CN108419676A - Iris tissue culture medium (TCM) and preparation method thereof - Google Patents
Iris tissue culture medium (TCM) and preparation method thereof Download PDFInfo
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- CN108419676A CN108419676A CN201810275141.2A CN201810275141A CN108419676A CN 108419676 A CN108419676 A CN 108419676A CN 201810275141 A CN201810275141 A CN 201810275141A CN 108419676 A CN108419676 A CN 108419676A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses a kind of iris tissue culture medium (TCM)s and preparation method thereof, it includes inducing culture, proliferated culture medium, strong seedling culture base and root media and preparation method thereof, wherein, inducing culture strong seedling culture base and root media are all on the basis of MS culture mediums, according to the nutrition of each cultivation stage and technical need feature, increase the biomass nutrients ingredients such as Tomato juice, mashed carrot, apple butter, banana puree, coconut juice, increases the steroids nutritional ingredients such as adenine, 6 benayl aminopurines, methyl α-naphthyl acetate.It is used for iris tissue culture, has many advantages, such as that hormone dosage is few, breeding is fast, quantity is big, goes out full stand, variety pure, at low cost, wide adaptability, the market competitiveness can be improved, promote iris industry development.
Description
Technical field
The invention belongs to field of plant tissue culture technique, especially a kind of iris tissue culture medium (TCM) and its preparation side
Method.
Background technology
Phalaenopsis orchid has the good reputation of " queen in blue ", and flower appearance is graceful, pattern is graceful, and the florescence is up to 3~4
A month, ornamental values and the economic values were higher, received consumers.Under field conditions (factors), iris is mainly by plant division, seed
Breeding, low reproduction rate, and also due to carrying out generative propagation for a long time, band Strain increases, gradually reduces kind of property, influence to grow.
Nowadays tissue cultures have become the most common method of asexual propagation of butterfly orchid, can both keep maternal merit, avoid
The generation of variation plant, and quickly to breed filial generation, be not subject to seasonal restrictions whole year production, the advantages that being easy virus removal, adapt to
Therefore the market demand has become the most effective means of Plant Tissue Culture of Phalaenopsis hybrid using explantation tissue's culture.
In recent years, there is the report of the research to induce protocorm using explant both at home and abroad, but growth rate and proliferation are
Number is not ideal, but also there is technical sophistications, easy to produce variation, and browning phenomenon can occur in culture.
Invention content
It is an object of the invention to overcome above-mentioned problems of the prior art, a kind of iris tissue culture medium (TCM) is provided
And preparation method thereof, it simplifies incubation step, and bennet survival rate is high, and plant aberration rate is low, efficiently solves iris group
Plant is easy the technical barrier of browning during training.
The technical proposal of the invention is realized in this way：
A kind of iris tissue culture medium (TCM), it includes inducing culture, proliferated culture medium, strong seedling culture base and root media,
It is characterized in that：
The inducing culture includes following components:1/4MS culture medium pulvis 1339mg/L, 2~4mg/L of thiadiazole phenylurea,
1900~2300mg/L of peptone, 25~35mg/L of citric acid, 25000~35000mg/L of sugar, 5500~6500mg/L of agar,
2~4mg/L of 6-benzyl aminopurine, 80~120mg/L of coconut juice；
The proliferated culture medium includes following components:It spends No. 1 400~600mg/L of treasured, spend precious No. 2 1200~1800mg/L, fleshes
80~120mg/L of alcohol, it is apt to deposit 280~320mg/L of piece, 900~1100mg/L of peptone, 16000~20000mg/ of Tomato juice
L, 16000~20000mg/L of mashed carrot, 16000~20000mg/L of apple butter, 16000~20000mg/L of banana puree, coconut juice
80~120mg/L, 7~9mg/L of Rosmarinus officinalis extract, 800~1200mg/L of polyvinylpyrrolidone, adenine 0.8~
1.2mg/L, 0.8~1.2mg/L of 6-benzyl aminopurine, 0.8~1.2mg/L of methyl α-naphthyl acetate, 18000~22000mg/L of sugar, agar
5500~6500mg/L；
The strong seedling culture base includes following components:1/2MS culture medium pulvis 2471mg/L, 80~120mg/L of inositol, lemon
