CN102657086A - Method for tissue culture and in-vitro rapid propagation of lamiophlomis rotata - Google Patents
Method for tissue culture and in-vitro rapid propagation of lamiophlomis rotata Download PDFInfo
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- CN102657086A CN102657086A CN201210143783XA CN201210143783A CN102657086A CN 102657086 A CN102657086 A CN 102657086A CN 201210143783X A CN201210143783X A CN 201210143783XA CN 201210143783 A CN201210143783 A CN 201210143783A CN 102657086 A CN102657086 A CN 102657086A
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Abstract
The invention relates to a method for tissue culture and in-vitro rapid propagation of lamiophlomis rotata. The method comprises the following steps of: selection and disinfection of explants, wherein lamiophlomis rotata seeds are soaked to be disinfected, washed and inoculated to obtain initial culture explants; inoculation of the explants, wherein the explants are cut into chunks and then are inoculated to form callus; multiplication of the callus, wherein the callus is inoculated to be multiplied so as to form dense callus; bud induction of the callus, wherein the dense callus is inoculated to form cluster buds; multiplication of the cluster buds, wherein the cluster buds are inoculated to form lamiophlomis rotata seedlings which grow vigorously; root induction, wherein the cluster lamiophlomis rotata seedlings are divided and then are inoculated and cultured to obtain complete regeneration plantlets; disinfection of nursery substrate; and transplantation of the plants, wherein the complete regeneration plantlets are directly transplanted in the disinfected nursery substrate and then grow to normal lamiophlomis rotata plants after exercising. The method solves the problems of overlong seedling period and low propagation coefficient of the lamiophlomis rotata, and has the beneficial effects of being easy to operate and capable of realizing rapid factory seedling culture.
Description
Technical field
The present invention relates to the tissue culture and the breeding fast of exsomatizing of a plant species, relate in particular to a kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation.
Background technology
Lamiophlomis rotata is that Labiatae (Lamiaceae) lamiophlomis rotata belongs to perennial acaulescence herbaceous plant; Have another name called unrivalled logical; " big bus ", " beating the cloth crust " also claimed in Tibetan language, originates in China Qinghai, Tibet, Sichuan, northwestern Yunnan Province, Gansu, is born in alpine meadow, the river shoal grassy marshland of height above sea level 2700 ~ 4500m.The lamiophlomis rotata bitter, be slightly cold; Root and rhizome or all herbal medicine; Medicinal material surface withered yellow or yellowish-brown, matter is hard, withered, gas stench, is one of national folks such as China Tibetan, illiteracy, Nahsi herbal medicine commonly used; Have effects such as hemostasis, analgesia, promoting blood circulation and removing blood stasis, anti-inflammation, enhancing immunity, the Tibetan medicine uses it for osteomyelitis, joint grasserie, fractures, gets injured by a fall, bullet wound etc.Just on the books in the Tibetanmedicine masterpiece Four-Volume Medical Code of China before more than 1,000 year and " brilliant pearl book on Chinese herbal medicine ".The Tibet medicine lamivphlomis root growth cycle is long, and along with a large amount of productions of national new drugs such as strange positive scorching pain subsides, lamiophlomis rotata sheet and capsule, supply falls short of demand for the lamiophlomis rotata medicinal material.And wild the excavating of the long-term dependence of lamiophlomis rotata medicinal material satisfied the domestic and international market demand, and germ plasm resource is destroyed seriously, reserves sharply reduce.Especially in recent years along with both at home and abroad to the increase of lamiophlomis rotata demand, a large amount of predatorinesses are excavated, and make lamiophlomis rotata become endangered plants gradually, face the danger of species forfeiture, resource exhaustion, therefore carry out plasm resource protection with enlarge lamiophlomis rotata breed imperative.
Plant tissue culture technique is that (cytothesis that certain promptly individual organ or tissue has broken up becomes the genetic potential of complete individuality to the totipotency of utilizing cell.), get cell mass or tissue on the plant individual, through the medium of manual work preparation, make these cell masses or tissue form thousands of plant.Breeding has following several respects significance to utilize tissue culture to exsomatize fast: the ⑴ reproduction speed is fast, not influenced by extraneous climatic factor, throughout the year in indoor all breedings in a large number fast; ⑵ be convenient to keep good biological property; ⑶ have very important meaning aspect the good mutant or the good first generation of hybrid preserving and breed, but a strain has the lamiophlomis rotata mutant of clear superiority or offspring's large tracts of land after tissue culture of a tool clear superiority is promoted or the like.
It is that explant carries out method for tissue culture that the present invention adopts the aseptic seedling young root first; Carry out the research work of wild lamiophlomis rotata germplasm in-vitro propagate; This carries out genetic improvement, promotes the factorial seedling growth of lamiophlomis rotata that a new way is provided for saving rare Tibet medicine lamivphlomis root germ plasm resource.
Summary of the invention
Technical problem to be solved by this invention provides a kind of easy operating, can carry out the lamiophlomis rotata tissue culture method for in-vitro rapid propagation of large-scale industrialized production.
