CN105918125A - Method and culture medium for obtaining lonicera macranthoides regeneration plants through tissue culture - Google Patents
Method and culture medium for obtaining lonicera macranthoides regeneration plants through tissue culture Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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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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Abstract
The invention discloses a method and culture medium for obtaining lonicera macranthoides regeneration plants through tissue culture. The culture medium for obtaining the lonicera macranthoides regeneration plants is prepared from, by mass, 1,650 parts of major elements (metered by NH4NO3), 0.83 part of trace elements (metered by KI), 37.3 parts of ferric salt (metered by FeSO4.7H2O), 0.5 part of organic ingredients (), 0.1-1.5 parts of zeatin, 0.1-0.5 part of indolebutyric acid and 30,000 parts of saccharose. The differential culture medium for obtaining the lonicera macranthoides regeneration plants is high in pertinence and good in applicability. The method for obtaining the lonicera macranthoides regeneration plants through the tissue culture has the advantages of being not limited by regions, weather and seasons and being beneficial for massively producing single plants with the fine properties.
Description
Technical field
The invention belongs to field of plant tissue culture technique, relate to a kind of obtaining largeflower-like honeysuckle flower regeneration plant by tissue culture
Method and culture medium thereof.
Technical background
Largeflower-like honeysuckle flower (Lonicera macranthoides Hand.-Mazz.) is that Caprifoliaceae Lonicera perennial evergreen is wound around or straight
Vertical undershrub plant." Chinese Pharmacopoeia " regulation largeflower-like honeysuckle flower is one of main plant source of Flos Lonicerae medical material, its alabastrum green
Determination of Chlorogenic Acid is the highest, for conventional medicinal part.
The a series of kinds formed for kind of source with largeflower-like honeysuckle flower, its alabastrum is large number of, alabastrum aggregates into umbrella or bulk inflorescence,
Corolla is neat, developmental stage is consistent and the florescence concentrates, flower opens hardly, can disposably pluck, and can significantly save on producing
About plucking time and labour cost, yield is significantly higher than the plant origin of Flos Lonicerae, i.e. with the kind of Radix Ophiopogonis origin, therefore, with
The continuous extension developing largeflower-like honeysuckle flower, the requirement to its quality and quantity the most constantly strengthens.
The breeding of largeflower-like honeysuckle flower is many based on asexual propagation such as cottage propagation at present, and the variation of different cultivars tree characteristics is relatively big,
And cuttage survival rate is low, it is impossible to meet the market demand；Additionally, cultivate largeflower-like honeysuckle flower improved seeds by molecular breeding means,
Make its excellent succedaneum as Radix Ophiopogonis (Flos Lonicerae), be also problem demanding prompt solution.And by the asexual propagation of tissue culture
Not only can realize Industrialization of seeds and seedlings at short notice, also provide certain basis for transgenic technology.Largeflower-like honeysuckle flower at present
Not having perfect regenerating system, these hamper the further research to largeflower-like honeysuckle flower, especially by the hands of molecular biology
Section studies its high chlorogenic acid content, spend more and alabastrum such as is kept closed at the study mechanism of phenomenon.Therefore, it is presently required and grinds
Study carefully and promote the regeneration techniques of a set of efficient largeflower-like honeysuckle flower.
Summary of the invention
It is an object of the present invention to provide a kind of culture medium for obtaining largeflower-like honeysuckle flower regeneration plant.