25~35mg/L of acid, 900~1100mg/L of peptone, it is kind deposit 280~320mg/L of piece, 16000~20000mg/L of Tomato juice,
16000~20000mg/L of mashed carrot, 16000~20000mg/L of apple butter, 16000~20000mg/L of banana puree, coconut juice 80
~120mg/L, 7~9mg/L of Rosmarinus officinalis extract, 800~1200mg/L of polyvinylpyrrolidone, 800~1200mg/ of activated carbon
L, 16000~20000mg/L of sugar, 550~650mg/L of agar；
The root media includes following components:1/2MS culture medium pulvis 2471mg/L, 25~35mg/L of citric acid, albumen
900~1100mg/L of peptone, 900~1100mg/L of lactoalbumin hydrolysate, it is kind deposit 280~320mg/L of piece, Tomato juice 16000~
20000mg/L, 16000~20000mg/L of mashed carrot, 16000~20000mg/L of apple butter, banana puree 16000~
20000mg/L, 80~120mg/L of coconut juice, 7~9mg/L of Rosmarinus officinalis extract, 800~1200mg/L of polyvinylpyrrolidone, work
800~1200mg/L of property charcoal, 2~4mg/L of methyl α-naphthyl acetate, 0.4~0.6mg/L of indolebutyric acid, 16000~20000mg/L of sugar, agar
5000~7000mg/L.
The present invention additionally provides a kind of iris tissue culture medium (TCM) on the basis of above-mentioned iris tissue cultures based formulas
Preparation method, it includes the preparation method of inducing culture, proliferated culture medium, strong seedling culture base and root media, special
Sign is：
The preparation method of the inducing culture weighs thiadiazole phenylurea, peptone, citric acid, sugar, agar by formula rate
In container, add distilled water, dissolves by heating；Formula rate is pressed again, and 1/4MS culture mediums pulvis, 6-benzyl aminopurine, coconut juice is added
It stirs evenly；Distilled water is added to be settled to 1L；Then it is 5.8 to adjust pH value；It is finally divided in 20 small triangular flasks, is sealed with sealed membrane
Tight bottleneck, in 121~126 DEG C of high temperature, 0.1~0.14 megapascal of high pressure sterilizes 15~20 minutes, and taking-up shakes up cooling be made while hot
Inducing culture；
The preparation method of the proliferated culture medium is weighed by formula rate and spends treasured 1, spend No. 2 precious, inositol, be apt to deposit piece, albumen
Peptone, Rosmarinus officinalis extract, polyvinylpyrrolidone, sugar, agar add distilled water in container, dissolve by heating；Formula rate is pressed again
Addition Tomato juice, mashed carrot, apple butter, banana puree, coconut juice, adenine, 6-benzyl aminopurine, methyl α-naphthyl acetate stir evenly；Add
Distilled water is settled to 1L；Then it is 5.8 to adjust pH value；It is finally divided in 12 iris special culture bottles, bottle is stoppered with rubber plug
Mouthful, then it is tamping bottleneck with sealed membrane, in 121~126 DEG C of high temperature, 0.1~0.14 megapascal of high pressure sterilizes 15~20 minutes, takes out
Cooling obtained proliferated culture medium is shaken up while hot；
The preparation method of the strong seedling culture base, by formula rate weigh inositol, citric acid, peptone, it is kind deposit piece, rosemary carries
Take object, polyvinylpyrrolidone, activated carbon, sugar, agar, in container, add distilled water, dissolve by heating；Add again by formula rate
Enter 1/2MS culture mediums pulvis, Tomato juice, mashed carrot, apple butter, banana puree, coconut juice stir evenly；Distilled water is added to be settled to
1L；Then it is 5.8 to adjust pH value；It is finally divided in 12 iris special culture bottles, bottleneck is stoppered with rubber plug, then with sealing
Film is tamping bottleneck, and in 121~126 DEG C of high temperature, 0.1~0.14 megapascal of high pressure sterilizes 15~20 minutes, and taking-up shakes up cooling while hot
Strong seedling culture base is made；
The preparation method of the root media weighs citric acid by formula rate, peptone, lactoalbumin hydrolysate, is apt to deposit piece, fan
Repeatedly fragrant extract, polyvinylpyrrolidone, activated carbon, sugar, agar add distilled water in container, dissolve by heating；Recipe ratio is pressed again
1/2MS culture mediums pulvis, Tomato juice, mashed carrot, apple butter, banana puree, coconut juice, methyl α-naphthyl acetate, indolebutyric acid stirring is added in example
Uniformly；Distilled water is added to be settled to 1L；Then it is 5.8 to adjust pH value；It is finally divided in 12 iris special culture bottles, uses glue
Bottleneck is stoppered, then bottleneck is tamping with sealed membrane, in 121~126 DEG C of high temperature, 0.1~0.14 megapascal of high pressure, sterilizing 15~20
Minute, taking-up shakes up cooling obtained root media while hot.
