CN108034630A - A kind of suspension culture method of cotton rose cell - Google Patents
A kind of suspension culture method of cotton rose cell Download PDFInfo
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- CN108034630A CN108034630A CN201711491079.2A CN201711491079A CN108034630A CN 108034630 A CN108034630 A CN 108034630A CN 201711491079 A CN201711491079 A CN 201711491079A CN 108034630 A CN108034630 A CN 108034630A
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Abstract
The invention discloses a kind of suspension culture method of cotton rose cell, is to take cotton rose axillary bud to carry out surface sterilization as explant；Then the explant after disinfection is placed on callus inducing medium, carries out culture and obtain callus；Cell preculture, cell suspension cultures and Subculture are carried out again, obtain substantial amounts of suspended culture cell.The present invention produces secondary metabolite for cotton rose cell suspension cultures and haves laid a good foundation by carrying out the suspension culture of cell to cotton rose, it can be achieved that quickly producing the high cotton rose cell culture of active constituent content on a large scale.
Description
(1) technical field
The present invention relates to biological technical field, more particularly to a kind of suspension culture method of cotton rose cell.
(2) background technology
Cotton rose (Hibiscus mutabilis Linn.) is the perennial dungarunga of Malvaceae Hibiscus or machaka.
There is Cultural distribution in China north and south.Cotton rose is both important landscape flower of viewing and admiring, while even more a kind of important medicinal plant
Thing, leaf, flower, the root of cotton rose can be used as medicine, and have important pharmaceutical value.Contain in cotton rose cell tissue rich in flavones it is intoxicated,
The drug ingedients such as hyperin, synanthrin glucoside, phenols, reduced sugar, cotton rose have clearing heat and detoxicating, detumescence and apocenosis, cooling blood and hemostasis and tune
Through anastalsis.Research shows that for the alcohol extract of cotton rose to staphylococcus aureus, hemolytic streptococcus has stronger suppression
Effect, the exploitation to the various cell secondary metabolites of cotton rose are also paid attention to further.
Cotton rose artificial growth mainly uses cutting propagation at present, and cotton rose is subject to infecting for virus and numerous in cuttage
Easy transmitted virus again in growing, so that the genetic resources of merit cannot be protected, cell secondary metabolism active material yield
Also can not be guaranteed, and the seminal propagation production cycle is very long, it is most important that gradually decreased by seasonal variations and arable land
Influence, lead to not the market demand for meeting cotton rose.Therefore there is an urgent need to find one kind safely, quickly can largely produce wood
The approaches and methods of lotus cell culture.And the breeding variation that cotton rose kind can not only be solved using tissue culture technique is asked
Topic, can also largely produce cotton rose secondary metabolite by cell culture.The foundation of the suspension cell line of cotton rose is current
Have not been reported, be to obtain cotton rose cell secondary metabolism medicine and extraction by cotton rose cell culture in the industrial production
One of effective way of the pharmacological activity factor, and the mechanism of cotton rose growth and development and differentiation, hereditary variation rule can be studied,
Acquire a special sense to the genetic breeding of cotton rose.
(3) content of the invention
It is an object of the invention to provide a kind of suspension culture method of cotton rose cell.
