. according to the method described in claim 1, it is characterised in that：Step 1）Described in the detailed process of sterilization treatment be：First
Surface sterilizing is carried out, i.e., carrying out surface through ethanol solution, liquor natrii hypochloritis, mercuric chloride solution successively to clean explant goes out
Bacterium, after cleaning up；Deep layer sterilizing is carried out again, i.e., the explant after surface sterilizing is shaken 20 in anti-browning deep layer sterilized solution
~40min.
3. method according to claim 2, it is characterised in that：The anti-browning deep layer sterilized solution be containing sucrose 1~6%,
100 ~ 400mg/L of penicillin, 100 ~ 400mg/L of streptomysin, 100 ~ 400mg/L of cephalosporin, 0.5 ~ 2g/L of carbendazim, L- are anti-bad
100 ~ 400mg/L of hematic acid, 100 ~ 400mg/L of citric acid, the 1/2MS culture mediums of 0.008 ‰ ~ 0.020 ‰ Tween-20s, the culture
Base pH is 5.0~5.9.
4. according to the method described in claim 1, it is characterised in that：Step 2）Described in explant 0.5~2mm of slice thick, contain
There is forming layer.
5. according to the method described in claim 1, it is characterised in that：Step 4）Described in section liquid to contain sucrose 1~6%, it is blue or green
100 ~ 400mg/L of mycin, 100 ~ 400mg/L of streptomysin, 100 ~ 400mg/L of cephalosporin, 100 ~ 400mg/L of L-AA,
100 ~ 400mg/L of citric acid 1/2MS culture mediums, the medium pH is 5.0~5.9.
6. according to the method described in claim 1, it is characterised in that：Step 4）The condition of culture and step 5 being separately cultured）Middle institute
The condition of culture for stating squamous subculture is：Temperature is 24 ~ 26 DEG C, humidity is 50 ~ 70%.
7. according to any described method of claim 1~6, it is characterised in that：Described archangel includes Radix Angelicae Sinensis
Angelica sinensis (Oliv.) Diels, root of Dahurain angelica Angelica dahurica（Fisch. ex Hoffm.）Benth.
Et Hook. f. ex Franch. et Sav, RADIX PEUCEDANI Angelica decursiva (Miq.) Franch. et
Savat, leaf Radix Angelicae Sinensis Angelica laxifoliata Diels are dredged, celery Angelica polymorpha Maxim, weight tooth is turned and works as
Return Angelica biserrata (Shan et Yuan) Yuan et Shan.
The method of culture 8. the cambial plant stem cell of archangel storage root suspends, it is characterised in that：Including following step
Suddenly：Plant stem cell prepared by any methods described of claim 1 ~ 7 is trained with 40 ~ 120g/L of weight in wet base suspensions for being inoculated in liquid
Support in base, be 23 DEG C ~ 27 DEG C in temperature, rotating speed is cultivated for 100 ~ 140rpm condition, cultivates 10 ~ 25 days, you can；
The suspension medium is containing 1~6% sucrose, 0.2 ~ 2mg/L 2,4-D, 0.01 ~ 0.5mg/L kinetins KT, 0.5 ~ 2mg/
L NAA B5 medium, the medium pH is 5.0 ~ 5.9.
9. the fermentation tank culture method of the cambial plant stem cell of archangel storage root, it is characterised in that：Including following
Step：Plant stem cell prepared by any methods described of claim 1 ~ 7 is inoculated in liquid subculture medium, and inoculum concentration is
The plant stem cell of 28 ~ 32g Han weight in wet base in every liter of culture medium, is 23 DEG C ~ 27 DEG C in temperature, dissolved oxygen rate is 0.20 ~ 0.40, rotating speed
For 100 ~ 140rpm CMC model 10 ~ 25 days；
The liquid subculture medium be containing 1~6% sucrose, 0.2 ~ 2mg/L 2,4-D, 0.01 ~ 0.5mg/L kinetins KT, 0.5 ~
2mg/L NAA, 0.08 ~ 0.12g/L activated carbons B5 medium, the medium pH are 5.0 ~ 5.9.
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| CN104920227A (en) * | 2015-07-15 | 2015-09-23 | 广东融和生态农业有限公司 | Stem cell tissue culture seedling raising technique |
| CN107119007A (en) * | 2017-05-03 | 2017-09-01 | 于荣敏 | The induction and identification of ginkgo root forming layer stem cell |
| CN109423471A (en) * | 2017-09-04 | 2019-03-05 | 厦门鹭港兆康生物科技有限公司 | A method of separation and culture Radix Glycyrrhizae shoot tip meristem stem cell |
| CO2017010115A1 (en) * | 2017-10-04 | 2018-01-05 | Univ Antioquia | Method of obtaining plant fibers from the isolation and culture of meristematic cells |
| CN107760645B (en) * | 2017-12-08 | 2021-06-15 | 新乡医学院 | Rehmannia stem cell derived from rehmannia vascular bundle cambium and preparation and culture method thereof |
| CN109234224A (en) * | 2018-09-30 | 2019-01-18 | 广州赛莱拉干细胞科技股份有限公司 | A kind of plant stem cell culture medium and cultural method |
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