The 1/2MS culture mediums pulvis has on the market by the prepared product of standard proportional, and here is to prepare 1L 1/
The dosage of each ingredient of 2MS culture medium nutrient solutions：Potassium nitrate KNO3950mg, ammonium nitrate NH4NO3825mg, potassium dihydrogen phosphate
KH2PO485mg, magnesium sulfate MgSO4·7H2O185mg, calcium chloride CaCl2·2H2O220mg, potassium iodide KI0.83mg, boric acid
H3BO36.2mg, manganese sulfate MnSO4·4H2O22.3mg, zinc sulfate ZnSO4·7H2O8.6mg, sodium molybdate Na2MoO4·
2H2O0.25mg, copper sulphate CuSO4·5H2O0.025mg, cobalt chloride CoCl2·6H2O0.025mg, disodium ethylene diamine tetraacetate
Na2EDTA37.25mg, ferrous sulfate FeSO4·7H2O27.85mg, inositol 100mg, glycine 2mg, thiamine hydrochloride
0.1mg, puridoxine hydrochloride 0.5mg, niacin 0.5mg.
The 1/4MS culture mediums pulvis has on the market by the prepared product of standard proportional, and here is to prepare 1L 1/
The dosage of each ingredient of 2MS culture medium nutrient solutions：Potassium nitrate KNO3475mg, ammonium nitrate NH4NO3412.5mg, potassium dihydrogen phosphate
KH2PO442.5mg, magnesium sulfate MgSO4·7H2O92.5mg, calcium chloride CaCl2·2H2O110mg, potassium iodide KI0.83mg, boric acid
H3BO36.2mg, manganese sulfate MnSO4·4H2O22.3mg, zinc sulfate ZnSO4·7H2O8.6mg, sodium molybdate Na2MoO4·
2H2O0.25mg, copper sulphate CuSO4·5H2O0.025mg, cobalt chloride CoCl2·6H2O0.025mg, disodium ethylene diamine tetraacetate
Na2EDTA37.25mg, ferrous sulfate FeSO4·7H2O27.85mg, inositol 100mg, glycine 2mg, thiamine hydrochloride
0.1mg, puridoxine hydrochloride 0.5mg, niacin 0.5mg.
The sugar is preferably sucrose.
The Tomato juice is to clean tomato, using alms bowl is smashed to pieces or tissue mashing machine smashs to pieces, is filtered out western red
The juice that persimmon skin and seed obtain.
The Rosmarinus officinalis extract is prepared with the following method：The stem and leaf of dry rosemary are taken, is crushed, with 40~95%
Alcohol steep 1~3 time 1~3 hour every time, merges extracting solution, and recycling ethyl alcohol is concentrated under reduced pressure, obtains concentrate；By concentrate
Add water to staticly settle, filter, take filtrate to carry out ultrafiltration membrance filter, ultrafiltrate is concentrated under reduced pressure, dry, as rosemary extracts
Object.Rosmarinus officinalis extract is a kind of antioxidant, has bactericidal effect, is conducive to that culture medium is fresh-keeping to guarantee the quality.
The kind piece of depositing is vitamin and minerals class non-prescribed medicine drug, it contains multivitamin and minerals member
Element, for preventing and treating the various diseases caused by vitamin and mineral deficiency.Kind on domestic market deposits piece
Have and is apt to deposit multivitamin and minerals tablet (29), is apt to deposit good dimension piece, Centrum Junior piece, is apt to deposit small good dimension, Centrum Silver.The present invention selects kind
It deposits piece and does good and deposit multivitamin and minerals tablet (29), kind deposit good dimension piece, Centrum Junior piece, kind one kind deposited in small good peacekeeping Centrum Silver；
It is preferred that kind deposit multivitamin and minerals tablet (29).