The technical solution adopted by the present invention is：
A kind of suspension culture method of cotton rose cell, the method carry out as follows：
(1) acquisition of aseptic explant：Healthy growth, the cotton rose axillary bud of no disease and pests harm are chosen as explant, with certainly
Water rinses 30~60min, then shakes 2~5min in volumetric concentration is the aqueous solution of 1% liquid detergent, is rushed with sterile water
Only；30~60s is soaked with 70~75% ethanol water of volumetric concentration on superclean bench, then with mass concentration 0.1%
HgCl2 8~12min of medicining liquid dipping, then with aseptic water washing 3~5 times, the suck dry moisture on the filter paper of sterilizing；
(2) Fiber differentiation of cotton rose callus：Axillary bud after disinfection is accessed in callus inducing medium, first
At 23~28 DEG C, light culture 10~15 days, when then daily illumination 8~12 is small, intensity of illumination be 1000~1500lx bar
Cultivated under part, the callus inducing medium for addition KT (i.e. 6-nonylaminopurine) 0.1~0.5mg/L,
6-BA (i.e. 6- benzyls aminoadenine) 0.1~1.0mg/L, 2,4-D (i.e. 2,4- dichlorophenoxyacetic acids) 1.0~3.0mg/L, sugarcane
The MS solid minimal mediums of sugared 30g/L, agar 6.0g/L, the pH value of culture medium is 5.8；
(3) preculture of cell：The yellow-white callus without browning obtained in step (2) is transferred into pre-culture solution
The preculture of 5~9 days is carried out in body culture medium, the preculture liq culture medium is 0.1~0.5mg/L of addition KT, 6-BA
0.5~1.5mg/L, 2,4-D, 1.0~4.0mg/L, the MS liquid minimal mediums of sucrose 30g/L, the pH value of culture medium are
5.8；
(4) the suspension culture of cell：The cotton rose in suspending nutrient solution middle part and top is single thin after choosing preculture
Born of the same parents or the small aggregation of loose cell, are transferred in cell suspension cultures base with the inoculum concentration of 20~50g/L, and the cell, which suspends, to be trained
Base is supported as 0.1~0.5mg/L of addition KT, 6-BA0.1~1.0mg/L, 2,4-D, 1.0~3.0mg/L, acid hydrolyzed casein 0.2
The MS fluid nutrient mediums of~0.8g/L, sucrose 30g/L, the pH value of culture medium is 5.8；
(5) squamous subculture of cell：Carry out first time squamous subculture within 15~25 days in cell suspension cultures, later every
15~25 days subcultures are once；
(6) cell Proliferation growth calculates：After taking the cell of squamous subculture to centrifuge 10min on the centrifuge of 4000r/min,
Outwell supernatant to weigh, be fresh weight；
Cell Proliferation multiple=(harvest yield fresh weight-inoculum concentration fresh weight)/inoculum concentration fresh weight；
The growth rate (i.e. daily increment in every liter of nutrient solution) of cell=(harvest fresh weight-inoculum concentration fresh weight)/culture
Number of days.
The condition of culture of cotton rose cell is 23~28 DEG C of temperature in above-mentioned (3), (4), (5) step, pH value 5.8, often
When its illumination 6~12 is small, intensity of illumination is 1000~1500lx, shaking speed 120r/min.
Further, step (2) described callus inducing medium is to add KT 0.2mg/L, 6-BA 0.5mg/L, 2,
The MS solid minimal mediums of 4-D 2.0mg/L.
Further, the preculture liq culture medium of step (3) the cotton rose cell is addition KT0.2mg/L, 6-BA
1.0mg/L, 2, the MS liquid minimal mediums of 4-D 2.0mg/L, pre-incubation time are 7 days.
Further, step (4) the cotton rose cell suspension cultures base is to add KT 0.2mg/L, 6-BA0.5mg/L, 2,
The MS liquid minimal mediums of 4-D 2.0mg/L, acid hydrolyzed casein 0.6g/L, pH value 5.8, sucrose 30g/L.
Further, the cell mass inoculum concentration of step (4) the cotton rose cell suspension cultures base is 30g/L.
MS minimal mediums of the present invention include a great number of elements, trace element, molysite, organic matter, sucrose.It is described a large amount of
Element composition is when configuring 1 liter of (1000 milliliters) culture medium, adds ammonium nitrate NH4NO31.65 grams, potassium nitrate (KNO3) 1.9 grams,
Calcium chloride (CaCl2·2H2O) 0.44 gram, magnesium sulfate (MgSO4·7H2O) 0.37 gram, potassium dihydrogen phosphate (KH2PO4) 0.17 gram.Institute
It is when configuring 1 liter of (1000 milliliters) culture medium to state trace element composition, adds 0.83 milligram of potassium iodide (KI), boric acid (H3BO3)
6.2 milligrams, manganese sulfate (MnSO4·4H2O) 22.3 milligrams, zinc sulfate (ZnSO4·7H2O) 8.6 milligrams, sodium molybdate (Na2MoO4·
2H2O) 0.25 milligram, copper sulphate (CuSO4·5H2O) 0.025 milligram, cobalt chloride (CoCl2·6H2O) 0.025 milligram.The iron
Salt element composition is when configuring 1 liter of (1000 milliliters) culture medium, adds disodium ethylene diamine tetraacetate (Na2EDTA) 37.3 milligrams,
Ferrous sulfate (FeSO4·7H2O) 27.8 milligrams.The organic matter composition is when configuring 1 liter of (1000 milliliters) culture medium, adds flesh
100 milligrams of alcohol, 2 milligrams of glycine, thiamine hydrochloride (VB1) 0.1 milligram, puridoxine hydrochloride (VB6) 0.5 milligram, nicotinic acid (VB5)
0.5 milligram.Sucrose concentration is 20~40 g/l, and solvent is water, and pH is 5.6~6.0.Wherein MS solid mediums include agar,
Agar is not contained in MS fluid nutrient mediums.