For addressing the above problem, a kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation of the present invention may further comprise the steps:
⑴ the selection of explant and disinfecting: the lamiophlomis rotata seed that selects full grains; Constant temperature bath 12min in 52 ℃ the water-bath; After placing the 500ppm Gibberellins solution to soak 1h, using volumetric concentration is 75% alcohol-pickled sterilization 15 ~ 30 seconds, then with aseptic deionized water flushing 2 ~ 3 times; It is 0.1% ~ 0.2% mercuric chloride that seed after will washing again places mass concentration; Soak sterilization 5 ~ 8 minutes, and do not have on the hormone culture-medium with being inoculated into MS after the aseptic deionized water flushing 3 ~ 6 times, the said MS of wherein every 25ml does not have the seed after 4 said sterilizations of inoculation in the hormone culture-medium; Seed begins to sprout after 3 ~ 4 days, after 10 days with the young root of the aseptic seedling of sprouting as the initial incubation explant;
⑵ explant inoculation: said explant is cut into the long segment of 0.3 ~ 0.5cm, is seeded on the callus inducing medium, the said explant of inoculation 2 segments in the said callus inducing medium of wherein every 25ml; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Condition under cultivated 10 ~ 25 days, incision is expanded the formation callus at the young root two ends;
⑶ callus propagation: said callus is inoculated on the callus proliferated culture medium 2 said callus of inoculation in the said callus proliferated culture medium of wherein every 25ml; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Condition under callus propagation 20 ~ 35 days, grow the callus light yellow, that graininess is fine and close;
⑷ the bud of callus is induced: the callus of said step ⑶ gained is inoculated into lures on the bud medium the said 2 said callus of bud inoculation of medium that lure of wherein every 25ml; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Condition under cultivated 30 ~ 35 days, form the bud of growing thickly;
⑸ the grow thickly propagation of bud: the said bud of growing thickly is inoculated on the bud proliferated culture medium 1 said bud of growing thickly of inoculation in the said bud proliferated culture medium of wherein every 25ml; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Condition under cultivated 30 ~ 35 days, form the lamiophlomis rotata seedling of robust growth;
⑹ root induction: the said lamiophlomis rotata seedling of growing thickly is carried out plant division, after change in the root media, in the said root media of wherein every 25ml the inoculation 1 said lamiophlomis rotata seedling of growing thickly; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Condition under cultivate 20 ~ 30 days after, white, sturdy short root appear in the seedling base portion, and grow complete root system, promptly get complete regeneration seedling;
⑺ seedling medium sterilization: seedling medium is carried out high-temperature sterilization under 115 ~ 121 ℃ of temperature, promptly get the seedling medium after sterilizing behind 15 ~ 20min; Said seedling medium is meant the mixed-matrix that humus and perlite mix by the 2:1 weight ratio;
⑻ plantlet of transplant: in the seedling medium of the little seedling direct transplantation of said complete regeneration after the said sterilization, promptly grow up to normal lamiophlomis rotata plant behind the refining seedling; A said complete regeneration seedling is planted in the seedling medium 10cm after the said sterilization
2Scope in.
Lamiophlomis rotata among the said step ⑴ belongs to herbaceos perennial for the Labiatae lamiophlomis rotata.
MS among the said step ⑴ does not have hormone culture-medium and is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml, sucrose 30 grams; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
Callus inducing medium among the said step ⑵ is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is the 2,4 dichlorophenoxyacetic acid (2 of 0.1mg/ml; 4-D) mother liquor 0 ~ 10ml, concentration is methyl (NAA) mother liquor 5 ~ 10ml of 0.1mg/ml, concentration is 6-benzyl aminoadenine (6-BA) mother liquor 5 ~ 20ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
Callus proliferated culture medium among the said step ⑶ is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is the 2,4 dichlorophenoxyacetic acid (2 of 0.1mg/ml; 4-D) mother liquor 5ml, concentration is methyl (NAA) the mother liquor 10ml of 0.1mg/ml, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 5ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
The bud medium that lures among the said step ⑷ is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is 6-benzyl aminoadenine (6-BA) mother liquor 10 ~ 30ml of 0.1mg/ml, concentration is methyl (NAA) mother liquor 0 ~ 10ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
Bud proliferated culture medium among the said step ⑸ is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 20ml of 0.1mg/ml, concentration is methyl (NAA) the mother liquor 5ml of 0.1mg/ml; Concentration be 0.1mg/ml 2,4 dichlorophenoxyacetic acid (2,4-D) mother liquor 2ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
Root media among the said step ⑹ is meant in the beaker of 1000ml, adds macroelement mother liquor 50ml successively, micro-mother liquor 10ml; Organic principle mother liquor 10ml, mother liquid of iron salt 5ml, citric acid mother liquor 4ml; Vc mother liquor 2ml, sucrose 30 grams, concentration is methyl (NAA) mother liquor 0 ~ 20ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
Seedling medium total porosity among the said step ⑺ is 70% ~ 75%, and the content of organic matter is 20%.
Said macroelement mother liquor is meant and contains ammonium nitrate (NH
4NO
3) 1650mg/L, potassium nitrate (KNO
3) 1900mg/L, magnesium sulfate (MgSO
4) 370mg/L, potassium dihydrogen phosphate (KH
2PO
4) 170mg/L, calcium chloride (CaCl
2 . 2H
2O) mixed liquor of 440mg/L.
Said micro-mother liquor is meant and contains manganese sulphate (MnSO
4 . 4H
2O) 22.3mg/L, zinc sulphate (ZnSO
4 . 7H
2O) 8.6mg/L, boric acid (H
3BO
3) 6.2mg/L, KI (KI) 0.83mg/L, sodium molybdate (NaMoO
4 . 2H
2O) 0.25mg/L, copper sulphate (CuSO
4 . 5H
2O) 0.025mg/L, cobalt chloride (CoCl
2 . 6H
2O) mixed liquor of 0.025mg/L.
Said organic principle mother liquor is meant and contains glycine 2mg/L, thiamine hydrochloride 0.1mg/L, puridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, the mixed liquor of inositol 100mg/L.
Said mother liquid of iron salt is meant and contains ferrous sulfate (FeSO
4 . 7H
2O) 27.8mg/L, Na
2 . EDTA
. 2H
2The mixed liquor of O 37.3 mg/L.
The present invention compared with prior art has the following advantages:
1, the present invention adopts the spire explant to pass through biotechnology first---and tissue culture technique carries out tissue culture, has overcome the problem that the lamiophlomis rotata growing-seedling period is long, reproduction coefficient is low; Carry out the research work of wild lamiophlomis rotata germ plasm resource in-vitro propagate simultaneously; For saving rare Tibet medicine lamivphlomis root germ plasm resource; Carry out genetic improvement, promote the factorial seedling growth of lamiophlomis rotata that a new way is provided, the tissue culture in-vitro propagation method of the high mutantion line of a kind of artificial propagation, germ plasm resource {in vitro} conservation, utilization and the seed selection pharmaceutical ingredient content that can carry out lamiophlomis rotata is provided.