The apple butter is to be enucleated apple peel, using smashing alms bowl to pieces or tissue mashing machine smashs to pieces to obtain apple butter.
The mashed carrot is that carrot is cleaned decaptitating to truncate, using smashing alms bowl to pieces or tissue mashing machine smashs to pieces to obtain recklessly
Radish mud.
The banana puree is to remove the peel banana, using smashing alms bowl to pieces or tissue mashing machine smashs to pieces to obtain banana puree.
The coconut juice is the juice inside fresh coconut.
The present invention compared to the prior art the advantages of be：
（1）It is directly inoculated into the induction for carrying out Multiple Buds in inducing culture using explant, Multiple Buds are cut and are inoculated into increasing
It grows progress shoot proliferation culture, last strengthening seedling and rooting in culture medium and cultivates the tissue-culture container seedling for meeting transplanting standard.Its advantage exists
In spanning the protocorm stage, seedling directly is grown from Multiple Buds, simplifies incubation step, reduce production cost, regeneration is planted
Strain aberration rate is low, can keep the good characteristic of maternal plant well.
（2）Using the present invention culture medium carry out iris tissue cultures, survival rate be 94~98%, pollution rate be 2~
3%, breeding coefficient is 8~10 times.
（3）Present invention uses asked browning common in iris tissue culture containing natural, synthesis additive culture medium
Topic plays effective inhibiting effect, forms a kind of method of compound anti-browning.Concrete measure is：Disappear using in explant
It is impregnated 10 minutes with ascorbic acid and sodium thiosulfate in poison；Addition polyphenol oxidase enzyme inhibitor citric acid, quinone in the medium
Substance activated carbon of sorbent and polyvinylpyrrolidone；Thiadiazole phenylurea is added in inducing culture；Proliferation, strong sprout, life
Rosmarinus officinalis extract is genial deposits piece for addition in root culture medium.Iris tissue culture medium browning degree more used at present is low 20~
30%。
（4）The organic matter added in the culture medium that the present invention uses for example coconut milk, caseinhydrolysate, peptone, banana puree,
Mashed carrot, Tomato juice, apple butter can be improved iris it is fast it is numerous during breeding coefficient, promote the seedling grown neat, strong
It is strong, it solves that the commercial seedling output that orchid in factory produces in existing method is low, florescence inconsistence problems, has greatly existing
Real application value.
（5）Using the present invention optimize after condition of culture, such as cut the size of explant, the process of disinfection, time length,
The control etc. that number of days, intensity of illumination, light application time, humiture are secretly trained when culture, can further increase Phalaenopsis Explant in Vitro
Bud ratio, proliferation rate and rooting rate.
（6）The present invention for iris tissue culture have hormone dosage is few, breeding is fast, quantity is big, go out full stand, variety pure, at
The market competitiveness can be improved in the advantages that this is low, wide adaptability, promotes iris industry development.Iris tissue training after optimization
The condition of supporting is suitable for the large-scale industrialized production of iris.
Specific implementation mode
To keep technical scheme of the present invention clearer, embodiment of the present invention is made into one below by specific embodiment
Step ground detailed description.