Cotton rose axillary bud of the present invention is selected from the growing fine of Zhejiang Hangzhou garden spot, the cotton rose of no disease and pests harm is planted
Strain.Acid hydrolyzed casein of the present invention is purchased from Qingdao Hai Bo biotech firms.
Compared with prior art, the beneficial effects are mainly as follows：
(1) present invention can quickly obtain substantial amounts of cotton rose suspended culture cell.
(2) present invention is by carrying out it before cell suspension cultures preculture and acid being added in suspension medium
Caseinhydrolysate, effectively increases vitro growth rates, adds cell fresh weight, realizes batch production large-scale production cotton rose
Cell.
(3) the cotton rose callus that the present invention obtains has the advantages that higher inductivity, grows vigorous, not browning, can
Long-term subculture is kept, and carries out the suspension culture of cell by cotton rose callus, it can be achieved that extensive quickly produce effectively
The high cotton rose cell culture of component content, so as to effectively solve current cotton rose plant resources and herb resource shortage
Problem.
(4) embodiment
With reference to specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in
This：
Embodiment 1
(1) acquisition of aseptic explant：Healthy growth, the cotton rose axillary bud of no disease and pests harm are chosen as explant, with certainly
Water rinses 30min, then shakes 2min in volumetric concentration is the aqueous solution of 1% liquid detergent, is washed down with sterile water；Ultra-clean
30s is soaked with 70~75% ethanol water of volumetric concentration on workbench, then with mass concentration 0.1%HgCl2Thimerosal soaks
8min is steeped, then with aseptic water washing 3 times, the suck dry moisture on the filter paper of sterilizing；
(2) Fiber differentiation of cotton rose callus：Axillary bud after disinfection is accessed in callus inducing medium, first
At 23 DEG C, light culture 10 days, when then daily illumination 8 is small, intensity of illumination be 1000lx under conditions of cultivated, it is described
Callus inducing medium is addition KT (i.e. 6-nonylaminopurine) 0.1mg/L, 6-BA (i.e. 6- benzyls aminoadenine)
0.1mg/L, 2,4-D (i.e. 2,4- dichlorophenoxyacetic acids) 1.0mg/L, sucrose 30g/L, the MS solids of agar 6.0g/L are trained substantially
Base is supported, the pH value of culture medium is 5.8；
(3) preculture of cell：The yellow-white callus without browning obtained in step (2) is transferred into pre-culture solution
The preculture of 5 days is carried out in body culture medium, the preculture liq culture medium is addition KT 0.1mg/L, 6-BA 0.5mg/
L, the MS liquid minimal mediums of 2,4-D 1.0mg/L, sucrose 30g/L, the pH value of culture medium is 5.8；
(4) the suspension culture of cell：The cotton rose in suspending nutrient solution middle part and top is single thin after choosing preculture
Born of the same parents or the small aggregation of loose cell, are transferred in cell suspension cultures base with the inoculum concentration of 20g/L, the cell suspension cultures base
To add the MS of KT 0.1mg/L, 6-BA 0.1mg/L, 2,4-D 1.0mg/L, acid hydrolyzed casein 0.2g/L, sucrose 30g/L
Fluid nutrient medium, the pH value of culture medium is 5.8；
(5) squamous subculture of cell：First time squamous subculture is carried out in cell suspension cultures within 15 days, later every 15 days
Subculture is once；
(6) cell Proliferation growth calculates：After taking the cell of squamous subculture to centrifuge 10min on the centrifuge of 4000r/min,
Outwell supernatant to weigh, the biomass proliferation times for calculating pomegranate cell are 1.56 times, and the growth rate of cell is
2.13g/g/(d·L)；
The condition of culture of cotton rose cell is 23 DEG C of temperature in above-mentioned (3), (4), (5) step, pH value 5.8, per the daylight
According to 6 it is small when, intensity of illumination 1000lx, shaking speed 120r/min.