2, owing to contain plant hormone 2 in the used medium of the present invention; 4-D and 6-BA, 2, the 4-D hormone belongs to the archusia class; The physiological action of this growth hormone mainly is to promote cell elongation growth and cell division, has the advantages that to induce injured tissue surface one to number confluent monolayer cells to recover splitting ability; The 6-BA hormone belongs to cytokinin, has the leading role of induced bud differentiation, characteristics such as the differentiation of promotion bud and sprouting of lateral bud growth; Therefore short, fast, the propagation frequency height of emerging of the time of bringing out, it is neat especially to emerge, and can directly become alive behind the refining seedling; Easy operating can carry out large-scale industrialized production.
3, the present invention utilizes tissue culture to exsomatize to breed fast has following several respects significance: the ⑴ reproduction speed is fast, not influenced by extraneous climatic factor, in indoor all breedings in a large number fast, promptly starts fast throughout the year; ⑵ be convenient to keep good biological property, and promptly synchronism is good; ⑶ have very important meaning aspect the good mutant or the good first generation of hybrid preserving and breed, but a strain has the lamiophlomis rotata mutant of clear superiority or offspring's large tracts of land after tissue culture of a tool clear superiority is promoted or the like.
Embodiment
Embodiment 1A kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation may further comprise the steps:
⑴ the selection of explant and disinfecting: select the lamiophlomis rotata seed of full grains, constant temperature bath 12min in 52 ℃ the water-bath places 500ppm gibberellin (GA
3) soak 1h in the solution after; On superclean bench, using volumetric concentration is 75% alcohol-pickled sterilization 15 seconds; Then with aseptic deionized water flushing 2 times, it is 0.1%% mercuric chloride that the seed after will wash again places mass concentration, soaks and sterilizes 8 minutes; And do not have on the hormone culture-medium with being inoculated into MS after the aseptic deionized water flushing 3 times, wherein every 25mlMS does not have the seed after 4 sterilizations of inoculation in the hormone culture-medium; Seed begins to sprout after 3 ~ 4 days, after 10 days with the young root of the aseptic seedling of sprouting as the initial incubation explant.
MS does not have hormone culture-medium and is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml, sucrose 30 grams; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑵ explant inoculation: explant is cut into the long segment of 0.3 ~ 0.5cm, is seeded on the callus inducing medium, inoculation 2 segment explants in wherein every 25ml callus inducing medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 10 ~ 25 days, incision is expanded the formation callus at the young root two ends.
Callus inducing medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is the 2,4 dichlorophenoxyacetic acid (2 of 0.1mg/ml; 4-D) mother liquor 0 ml, concentration is methyl (NAA) mother liquor 5 ml of 0.1mg/ml, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 10ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑶ callus propagation: callus is inoculated on the callus proliferated culture medium 2 callus of inoculation in wherein every 25ml callus proliferated culture medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in callus propagation 20 ~ 35 days, grow the callus light yellow, that graininess is fine and close.
The callus proliferated culture medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is the 2,4 dichlorophenoxyacetic acid (2 of 0.1mg/ml; 4-D) mother liquor 5ml, concentration is methyl (NAA) the mother liquor 10ml of 0.1mg/ml, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 5ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑷ the bud of callus is induced: the callus of step ⑶ gained is inoculated into lures on the bud medium, wherein every 25ml lures 2 callus of bud inoculation of medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 30 ~ 35 days, form the bud of growing thickly.
Lure the bud medium to be meant in the beaker of 1000ml, add macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 10ml of 0.1mg/ml, concentration is methyl (NAA) the mother liquor 0ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑸ the grow thickly propagation of bud: the bud of will growing thickly is inoculated on the bud proliferated culture medium, 1 bud of growing thickly of inoculation in wherein every 25ml bud proliferated culture medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 30 ~ 35 days, form the lamiophlomis rotata seedling of robust growth.
The bud proliferated culture medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 20ml of 0.1mg/ml, concentration is methyl (NAA) the mother liquor 5ml of 0.1mg/ml; Concentration be 0.1mg/ml 2,4 dichlorophenoxyacetic acid (2,4-D) mother liquor 2ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑹ root induction: the lamiophlomis rotata seedling of will growing thickly carries out plant division; Promptly in aseptic superclean bench; Open the bottle mouth sealing film; With sterile tweezers eugonic seedling is peeled from the Miao Zhongfen of growing thickly, change over to then in the root media, 1 lamiophlomis rotata seedling of growing thickly of inoculation in wherein every 25ml root media; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivate after 20 ~ 30 days, white, sturdy short root appear in the seedling base portion, and grow complete root system; Promptly get complete regeneration seedling; This complete regeneration seedling is meant the differentiation of having accomplished bud and root, and has grown vanelets, can independently carry out the immature plant of autophyting growth.
Root media is meant in the beaker of 1000ml, adds macroelement mother liquor 50ml successively, micro-mother liquor 10ml; Organic principle mother liquor 10ml, mother liquid of iron salt 5ml, citric acid mother liquor 4ml; Vc mother liquor 2ml, sucrose 30 grams, concentration is methyl (NAA) mother liquor 0 ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑺ seedling medium sterilization: seedling medium is carried out high-temperature sterilization under 115 ~ 121 ℃ of temperature, promptly get the seedling medium after sterilizing behind 15 ~ 20min.
Wherein seedling medium is meant the mixed-matrix that humus and perlite mix by the 2:1 weight ratio, and the total porosity of this seedling medium is 70% ~ 75%, and the content of organic matter is 20%.
⑻ plantlet of transplant: in the seedling medium of the little seedling direct transplantation of complete regeneration after the sterilization, refining promptly grows up to normal lamiophlomis rotata plant behind the seedling, and general survival rate is more than 70%.A complete regeneration seedling is planted in the seedling medium 10cm after the sterilization
2Scope in.