Embodiment 1：A kind of iris tissue culture medium (TCM), it include inducing culture, proliferated culture medium, strong seedling culture base and
Root media；
The inducing culture specific formula is as follows:1/4MS culture mediums 1339mg/L, thiadiazole phenylurea 3mg/L, peptone
2000mg/L, citric acid 30mg/L, sucrose 30000mg/L, agar 6000mg/L, 6-benzyl aminopurine 3mg/L, coconut juice 100mg/
L, it is settled to the distilled water needed for 1L；
The proliferated culture medium consists of the following compositions:Spend No. 1 500mg/L of treasured, spend No. 2 1500mg/L of treasured, inositol 100mg/L,
Multivitamin and minerals tablet (29) 300mg/L, peptone 1000mg/L, Tomato juice 18000mg/L, mashed carrot 18000mg/L, apple
Mud 18000mg/L, banana puree 18000mg/L, coconut juice 100mg/L, Rosmarinus officinalis extract 8mg/L, polyvinylpyrrolidone
1000mg/L, adenine 1mg/L, 6-benzyl aminopurine 1mg/L, methyl α-naphthyl acetate 1mg/L, sucrose 20000mg/L, agar 6000mg/
L, it is settled to the distilled water needed for 1L；
The strong seedling culture base consists of the following compositions:1/2MS culture mediums 2471mg/L, inositol 100mg/L, citric acid 30mg/
L, peptone 1000mg/L, multivitamin and minerals tablet (29) 300mg/L, Tomato juice 18000mg/L, mashed carrot 18000mg/L, apple
Puree 18000mg/L, banana puree 18000mg/L, coconut juice 100mg/L, Rosmarinus officinalis extract 8mg/L, polyvinylpyrrolidone
1000mg/L, activated carbon 1000mg/L, sucrose 18000mg/L, agar 600mg/L, it is settled to distilled water needed for 1L；
The root media consists of the following compositions:1/2MS culture mediums 2471mg/L, citric acid 30mg/L, peptone
1000mg/L, lactoalbumin hydrolysate 1000mg/L, multivitamin and minerals tablet (29) 300mg/L, Tomato juice 18000mg/L, mashed carrot
18000mg/L, apple butter 18000mg/L, banana puree 18000mg/L, coconut juice 100mg/L, Rosmarinus officinalis extract 8mg/L, poly- second
Alkene pyrrolidone 1000mg/L, activated carbon 1000mg/L, methyl α-naphthyl acetate 3mg/L, indolebutyric acid 0.5mg/L, sucrose 18000mg/L,
Agar 6000mg/L, it is settled to distilled water needed for 1L.
Embodiment 2：A kind of preparation method of iris tissue culture medium (TCM) is pressed such as respectively using the formula rate of embodiment 1
Lower step and method prepare inducing culture, proliferated culture medium, strong seedling culture base and root media；
The preparation method of inducing culture weighs thiadiazole phenylurea, peptone, citric acid, sucrose, agar in container in proportion
In, add distilled water, dissolves by heating；1/4MS culture mediums, 6-benzyl aminopurine, coconut juice is proportionally added into again to stir evenly；Add distillation
Water is settled to 1L；Then it is 5.8 to adjust pH value；It is finally divided in 20 small triangular flasks, bottleneck is tamping with sealed membrane, in high temperature
123 DEG C, 0.12 megapascal of high pressure sterilizes 18 minutes, and taking-up shakes up cooling obtained inducing culture while hot；
The preparation method of proliferated culture medium weighs and spends treasured 1, spends No. 2 precious, inositol, multivitamin and minerals tablet (29), albumen in proportion
Peptone, Rosmarinus officinalis extract, polyvinylpyrrolidone, sucrose, agar add distilled water in container, dissolve by heating；Add in proportion again
Enter Tomato juice, mashed carrot, apple butter, banana puree, coconut juice, adenine, 6-benzyl aminopurine, methyl α-naphthyl acetate stir evenly；Add steaming
Distilled water is settled to 1L；Then it is 5.8 to adjust pH value；It is finally divided in 12 iris special culture bottles, bottleneck is stoppered with rubber plug,
It is tamping bottleneck with sealed membrane again, in 123 DEG C of high temperature, 0.12 megapascal of high pressure sterilizes 18 minutes, and taking-up shakes up cooling be made and increases while hot
Grow culture medium；
The preparation method of strong seedling culture base weighs inositol, citric acid, peptone, multivitamin and minerals tablet (29), rosemary and carries in proportion
Take object, polyvinylpyrrolidone, activated carbon, sucrose, agar, in container, add distilled water, dissolve by heating；It is proportionally added into again
1/2MS culture mediums, Tomato juice, mashed carrot, apple butter, banana puree, coconut juice stir evenly；Distilled water is added to be settled to 1L；Then
It is 5.8 to adjust pH value；It is finally divided in 12 iris special culture bottles, stoppers bottleneck with rubber plug, then bottle is tamping with sealed membrane
Mouthful, in 123 DEG C of high temperature, 0.12 megapascal of high pressure sterilizes 18 minutes, and taking-up shakes up cooling obtained strong seedling culture base while hot；
The preparation method of root media weighs citric acid, peptone, lactoalbumin hydrolysate, multivitamin and minerals tablet (29), fan in proportion
Repeatedly fragrant extract, polyvinylpyrrolidone, activated carbon, sucrose, agar add distilled water in container, dissolve by heating；Again in proportion
Addition 1/2MS culture mediums, Tomato juice, mashed carrot, apple butter, banana puree, coconut juice, methyl α-naphthyl acetate, indolebutyric acid stir evenly；
Distilled water is added to be settled to 1L；Then it is 5.8 to adjust pH value；It is finally divided in 12 iris special culture bottles, is stoppered with rubber plug
Bottleneck, then it is tamping bottleneck with sealed membrane, in 123 DEG C of high temperature, 0.12 megapascal of high pressure sterilizes 18 minutes, and taking-up shakes up cooling while hot
Root media is made.