Influence of 2 acid hydrolyzed casein of embodiment to cotton rose cell suspension cultures
Cotton rose individual cells or the small aggregation of loose cell in the middle part of suspending nutrient solution with top are in after choosing preculture
Body is transferred in the cell suspension cultures base of addition hormon, and other operations are the same as embodiment 1, cotton rose cell suspension cultures knot
Fruit is shown in Table 1.The result shows that the culture medium of different hormone combinations can induce cotton rose cell Proliferation to some extent, but
It is that cell melting brown rate is reduced after acid hydrolyzed casein is added in culture medium, the increase of cell growth multiple, cell growth rate
Also increase, wherein addition 0.6g/L acid hydrolyzed caseins, cell growth rate is most fast, reaches 3.37g/ (dL), cell Proliferation
Multiple reaches 2.98.
Influence of 1 acid hydrolyzed casein of table to cotton rose cell suspension cultures
Influence of the preculture of 3 cell of embodiment to cotton rose cell suspension cultures
Yellow-white callus of the cotton rose without browning obtained in 1 step of embodiment (2) is transferred into preculture liq
The preculture of different number of days is carried out in culture medium, for other steps with embodiment 1, cotton rose cell suspension cultures the results are shown in Table 2 institutes
Show.The result shows that preculture, after 7 days, the cotton rose cellular biomass propagation in 3 generation of squamous subculture is maximum, and cell Proliferation multiple is
3.24 times, the growth rate of cell is 3.15g/ (dL).
Influence of 2 preculture of table to cotton rose cell suspension cultures
| Pre-incubation time (my god) | Cell Proliferation multiple (again) | Cell growth rate g/ (dL) |
| 0 | 0.95 | 1.29 |
| 5 | 2.89 | 2.56 |
| 7 | 3.24 | 3.15 |
| 9 | 2.86 | 3.06 |
Influence of 4 inoculum concentration of embodiment to cotton rose cell suspension cultures
Cotton rose individual cells or the small aggregation of loose cell in the middle part of suspending nutrient solution with top are in after choosing preculture
Body, is transferred in cell suspension cultures base with different inoculum concentrations, and other steps are the same as embodiment 1, cotton rose cell suspension cultures
It the results are shown in Table shown in 3.The result shows that when inoculum concentration is 30g/L, the cotton rose cell growth rate in 3 generation of squamous subculture is maximum, carefully
Born of the same parents' proliferation times are 3.07 times, and the growth rate of cell is 3.28g/ (dL).
Influence of 3 inoculum concentration of table to cotton rose cell suspension cultures
| Inoculum concentration (g/L) | Cell Proliferation multiple (again) | Cell growth rate (g/ ((dL) |
| 20 | 2.45 | 2.68 |
| 30 | 3.07 | 3.28 |
| 50 | 2.19 | 2.43 |
Embodiment 5
(1) acquisition of aseptic explant：Healthy growth, the cotton rose axillary bud of no disease and pests harm are chosen as explant, with certainly
Water rinses 50min, then shakes 3min in volumetric concentration is the aqueous solution of 1% liquid detergent, is washed down with sterile water；Ultra-clean
50s is soaked with 70~75% ethanol water of volumetric concentration on workbench, then with mass concentration 0.1%HgCl2Thimerosal soaks
10min is steeped, then with aseptic water washing 4 times, the suck dry moisture on the filter paper of sterilizing；
(2) Fiber differentiation of cotton rose callus：Axillary bud after disinfection is accessed in callus inducing medium, first
At 25 DEG C, light culture 12 days, when then daily illumination 10 is small, intensity of illumination be 1200lx under conditions of cultivated, it is described
Callus inducing medium for addition KT (i.e. 6-nonylaminopurine) 0.2mg/L, 6-BA (i.e. 6- benzyls aminoadenine)
0.5mg/L, 2,4-D (i.e. 2,4- dichlorophenoxyacetic acids) 2.0mg/L, sucrose 30g/L, the MS solids of agar 6.0g/L are trained substantially
Base is supported, the pH value of culture medium is 5.8；
(3) preculture of cell：The yellow-white callus without browning obtained in step (2) is transferred into pre-culture solution