Embodiment 2A kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation may further comprise the steps:
⑴ the selection of explant and disinfecting: select the lamiophlomis rotata seed of full grains, constant temperature bath 12min in 52 ℃ the water-bath places 500ppm gibberellin (GA
3) soak 1h in the solution after; On superclean bench, using volumetric concentration is 75% alcohol-pickled sterilization 20 seconds; Then with aseptic deionized water flushing 2 times, it is 0.15% mercuric chloride that the seed after will wash again places mass concentration, soaks and sterilizes 6 minutes; And do not have on the hormone culture-medium with being inoculated into MS after the aseptic deionized water flushing 4 times, wherein every 25mlMS does not have the seed after 4 sterilizations of inoculation in the hormone culture-medium; Seed begins to sprout after 3 ~ 4 days, after 10 days with the young root of the aseptic seedling of sprouting as the initial incubation explant.
MS does not have hormone culture-medium and is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml, sucrose 30 grams; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑵ explant inoculation: explant is cut into the long segment of 0.3 ~ 0.5cm, is seeded on the callus inducing medium, inoculation 2 segment explants in wherein every 25ml callus inducing medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 10 ~ 25 days, incision is expanded the formation callus at the young root two ends.
Callus inducing medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is the 2,4 dichlorophenoxyacetic acid (2 of 0.1mg/ml; 4-D) mother liquor 2ml, concentration is methyl (NAA) the mother liquor 5ml of 0.1mg/ml, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 5ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑶ callus propagation: callus is inoculated on the callus proliferated culture medium 2 callus of inoculation in wherein every 25ml callus proliferated culture medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in callus propagation 20 ~ 35 days, grow the callus light yellow, that graininess is fine and close.
The callus proliferated culture medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is the 2,4 dichlorophenoxyacetic acid (2 of 0.1mg/ml; 4-D) mother liquor 5ml, concentration is methyl (NAA) the mother liquor 10ml of 0.1mg/ml, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 5ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑷ the bud of callus is induced: the callus of step ⑶ gained is inoculated into lures on the bud medium, wherein every 25ml lures 2 callus of bud inoculation of medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 30 ~ 35 days, form the bud of growing thickly.
Lure the bud medium to be meant in the beaker of 1000ml, add macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 20ml of 0.1mg/ml, concentration is methyl (NAA) the mother liquor 5ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑸ the grow thickly propagation of bud: the bud of will growing thickly is inoculated on the bud proliferated culture medium, 1 bud of growing thickly of inoculation in wherein every 25ml bud proliferated culture medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 30 ~ 35 days, form the lamiophlomis rotata seedling of robust growth.
The bud proliferated culture medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 20ml of 0.1mg/ml, concentration is methyl (NAA) the mother liquor 5ml of 0.1mg/ml; Concentration be 0.1mg/ml 2,4 dichlorophenoxyacetic acid (2,4-D) mother liquor 2ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑹ root induction: the lamiophlomis rotata seedling of will growing thickly carries out plant division; Promptly in aseptic superclean bench; Open the bottle mouth sealing film; With sterile tweezers eugonic seedling is peeled from the Miao Zhongfen of growing thickly, change over to then in the root media, 1 lamiophlomis rotata seedling of growing thickly of inoculation in wherein every 25ml root media; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivate after 20 ~ 30 days, white, sturdy short root appear in the seedling base portion, and grow complete root system; Promptly get complete regeneration seedling; This complete regeneration seedling is meant the differentiation of having accomplished bud and root, and has grown vanelets, can independently carry out the immature plant of autophyting growth.
Root media is meant in the beaker of 1000ml, adds macroelement mother liquor 50ml successively, micro-mother liquor 10ml; Organic principle mother liquor 10ml, mother liquid of iron salt 5ml, citric acid mother liquor 4ml; Vc mother liquor 2ml, sucrose 30 grams, concentration is methyl (NAA) the mother liquor 2ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑺ seedling medium sterilization: seedling medium is carried out high-temperature sterilization under 115 ~ 121 ℃ of temperature, promptly get the seedling medium after sterilizing behind 15 ~ 20min.
Wherein seedling medium is meant the mixed-matrix that humus and perlite mix by the 2:1 weight ratio, and the total porosity of this seedling medium is 70% ~ 75%, and the content of organic matter is 20%.
⑻ plantlet of transplant: in the seedling medium of the little seedling direct transplantation of complete regeneration after the sterilization, refining promptly grows up to normal lamiophlomis rotata plant behind the seedling, and general survival rate is more than 70%.A complete regeneration seedling is planted in the seedling medium 10cm after the sterilization
2Scope in.
Embodiment 3A kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation may further comprise the steps:
⑴ the selection of explant and disinfecting: select the lamiophlomis rotata seed of full grains, constant temperature bath 12min in 52 ℃ the water-bath places 500ppm gibberellin (GA
3) soak 1h in the solution after; On superclean bench, using volumetric concentration is 75% alcohol-pickled sterilization 25 seconds; Then with aseptic deionized water flushing 3 times, it is 0.2% mercuric chloride that the seed after will wash again places mass concentration, soaks and sterilizes 5 minutes; And do not have on the hormone culture-medium with being inoculated into MS after the aseptic deionized water flushing 6 times, wherein every 25mlMS does not have the seed after 4 sterilizations of inoculation in the hormone culture-medium; Seed begins to sprout after 3 ~ 4 days, after 10 days with the young root of the aseptic seedling of sprouting as the initial incubation explant.
MS does not have hormone culture-medium and is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml, sucrose 30 grams; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑵ explant inoculation: explant is cut into the long segment of 0.3 ~ 0.5cm, is seeded on the callus inducing medium, inoculation 2 segment explants in wherein every 25ml callus inducing medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 10 ~ 25 days, incision is expanded the formation callus at the young root two ends.
Callus inducing medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is the 2,4 dichlorophenoxyacetic acid (2 of 0.1mg/ml; 4-D) mother liquor 5ml, concentration is methyl (NAA) the mother liquor 8ml of 0.1mg/ml, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 5ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑶ callus propagation: callus is inoculated on the callus proliferated culture medium 2 callus of inoculation in wherein every 25ml callus proliferated culture medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in callus propagation 20 ~ 35 days, grow the callus light yellow, that graininess is fine and close.