Embodiment 3：A kind of iris tissue culture medium (TCM), it include inducing culture, proliferated culture medium, strong seedling culture base and
Root media；
The inducing culture specific formula is as follows:1/4MS culture mediums 1339mg/L, thiadiazole phenylurea 2mg/L, peptone
1950mg/L, citric acid 30mg/L, sucrose 32000mg/L, agar 6300mg/L, 6-benzyl aminopurine 2mg/L, coconut juice 120mg/
L, it is settled to the distilled water needed for 1L；
The proliferated culture medium consists of the following compositions:Spend No. 1 420mg/L of treasured, spend No. 2 1300mg/L of treasured, inositol 120mg/L,
Multivitamin and minerals tablet (29) 320mg/L, peptone 900mg/L, Tomato juice 20000mg/L, mashed carrot 16000mg/L, apple butter
17000mg/L, banana puree 19000mg/L, coconut juice 80mg/L, Rosmarinus officinalis extract 9mg/L, polyvinylpyrrolidone 900mg/L,
Adenine 1.2mg/L, 6-benzyl aminopurine 0.8mg/L, methyl α-naphthyl acetate 1.2mg/L, sucrose 19000mg/L, agar 5800mg/L, determine
Hold to the distilled water needed for 1L；
The strong seedling culture base consists of the following compositions:1/2MS culture mediums 2471mg/L, inositol 80mg/L, citric acid 25mg/
L, peptone 1100mg/L, multivitamin and minerals tablet (29) 310mg/L, Tomato juice 20000mg/L, mashed carrot 16000mg/L, apple
Puree 17000mg/L, banana puree 19000mg/L, coconut juice 120mg/L, Rosmarinus officinalis extract 7mg/L, polyvinylpyrrolidone
900mg/L, activated carbon 1100mg/L, sucrose 17000mg/L, agar 580mg/L, it is settled to distilled water needed for 1L；
The root media consists of the following compositions:1/2MS culture mediums 2471mg/L, citric acid 35mg/L, peptone
900mg/L, lactoalbumin hydrolysate 1100mg/L, multivitamin and minerals tablet (29) 290mg/L, Tomato juice 16000mg/L, mashed carrot
20000mg/L, apple butter 19000mg/L, banana puree 17000mg/L, coconut juice 120mg/L, Rosmarinus officinalis extract 7mg/L, poly- second
Alkene pyrrolidone 900mg/L, activated carbon 1100mg/L, methyl α-naphthyl acetate 4mg/L, indolebutyric acid 0.4mg/L, sucrose 17000mg/L, fine jade
Fat 5500mg/L, it is settled to distilled water needed for 1L.