Carry out the preculture of 7 days in body culture medium, the preculture liq culture medium is addition KT0.2mg/L, 6-BA1.0mg/L,
2,4-D (i.e. 2,4- dichlorophenoxyacetic acids) 2.0mg/L, the MS liquid minimal mediums of sucrose 30g/L, the pH value of culture medium are
5.8；
(4) the suspension culture of cell：The cotton rose in suspending nutrient solution middle part and top is single thin after choosing preculture
Born of the same parents or the small aggregation of loose cell, are transferred in cell suspension cultures base with the inoculum concentration of 30g/L, the cell suspension cultures base
To add the MS liquid of KT0.2mg/L, 6-BA 0.5mg/L, 2,4-D 2.0mg/L, acid hydrolyzed casein 0.6g/L, sucrose 30g/L
Body culture medium, the pH value of culture medium is 5.8；
(5) squamous subculture of cell：First time squamous subculture is carried out in cell suspension cultures within 20 days, later every 20 days
Subculture is once；
(6) cell Proliferation growth calculates：After taking the cell of squamous subculture to centrifuge 10min on the centrifuge of 4000r/min,
Outwell supernatant to weigh, the biomass proliferation times for calculating pomegranate cell are 2.88 times, and the growth rate of cell is
3.15g/g/(d·L)；
The condition of culture of cotton rose cell is 25 DEG C of temperature in above-mentioned (3), (4), (5) step, pH value 5.8, per the daylight
According to 10 it is small when, intensity of illumination 1200lx, shaking speed 120r/min.
Embodiment 6
(1) acquisition of aseptic explant：Healthy growth, the cotton rose axillary bud of no disease and pests harm are chosen as explant, with certainly
Water rinses 60min, then shakes 5min in volumetric concentration is the aqueous solution of 1% liquid detergent, is washed down with sterile water；Ultra-clean
60s is soaked with 70~75% ethanol water of volumetric concentration on workbench, then with mass concentration 0.1%HgCl2Thimerosal soaks
12min is steeped, then with aseptic water washing 5 times, the suck dry moisture on the filter paper of sterilizing；
(2) Fiber differentiation of cotton rose callus：Axillary bud after disinfection is accessed in callus inducing medium, first
At 28 DEG C, light culture 15 days, when then daily illumination 12 is small, intensity of illumination be 1500lx under conditions of cultivated, it is described
Callus inducing medium for addition KT0.5mg/L, 6-BA 1.0mg/L, 2,4-D (i.e. 2,4- dichlorophenoxyacetic acids)
3.0mg/L, sucrose 30g/L, the MS solid minimal mediums of agar 6.0g/L, the pH value of culture medium is 5.8；
(3) preculture of cell：The yellow-white callus without browning obtained in step (2) is transferred into pre-culture solution
The preculture of 9 days is carried out in body culture medium, the preculture liq culture medium is addition KT0.5mg/L, 6-BA (i.e. 6- benzyls ammonia
Base adenine) 1.5mg/L, 2,4-D (i.e. 2,4- dichlorophenoxyacetic acids) 4.0mg/L, the MS liquid of sucrose 30g/L cultivates substantially
Base, the pH value of culture medium is 5.8；
(4) the suspension culture of cell：The cotton rose in suspending nutrient solution middle part and top is single thin after choosing preculture
Born of the same parents or the small aggregation of loose cell, are transferred in cell suspension cultures base with the inoculum concentration of 50g/L, the cell suspension cultures base
To add the MS liquid of KT0.5mg/L, 6-BA 1.0mg/L, 2,4-D 3.0mg/L, acid hydrolyzed casein 0.8g/L, sucrose 30g/L
Body culture medium, the pH value of culture medium is 5.8；
(5) squamous subculture of cell：First time squamous subculture is carried out in cell suspension cultures within 25 days, later every 25 days
Subculture is once；
(6) cell Proliferation growth calculates：After taking the cell of squamous subculture to centrifuge 10min on the centrifuge of 4000r/min,
Outwell supernatant to weigh, the biomass proliferation times for calculating pomegranate cell are 2.46 times, and the growth rate of cell is
2.83g/g/(d·L)；
The condition of culture of cotton rose cell is 28 DEG C of temperature in above-mentioned (3), (4), (5) step, pH value 5.8, per the daylight
According to 12 it is small when, intensity of illumination 1500lx, shaking speed 120r/min.