The callus proliferated culture medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is the 2,4 dichlorophenoxyacetic acid (2 of 0.1mg/ml; 4-D) mother liquor 5ml, concentration is methyl (NAA) the mother liquor 10ml of 0.1mg/ml, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 5ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑷ the bud of callus is induced: the callus of step ⑶ gained is inoculated into lures on the bud medium, wherein every 25ml lures 2 callus of bud inoculation of medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 30 ~ 35 days, form the bud of growing thickly.
Lure the bud medium to be meant in the beaker of 1000ml, add macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 10ml of 0.1mg/ml, concentration is methyl (NAA) the mother liquor 5ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑸ the grow thickly propagation of bud: the bud of will growing thickly is inoculated on the bud proliferated culture medium, 1 bud of growing thickly of inoculation in wherein every 25ml bud proliferated culture medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 30 ~ 35 days, form the lamiophlomis rotata seedling of robust growth.
The bud proliferated culture medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 20ml of 0.1mg/ml, concentration is methyl (NAA) the mother liquor 5ml of 0.1mg/ml; Concentration be 0.1mg/ml 2,4 dichlorophenoxyacetic acid (2,4-D) mother liquor 2ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑹ root induction: the lamiophlomis rotata seedling of will growing thickly carries out plant division; Promptly in aseptic superclean bench; Open the bottle mouth sealing film; With sterile tweezers eugonic seedling is peeled from the Miao Zhongfen of growing thickly, change over to then in the root media, 1 lamiophlomis rotata seedling of growing thickly of inoculation in wherein every 25ml root media; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivate after 20 ~ 30 days, white, sturdy short root appear in the seedling base portion, and grow complete root system; Promptly get complete regeneration seedling; This complete regeneration seedling is meant the differentiation of having accomplished bud and root, and has grown vanelets, can independently carry out the immature plant of autophyting growth.
Root media is meant in the beaker of 1000ml, adds macroelement mother liquor 50ml successively, micro-mother liquor 10ml; Organic principle mother liquor 10ml, mother liquid of iron salt 5ml, citric acid mother liquor 4ml; Vc mother liquor 2ml, sucrose 30 grams, concentration is methyl (NAA) the mother liquor 5ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑺ seedling medium sterilization: seedling medium is carried out high-temperature sterilization under 115 ~ 121 ℃ of temperature, promptly get the seedling medium after sterilizing behind 15 ~ 20min.
Wherein seedling medium is meant the mixed-matrix that humus and perlite mix by the 2:1 weight ratio, and the total porosity of this seedling medium is 70% ~ 75%, and the content of organic matter is 20%.
⑻ plantlet of transplant: in the seedling medium of the little seedling direct transplantation of complete regeneration after the sterilization, refining promptly grows up to normal lamiophlomis rotata plant behind the seedling, and general survival rate is more than 70%.A complete regeneration seedling is planted in the seedling medium 10cm after the sterilization
2Scope in.
Embodiment 4A kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation may further comprise the steps:
⑴ the selection of explant and disinfecting: select the lamiophlomis rotata seed of full grains, constant temperature bath 12min in 52 ℃ the water-bath places 500ppm gibberellin (GA
3) soak 1h in the solution after; On superclean bench, using volumetric concentration is 75% alcohol-pickled sterilization 30 seconds; Then with aseptic deionized water flushing 3 times, it is 0.2% mercuric chloride that the seed after will wash again places mass concentration, soaks and sterilizes 6 minutes; And do not have on the hormone culture-medium with being inoculated into MS after the aseptic deionized water flushing 6 times, wherein every 25mlMS does not have the seed after 4 sterilizations of inoculation in the hormone culture-medium; Seed begins to sprout after 3 ~ 4 days, after 10 days with the young root of the aseptic seedling of sprouting as the initial incubation explant.
MS does not have hormone culture-medium and is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml, sucrose 30 grams; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑵ explant inoculation: explant is cut into the long segment of 0.3 ~ 0.5cm, is seeded on the callus inducing medium, inoculation 2 segment explants in wherein every 25ml callus inducing medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 10 ~ 25 days, incision is expanded the formation callus at the young root two ends.
Callus inducing medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is the 2,4 dichlorophenoxyacetic acid (2 of 0.1mg/ml; 4-D) mother liquor 5ml, concentration is methyl (NAA) the mother liquor 10ml of 0.1mg/ml, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 10ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑶ callus propagation: callus is inoculated on the callus proliferated culture medium 2 callus of inoculation in wherein every 25ml callus proliferated culture medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in callus propagation 20 ~ 35 days, grow the callus light yellow, that graininess is fine and close.
The callus proliferated culture medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is the 2,4 dichlorophenoxyacetic acid (2 of 0.1mg/ml; 4-D) mother liquor 5ml, concentration is methyl (NAA) the mother liquor 10ml of 0.1mg/ml, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 5ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑷ the bud of callus is induced: the callus of step ⑶ gained is inoculated into lures on the bud medium, wherein every 25ml lures 2 callus of bud inoculation of medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 30 ~ 35 days, form the bud of growing thickly.
Lure the bud medium to be meant in the beaker of 1000ml, add macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 30ml of 0.1mg/ml, concentration is methyl (NAA) the mother liquor 5ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑸ the grow thickly propagation of bud: the bud of will growing thickly is inoculated on the bud proliferated culture medium, 1 bud of growing thickly of inoculation in wherein every 25ml bud proliferated culture medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 30 ~ 35 days, form the lamiophlomis rotata seedling of robust growth.