Embodiment 4：A kind of iris tissue culture medium (TCM), it include inducing culture, proliferated culture medium, strong seedling culture base and
Root media；
The inducing culture specific formula is as follows:1/4MS culture mediums 1339mg/L, thiadiazole phenylurea 4mg/L, peptone
2100mg/L, citric acid 25mg/L, sucrose 28000mg/L, agar 5700mg/L, 6-benzyl aminopurine 4mg/L, coconut juice 80mg/
L, it is settled to the distilled water needed for 1L；
The proliferated culture medium consists of the following compositions:No. 1 580mg/L of treasured is spent, No. 2 1700mg/L of treasured is spent, is inositol 80mg/L, more
Tie up member plain piece (29) 280mg/L, peptone 1100mg/L, Tomato juice 16000mg/L, mashed carrot 20000mg/L, apple butter
19000mg/L, banana puree 17000mg/L, coconut juice 120mg/L, Rosmarinus officinalis extract 7mg/L, polyvinylpyrrolidone 1100mg/
L, adenine 0.8mg/L, 6-benzyl aminopurine 1.2mg/L, methyl α-naphthyl acetate 0.8mg/L, sucrose 21000mg/L, agar 6200mg/L,
It is settled to the distilled water needed for 1L；
The strong seedling culture base consists of the following compositions:1/2MS culture mediums 2471mg/L, inositol 120mg/L, citric acid 35mg/
L, peptone 900mg/L, multivitamin and minerals tablet (29) 290mg/L, Tomato juice 16000mg/L, mashed carrot 20000mg/L, apple
Mud 19000mg/L, banana puree 17000mg/L, coconut juice 80mg/L, Rosmarinus officinalis extract 9mg/L, polyvinylpyrrolidone
1100mg/L, activated carbon 900mg/L, sucrose 19000mg/L, agar 620mg/L, it is settled to distilled water needed for 1L；
The root media consists of the following compositions:1/2MS culture mediums 2471mg/L, citric acid 25mg/L, peptone
1100mg/L, lactoalbumin hydrolysate 900mg/L, multivitamin and minerals tablet (29) 310mg/L, Tomato juice 20000mg/L, mashed carrot
16000mg/L, apple butter 17000mg/L, banana puree 19000mg/L, coconut juice 80mg/L, Rosmarinus officinalis extract 9mg/L, polyethylene
Pyrrolidones 1100mg/L, activated carbon 900mg/L, methyl α-naphthyl acetate 2mg/L, indolebutyric acid 0.6mg/L, sucrose 19000mg/L, agar
6500mg/L, it is settled to distilled water needed for 1L.
Claims (3)
1. a kind of iris tissue culture medium (TCM), it includes inducing culture, proliferated culture medium, strong seedling culture base and culture of rootage
Base, it is characterised in that：
The inducing culture includes following components:1/4MS culture medium pulvis 1339mg/L, 2~4mg/L of thiadiazole phenylurea,
1900~2300mg/L of peptone, 25~35mg/L of citric acid, 25000~35000mg/L of sugar, 5500~6500mg/L of agar,
2~4mg/L of 6-benzyl aminopurine, 80~120mg/L of coconut juice；
The proliferated culture medium includes following components:It spends No. 1 400~600mg/L of treasured, spend precious No. 2 1200~1800mg/L, fleshes
80~120mg/L of alcohol, it is apt to deposit 280~320mg/L of piece, 900~1100mg/L of peptone, 16000~20000mg/ of Tomato juice
L, 16000~20000mg/L of mashed carrot, 16000~20000mg/L of apple butter, 16000~20000mg/L of banana puree, coconut juice
80~120mg/L, 7~9mg/L of Rosmarinus officinalis extract, 800~1200mg/L of polyvinylpyrrolidone, adenine 0.8~
1.2mg/L, 0.8~1.2mg/L of 6-benzyl aminopurine, 0.8~1.2mg/L of methyl α-naphthyl acetate, 18000~22000mg/L of sugar, agar
5500~6500mg/L；
The strong seedling culture base includes following components:1/2MS culture medium pulvis 2471mg/L, 80~120mg/L of inositol, lemon
25~35mg/L of acid, 900~1100mg/L of peptone, it is kind deposit 280~320mg/L of piece, 16000~20000mg/L of Tomato juice,
16000~20000mg/L of mashed carrot, 16000~20000mg/L of apple butter, 16000~20000mg/L of banana puree, coconut juice 80
~120mg/L, 7~9mg/L of Rosmarinus officinalis extract, 800~1200mg/L of polyvinylpyrrolidone, 800~1200mg/ of activated carbon
L, 16000~20000mg/L of sugar, 550~650mg/L of agar；
The root media includes following components:1/2MS culture medium pulvis 2471mg/L, 25~35mg/L of citric acid, albumen
900~1100mg/L of peptone, 900~1100mg/L of lactoalbumin hydrolysate, it is kind deposit 280~320mg/L of piece, Tomato juice 16000~
20000mg/L, 16000~20000mg/L of mashed carrot, 16000~20000mg/L of apple butter, banana puree 16000~
20000mg/L, 80~120mg/L of coconut juice, 7~9mg/L of Rosmarinus officinalis extract, 800~1200mg/L of polyvinylpyrrolidone, work
800~1200mg/L of property charcoal, 2~4mg/L of methyl α-naphthyl acetate, 0.4~0.6mg/L of indolebutyric acid, 16000~20000mg/L of sugar, agar
5000~7000mg/L.