Claims (4)
1. a kind of suspension culture method of cotton rose cell, it is characterised in that include the following steps：
(1) acquisition of aseptic explant：Healthy growth, the cotton rose axillary bud of no disease and pests harm are chosen as explant, uses tap water
30~60min is rinsed, then 2~5min is shaken in volumetric concentration is the aqueous solution of 1% liquid detergent, is washed down with sterile water,
30~60s is soaked with 70~75% ethanol water of volumetric concentration on superclean bench, then with mass concentration 0.1%HgCl2
8~12min of medicining liquid dipping, then with aseptic water washing 3~5 times, the suck dry moisture on the filter paper of sterilizing；
(2) Fiber differentiation of cotton rose callus：Axillary bud after disinfection is accessed in callus inducing medium, first 23
At~28 DEG C, light culture 10~15 days, when then daily illumination 8~12 is small, intensity of illumination be 1000~1500lx under conditions of
Cultivated, the callus inducing medium is addition KT (i.e. 6-nonylaminopurine) 0.1~0.5mg/L, 6-BA
(i.e. 6- benzyls aminoadenine) 0.1~1.0mg/L, 2,4-D (i.e. 2,4- dichlorophenoxyacetic acids) 1.0~3.0mg/L, sucrose
The MS solid minimal mediums of 30g/L, agar 6.0g/L, the pH value of culture medium is 5.8；
(3) preculture of cell：The yellow-white callus without browning obtained in step (2) is transferred and is trained into preculture liq
The preculture carried out in base 5~9 days is supported, the preculture liq culture medium is 0.1~0.5mg/L of addition KT, 6-BA 0.5
~1.5mg/L, 2,4-D, 1.0~4.0mg/L, the MS liquid minimal mediums of sucrose 30g/L, the pH value of culture medium is 5.8；
(4) the suspension culture of cell：Choose after preculture in the middle part of the suspending nutrient solution and the cotton rose individual cells on top or
The loose small aggregation of cell, is transferred in cell suspension cultures base with the inoculum concentration of 20~50g/L, the cell suspension cultures base
For addition 0.1~0.5mg/L of KT, 0.1~1.0mg/L of 6-BA, 2,4-D, 1.0~3.0mg/L, acid hydrolyzed casein 0.2~
The MS fluid nutrient mediums of 0.8g/L, sucrose 30g/L, the pH value of culture medium is 5.8；
(5) squamous subculture of cell：Carry out first time squamous subculture within 15~25 days in cell suspension cultures, later every 15~
25 days subcultures are once；
The condition of culture of cotton rose cell is 23~28 DEG C of temperature in above-mentioned (3), (4), (5) step, pH value 5.8, per the daylight
According to 6~12 it is small when, intensity of illumination is 1000~1500lx, shaking speed 120r/min.
2. the suspension culture method of cotton rose cell according to claim 1, it is characterised in that：Step (2) described callus
Inducing culture is organized as addition KT 0.2mg/L, 6-BA 0.5mg/L, the MS solid minimal mediums of 2,4-D 2.0mg/L.
3. the suspension culture method of cotton rose cell according to claim 1, it is characterised in that：Step (3) the wooden cottonrose hibiscus
The preculture liq culture medium of Rong's cell is addition KT 0.2mg/L, 6-BA 1.0mg/L, 2, the MS liquid bases of 4-D 2.0mg/L
Basal culture medium, pre-incubation time are 7 days.
4. the suspension culture method of cotton rose cell according to claim 1, it is characterised in that：Step (4) the wooden cottonrose hibiscus
Rong's cell suspension cultures base is addition KT 0.2mg/L, 6-BA0.5mg/L, 2,4-D 2.0mg/L, acid hydrolyzed casein 0.6g/L
MS liquid minimal mediums, pH value 5.8, sucrose 30g/L, cell mass inoculum concentration is 30g/L.
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| CN107937331A (en) * | 2017-12-30 | 2018-04-20 | 杭州纽贝生物科技有限公司 | A kind of suspension culture method of campanulaceae cell |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102994444A (en) * | 2012-11-20 | 2013-03-27 | 成都大学 | Pseudolarix amabilis cell suspension culture method |
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| Publication number | Priority date | Publication date | Assignee | Title |
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