The bud proliferated culture medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 20ml of 0.1mg/ml, concentration is methyl (NAA) the mother liquor 5ml of 0.1mg/ml; Concentration be 0.1mg/ml 2,4 dichlorophenoxyacetic acid (2,4-D) mother liquor 2ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑹ root induction: the lamiophlomis rotata seedling of will growing thickly carries out plant division; Promptly in aseptic superclean bench; Open the bottle mouth sealing film; With sterile tweezers eugonic seedling is peeled from the Miao Zhongfen of growing thickly, change over to then in the root media, 1 lamiophlomis rotata seedling of growing thickly of inoculation in wherein every 25ml root media; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivate after 20 ~ 30 days, white, sturdy short root appear in the seedling base portion, and grow complete root system; Promptly get complete regeneration seedling; This complete regeneration seedling is meant the differentiation of having accomplished bud and root, and has grown vanelets, can independently carry out the immature plant of autophyting growth.
Root media is meant in the beaker of 1000ml, adds macroelement mother liquor 50ml successively, micro-mother liquor 10ml; Organic principle mother liquor 10ml, mother liquid of iron salt 5ml, citric acid mother liquor 4ml; Vc mother liquor 2ml, sucrose 30 grams, concentration is methyl (NAA) the mother liquor 10ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑺ seedling medium sterilization: seedling medium is carried out high-temperature sterilization under 115 ~ 121 ℃ of temperature, promptly get the seedling medium after sterilizing behind 15 ~ 20min.
Wherein seedling medium is meant the mixed-matrix that humus and perlite mix by the 2:1 weight ratio, and the total porosity of this seedling medium is 70% ~ 75%, and the content of organic matter is 20%.
⑻ plantlet of transplant: in the seedling medium of the little seedling direct transplantation of complete regeneration after the sterilization, refining promptly grows up to normal lamiophlomis rotata plant behind the seedling, and general survival rate is more than 70%.A complete regeneration seedling is planted in the seedling medium 10cm after the sterilization
2Scope in.
Embodiment 5A kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation may further comprise the steps:
⑴ the selection of explant and disinfecting: select the lamiophlomis rotata seed of full grains, constant temperature bath 12min in 52 ℃ the water-bath places 500ppm gibberellin (GA
3) soak 1h in the solution after; On superclean bench, using volumetric concentration is 75% alcohol-pickled sterilization 30 seconds; Then with aseptic deionized water flushing 3 times, it is 0.2% mercuric chloride that the seed after will wash again places mass concentration, soaks and sterilizes 8 minutes; And do not have on the hormone culture-medium with being inoculated into MS after the aseptic deionized water flushing 6 times, wherein every 25mlMS does not have the seed after 4 sterilizations of inoculation in the hormone culture-medium; Seed begins to sprout after 3 ~ 4 days, after 10 days with the young root of the aseptic seedling of sprouting as the initial incubation explant.
MS does not have hormone culture-medium and is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml, sucrose 30 grams; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑵ explant inoculation: explant is cut into the long segment of 0.3 ~ 0.5cm, is seeded on the callus inducing medium, inoculation 2 segment explants in wherein every 25ml callus inducing medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 10 ~ 25 days, incision is expanded the formation callus at the young root two ends.
Callus inducing medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is the 2,4 dichlorophenoxyacetic acid (2 of 0.1mg/ml; 4-D) mother liquor 10ml, concentration is methyl (NAA) the mother liquor 10ml of 0.1mg/ml, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 20ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑶ callus propagation: callus is inoculated on the callus proliferated culture medium 2 callus of inoculation in wherein every 25ml callus proliferated culture medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in callus propagation 20 ~ 35 days, grow the callus light yellow, that graininess is fine and close.
The callus proliferated culture medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is the 2,4 dichlorophenoxyacetic acid (2 of 0.1mg/ml; 4-D) mother liquor 5ml, concentration is methyl (NAA) the mother liquor 10ml of 0.1mg/ml, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 5ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑷ the bud of callus is induced: the callus of step ⑶ gained is inoculated into lures on the bud medium, wherein every 25ml lures 2 callus of bud inoculation of medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 30 ~ 35 days, form the bud of growing thickly.
Lure the bud medium to be meant in the beaker of 1000ml, add macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 30ml of 0.1mg/ml, concentration is methyl (NAA) the mother liquor 10ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑸ the grow thickly propagation of bud: the bud of will growing thickly is inoculated on the bud proliferated culture medium, 1 bud of growing thickly of inoculation in wherein every 25ml bud proliferated culture medium; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivated 30 ~ 35 days, form the lamiophlomis rotata seedling of robust growth.
The bud proliferated culture medium is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml, organic principle mother liquor 10ml; Mother liquid of iron salt 5ml, citric acid mother liquor 4ml, Vc mother liquor 2ml; Sucrose 30 grams, concentration is 6-benzyl aminoadenine (6-BA) the mother liquor 20ml of 0.1mg/ml, concentration is methyl (NAA) the mother liquor 5ml of 0.1mg/ml; Concentration be 0.1mg/ml 2,4 dichlorophenoxyacetic acid (2,4-D) mother liquor 2ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑹ root induction: the lamiophlomis rotata seedling of will growing thickly carries out plant division; Promptly in aseptic superclean bench; Open the bottle mouth sealing film; With sterile tweezers eugonic seedling is peeled from the Miao Zhongfen of growing thickly, change over to then in the root media, 1 lamiophlomis rotata seedling of growing thickly of inoculation in wherein every 25ml root media; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Illumination box in cultivate after 20 ~ 30 days, white, sturdy short root appear in the seedling base portion, and grow complete root system; Promptly get complete regeneration seedling; This complete regeneration seedling is meant the differentiation of having accomplished bud and root, and has grown vanelets, can independently carry out the immature plant of autophyting growth.
Root media is meant in the beaker of 1000ml, adds macroelement mother liquor 50ml successively, micro-mother liquor 10ml; Organic principle mother liquor 10ml, mother liquid of iron salt 5ml, citric acid mother liquor 4ml; Vc mother liquor 2ml, sucrose 30 grams, concentration is methyl (NAA) the mother liquor 20ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
⑺ seedling medium sterilization: seedling medium is carried out high-temperature sterilization under 115 ~ 121 ℃ of temperature, promptly get the seedling medium after sterilizing behind 15 ~ 20min.