2. iris tissue culture medium (TCM) according to claim 1, it is characterised in that：The Rosmarinus officinalis extract is using as follows
It is prepared by method：The stem and leaf of dry rosemary are taken, is crushed, with 40~95% alcohol steep 1~3 time, 1~3 hour every time, is closed
And extracting solution, recycling ethyl alcohol is concentrated under reduced pressure, obtains concentrate；Concentrate plus water are staticly settled, filtered, filtrate is taken to carry out ultrafiltration
Ultrafiltrate is concentrated under reduced pressure membrane filtration, dry, as Rosmarinus officinalis extract.
3. the Preparation Method of iris tissue culture medium (TCM) described in claims 1 or 2, it include inducing culture, proliferated culture medium,
The preparation method of strong seedling culture base and root media, it is characterised in that：
The preparation method of the inducing culture weighs thiadiazole phenylurea, peptone, citric acid, sugar, agar by formula rate
In container, add distilled water, dissolves by heating；Formula rate is pressed again, and 1/4MS culture mediums pulvis, 6-benzyl aminopurine, coconut juice is added
It stirs evenly；Distilled water is added to be settled to 1L；Then it is 5.8 to adjust pH value；It is finally divided in 20 small triangular flasks, is sealed with sealed membrane
Tight bottleneck, in 121~126 DEG C of high temperature, 0.1~0.14 megapascal of high pressure sterilizes 15~20 minutes, and taking-up shakes up cooling be made while hot
Inducing culture；
The preparation method of the proliferated culture medium is weighed by formula rate and spends treasured 1, spend No. 2 precious, inositol, be apt to deposit piece, albumen
Peptone, Rosmarinus officinalis extract, polyvinylpyrrolidone, sugar, agar add distilled water in container, dissolve by heating；Formula rate is pressed again
Addition Tomato juice, mashed carrot, apple butter, banana puree, coconut juice, adenine, 6-benzyl aminopurine, methyl α-naphthyl acetate stir evenly；Add
Distilled water is settled to 1L；Then it is 5.8 to adjust pH value；It is finally divided in 12 iris special culture bottles, bottle is stoppered with rubber plug
Mouthful, then it is tamping bottleneck with sealed membrane, in 121~126 DEG C of high temperature, 0.1~0.14 megapascal of high pressure sterilizes 15~20 minutes, takes out
Cooling obtained proliferated culture medium is shaken up while hot；
The preparation method of the strong seedling culture base, by formula rate weigh inositol, citric acid, peptone, it is kind deposit piece, rosemary carries
Take object, polyvinylpyrrolidone, activated carbon, sugar, agar, in container, add distilled water, dissolve by heating；Add again by formula rate
Enter 1/2MS culture mediums pulvis, Tomato juice, mashed carrot, apple butter, banana puree, coconut juice stir evenly；Distilled water is added to be settled to
1L；Then it is 5.8 to adjust pH value；It is finally divided in 12 iris special culture bottles, bottleneck is stoppered with rubber plug, then with sealing
Film is tamping bottleneck, and in 121~126 DEG C of high temperature, 0.1~0.14 megapascal of high pressure sterilizes 15~20 minutes, and taking-up shakes up cooling while hot
Strong seedling culture base is made；
The preparation method of the root media weighs citric acid by formula rate, peptone, lactoalbumin hydrolysate, is apt to deposit piece, fan
Repeatedly fragrant extract, polyvinylpyrrolidone, activated carbon, sugar, agar add distilled water in container, dissolve by heating；Recipe ratio is pressed again
1/2MS culture mediums pulvis, Tomato juice, mashed carrot, apple butter, banana puree, coconut juice, methyl α-naphthyl acetate, indolebutyric acid stirring is added in example
Uniformly；Distilled water is added to be settled to 1L；Then it is 5.8 to adjust pH value；It is finally divided in 12 iris special culture bottles, uses glue
Bottleneck is stoppered, then bottleneck is tamping with sealed membrane, in 121~126 DEG C of high temperature, 0.1~0.14 megapascal of high pressure, sterilizing 15~20
Minute, taking-up shakes up cooling obtained root media while hot.
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Application publication date: 20180821 |