Wherein seedling medium is meant the mixed-matrix that humus and perlite mix by the 2:1 weight ratio, and the total porosity of this seedling medium is 70% ~ 75%, and the content of organic matter is 20%.
⑻ plantlet of transplant: in the seedling medium of the little seedling direct transplantation of complete regeneration after the sterilization, refining promptly grows up to normal lamiophlomis rotata plant behind the seedling, and general survival rate is more than 70%.A complete regeneration seedling is planted in the seedling medium 10cm after the sterilization
2Scope in.
Above-mentioned
Embodiment 1 ~ 5In lamiophlomis rotata belong to the herbaceos perennial lamiophlomis rotata for the Labiatae lamiophlomis rotata
(Lamiophlomis rotate (Benth.) Kudo)Superclean bench, model are BCM-1000A, are produced by SuZhou Antai Air Tech Co., Ltd..
MS do not have hormone culture-medium, callus inducing medium, callus proliferated culture medium, lure in bud medium, bud proliferated culture medium, the root media macroelement mother liquor to be meant contains ammonium nitrate (NH
4NO
3) 1650mg/L, potassium nitrate (KNO
3) 1900mg/L, magnesium sulfate (MgSO
4) 370mg/L, potassium dihydrogen phosphate (KH
2PO
4) 170mg/L, calcium chloride (CaCl
2 . 2H
2O) mixed liquor of 440mg/L; The trace element mother liquor is meant and contains manganese sulphate (MnSO
4 . 4H
2O) 22.3mg/L, zinc sulphate (ZnSO
4 . 7H
2O) 8.6mg/L, boric acid (H
3BO
3) 6.2mg/L, KI (KI) 0.83mg/L, sodium molybdate (NaMoO
4 . 2H
2O) 0.25mg/L, copper sulphate (CuSO
4 . 5H
2O) 0.025mg/L, cobalt chloride (CoCl
2 . 6H
2O) mixed liquor of 0.025mg/L; The organic principle mother liquor is meant and contains glycine 2mg/L, thiamine hydrochloride 0.1mg/L, puridoxine hydrochloride 0.5mg/L, nicotinic acid 0.5mg/L, the mixed liquor of inositol 100mg/L; Mother liquid of iron salt is meant and contains ferrous sulfate (FeSO
4 . 7H
2O) 27.8mg/L, Na
2 . EDTA
. 2H
2The mixed liquor of O 37.3 mg/L.
Claims (9)
1. lamiophlomis rotata tissue culture method for in-vitro rapid propagation may further comprise the steps:
⑴ the selection of explant and disinfecting: the lamiophlomis rotata seed that selects full grains; Constant temperature bath 12min in 52 ℃ the water-bath; After placing the 500ppm Gibberellins solution to soak 1h, using volumetric concentration is 75% alcohol-pickled sterilization 15 ~ 30 seconds, then with aseptic deionized water flushing 2 ~ 3 times; It is 0.1% ~ 0.2% mercuric chloride that seed after will washing again places mass concentration; Soak sterilization 5 ~ 8 minutes, and do not have on the hormone culture-medium with being inoculated into MS after the aseptic deionized water flushing 3 ~ 6 times, the said MS of wherein every 25ml does not have the seed after 4 said sterilizations of inoculation in the hormone culture-medium; Seed begins to sprout after 3 ~ 4 days, after 10 days with the young root of the aseptic seedling of sprouting as the initial incubation explant;
⑵ explant inoculation: said explant is cut into the long segment of 0.3 ~ 0.5cm, is seeded on the callus inducing medium, the said explant of inoculation 2 segments in the said callus inducing medium of wherein every 25ml; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Condition under cultivated 10 ~ 25 days, incision is expanded the formation callus at the young root two ends;
⑶ callus propagation: said callus is inoculated on the callus proliferated culture medium 2 said callus of inoculation in the said callus proliferated culture medium of wherein every 25ml; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Condition under callus propagation 20 ~ 35 days, grow the callus light yellow, that graininess is fine and close;
⑷ the bud of callus is induced: the callus of said step ⑶ gained is inoculated into lures on the bud medium the said 2 said callus of bud inoculation of medium that lure of wherein every 25ml; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Condition under cultivated 30 ~ 35 days, form the bud of growing thickly;
⑸ the grow thickly propagation of bud: the said bud of growing thickly is inoculated on the bud proliferated culture medium 1 said bud of growing thickly of inoculation in the said bud proliferated culture medium of wherein every 25ml; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Condition under cultivated 30 ~ 35 days, form the lamiophlomis rotata seedling of robust growth;
⑹ root induction: the said lamiophlomis rotata seedling of growing thickly is carried out plant division, after change in the root media, in the said root media of wherein every 25ml the inoculation 1 said lamiophlomis rotata seedling of growing thickly; In temperature is that 25 ℃ ± 2 ℃, intensity of illumination are 30 ~ 60 umol. m
-2. s
-1, light application time is 12 ~ 14 h .d
-1Condition under cultivate 20 ~ 30 days after, white, sturdy short root appear in the seedling base portion, and grow complete root system, promptly get complete regeneration seedling;
⑺ seedling medium sterilization: seedling medium is carried out high-temperature sterilization under 115 ~ 121 ℃ of temperature, promptly get the seedling medium after sterilizing behind 15 ~ 20min; Said seedling medium is meant the mixed-matrix that humus and perlite mix by the 2:1 weight ratio;
⑻ plantlet of transplant: in the seedling medium of the little seedling direct transplantation of said complete regeneration after the said sterilization, promptly grow up to normal lamiophlomis rotata plant behind the refining seedling; A said complete regeneration seedling is planted in the seedling medium 10cm after the said sterilization
2Scope in.
2. a kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation as claimed in claim 1, it is characterized in that: the lamiophlomis rotata among the said step ⑴ belongs to herbaceos perennial for the Labiatae lamiophlomis rotata.
3. a kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation as claimed in claim 1 is characterized in that: the MS among the said step ⑴ does not have hormone culture-medium and is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively; Trace element mother liquor 10ml, organic principle mother liquor 10ml, mother liquid of iron salt 5ml; Citric acid mother liquor 4ml, Vc mother liquor 2ml, sucrose 30 grams; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
4. a kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation as claimed in claim 1 is characterized in that: the callus inducing medium among the said step ⑵ is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml; Organic principle mother liquor 10ml, mother liquid of iron salt 5ml, citric acid mother liquor 4ml; Vc mother liquor 2ml, sucrose 30 grams, concentration is 2 of 0.1mg/ml; 4-dichlorphenoxyacetic acid mother liquor 0 ~ 10ml, concentration is methyl mother liquor 5 ~ 10ml of 0.1mg/ml, concentration is 6-benzyl aminoadenine mother liquor 5 ~ 20ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
5. a kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation as claimed in claim 1 is characterized in that: the callus proliferated culture medium among the said step ⑶ is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml; Organic principle mother liquor 10ml, mother liquid of iron salt 5ml, citric acid mother liquor 4ml; Vc mother liquor 2ml, sucrose 30 grams, concentration is 2 of 0.1mg/ml; 4-dichlorphenoxyacetic acid mother liquor 5ml, concentration is the methyl mother liquor 10ml of 0.1mg/ml, concentration is the 6-benzyl aminoadenine mother liquor 5ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
6. a kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation as claimed in claim 1 is characterized in that: the bud medium that lures among the said step ⑷ is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively; Trace element mother liquor 10ml, organic principle mother liquor 10ml, mother liquid of iron salt 5ml; Citric acid mother liquor 4ml, Vc mother liquor 2ml, sucrose 30 grams; Concentration is 6-benzyl aminoadenine mother liquor 10 ~ 30ml of 0.1mg/ml, and concentration is methyl mother liquor 0 ~ 10ml of 0.1mg/ml, after add DDW to 1000ml scale place; After stirring, add 1mM NaOH solution adjustment pH value to 5.8, add agar powder 6.0 grams at last; Every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
7. a kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation as claimed in claim 1 is characterized in that: the bud proliferated culture medium among the said step ⑸ is meant in the beaker of 1000ml, adds macroelement mother liquor 100ml successively, micro-mother liquor 10ml; Organic principle mother liquor 10ml, mother liquid of iron salt 5ml, citric acid mother liquor 4ml; Vc mother liquor 2ml, sucrose 30 grams, concentration is the 6-benzyl aminoadenine mother liquor 20ml of 0.1mg/ml; Concentration is the methyl mother liquor 5ml of 0.1mg/ml, and concentration is the 2,4 dichlorophenoxyacetic acid mother liquor 2ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
8. a kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation as claimed in claim 1 is characterized in that: the root media among the said step ⑹ is meant in the beaker of 1000ml, adds macroelement mother liquor 50ml successively, micro-mother liquor 10ml; Organic principle mother liquor 10ml, mother liquid of iron salt 5ml, citric acid mother liquor 4ml; Vc mother liquor 2ml, sucrose 30 grams, concentration is methyl mother liquor 0 ~ 20ml of 0.1mg/ml; After add DDW to 1000ml scale place, after stirring, add 1mM NaOH solution adjustment pH value to 5.8; Add agar powder 6.0 grams at last, every 25ml is packed as one bottle in the 100ml triangular flask, promptly gets through 121 ℃ of high-temperature sterilizations.
9. a kind of lamiophlomis rotata tissue culture method for in-vitro rapid propagation as claimed in claim 1, it is characterized in that: the seedling medium total porosity among the said step ⑺ is 70% ~ 75%, the content of organic matter is 20%.
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| CN103348914A (en) * | 2013-06-28 | 2013-10-16 | 西藏自治区农牧科学院蔬菜研究所 | Plant cultivation method |
| CN103444551A (en) * | 2013-09-23 | 2013-12-18 | 成都大学 | Tissue culture test-tube plantlet obtaining method achieved through lamiophlomis rotata seed induced to sprout to be explant |
| CN103461138A (en) * | 2013-09-23 | 2013-12-25 | 四川农业大学 | One-step plantlet formation medium and method for isolated culture of lamiophlomis rotata |
| CN103636474A (en) * | 2012-11-26 | 2014-03-19 | 李敏 | Rapid seedling culturing method for lamiophlomis rotata |
| CN105875405A (en) * | 2014-10-29 | 2016-08-24 | 四川深达生物科技有限公司 | Culture medium inducing proliferation of lamiophlomis rotata and preparation method thereof |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103636474A (en) * | 2012-11-26 | 2014-03-19 | 李敏 | Rapid seedling culturing method for lamiophlomis rotata |
| CN103636474B (en) * | 2012-11-26 | 2016-07-06 | 李敏 | A kind of Radix Lamiophlomidis Rotatae fast seedling-cultivating method |
| CN103348914A (en) * | 2013-06-28 | 2013-10-16 | 西藏自治区农牧科学院蔬菜研究所 | Plant cultivation method |
| CN103348914B (en) * | 2013-06-28 | 2016-03-09 | 西藏自治区农牧科学院蔬菜研究所 | A kind of lamiophlomis rotata breeding method |
| CN103444551A (en) * | 2013-09-23 | 2013-12-18 | 成都大学 | Tissue culture test-tube plantlet obtaining method achieved through lamiophlomis rotata seed induced to sprout to be explant |
| CN103461138A (en) * | 2013-09-23 | 2013-12-25 | 四川农业大学 | One-step plantlet formation medium and method for isolated culture of lamiophlomis rotata |
| CN103461138B (en) * | 2013-09-23 | 2015-10-07 | 四川农业大学 | A kind of lamiophlomis rotata isolated culture one-step seedling medium and method thereof |
| CN105875405A (en) * | 2014-10-29 | 2016-08-24 | 四川深达生物科技有限公司 | Culture medium inducing proliferation of lamiophlomis rotata and preparation method thereof |
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