Culture medium for obtaining largeflower-like honeysuckle flower regeneration plant provided by the present invention, is as division culture medium, by following matter
The nutritional labeling composition of amount part:
A great number of elements is with NH4NO3Count 1650 parts, trace element in terms of KI 0.83 part, iron salt is with FeSO4·7H2O meter 37.3
Part, organic principle in terms of nicotinic acid 0.5 part, zeatin 0.1 part-1.5 parts, indolebutyric acid 0.1 part-0.5 part and sucrose 30000 parts；
Described a great number of elements is made up of the material of following mass parts:
NH4NO31650 parts, KNO31900 parts, KH2PO4170 parts, CaCl2·2H2O 440 parts and MgSO4·7H2O
370 parts；
Described trace element is made up of the material of following mass parts:
KI 0.83 part, Na2MoO4·2H2O 0.25 part, H3BO36.2 parts, CuSO4·5H2O 0.025 part, MnSO4·H2O
16.9 parts, CoCl2·6H2O 0.025 part and ZnSO4·7H2O 8.6 parts；
Described iron salt is made up of the material of following mass parts:
Na2EDTA·2H2O 27.8 parts and FeSO4·7H2O 37.3 parts；
Described organic principle is made up of the material of following mass parts:
0.5 part of nicotinic acid, inositol 100 parts, thiamine hydrochloride 0.1 part, pyridoxine hydrochloride 0.5 part and glycine 2.0 parts.
Described culture medium consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, zeatin, indolebutyric acid, sucrose and water；
Above composition concentration in described culture medium is respectively as follows:
NH4NO3 1.65g/L、KNO3 1.90g/L、KH2PO4 0.17g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O
0.37g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、CuSO4·5H2O 0.025
mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L, hydrochloric acid
Thiamine 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, zeatin 0.1mg/L-1.5mg/L, Yin
Diindyl butanoic acid 0.1mg/L-0.5mg/L and sucrose 30g/L.
It is a further object to provide a kind of solid medium for obtaining largeflower-like honeysuckle flower regeneration plant.
Solid medium for obtaining largeflower-like honeysuckle flower regeneration plant provided by the present invention, is by coagulator and above-mentioned for obtaining
Obtain the solid medium that the culture medium of largeflower-like honeysuckle flower regeneration plant is made into.
Described coagulator concentration in described solid medium is 6g/L-8g/L；
Described coagulator is agar.
The pH value of described culture medium is 5.8-6.2.
A further object of the present invention is to provide a kind of method obtaining largeflower-like honeysuckle flower regeneration plant.
The method of acquisition largeflower-like honeysuckle flower regeneration plant provided by the present invention, comprises the steps:
It is used for obtaining on the solid medium of largeflower-like honeysuckle flower regeneration plant described in largeflower-like honeysuckle flower callus is inoculated into and carrying out point
Change and cultivate, obtain largeflower-like honeysuckle flower regeneration bud；Described largeflower-like honeysuckle flower regeneration bud is inoculated on root media and carries out training of taking root
Support, i.e. obtain largeflower-like honeysuckle flower regeneration plant.
Described largeflower-like honeysuckle flower callus is to be obtained by the method comprised the steps:
Outer for largeflower-like honeysuckle flower implant is inoculated on calli induction media and carries out inducing culture, obtain largeflower-like honeysuckle flower callus；
Described calli induction media consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, 6-benzyladenine, kinetins, indolebutyric acid, sugarcane
Sugar, agar and water；
Above composition concentration in described calli induction media is respectively as follows:
NH4NO3 1.65g/L、KNO3 1.90g/L、KH2PO4 0.17g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O
0.37g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、CuSO4·5H2O 0.025
mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L, hydrochloric acid
Thiamine 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, 6-benzyladenine 0mg/L-3.0mg/L, swash
Therbligs 0mg/L-1.5mg/L, zeatin 0-1.5mg/L, indolebutyric acid 0.1mg/L-0.5mg/L, sucrose 30g/L and agar
6g/L-8g/L；
The pH value of described inducing culture is 5.8-6.2.
Described root media consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, indolebutyric acid, sucrose, agar and water；
Above composition concentration in described root media is respectively as follows:
NH4NO3 0.825g/L、KNO3 0.95g/L、KH2PO4 0.085g/L、CaCl2·2H2O 0.22g/L、
MgSO4·7H2O 0.185g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、
CuSO4·5H2O 0.025mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6
mg/L、Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L,
Thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, indolebutyric acid 0.2mg/L-1.0mg/L,
Sucrose 10g/L and agar 5g/L-8g/L；
The pH value of described root media is 5.8-6.2.
Described inducing culture be temperature be 20 DEG C-25 DEG C, light intensity 2000Lux-4000Lux, photoperiod be 12 hours illumination/12
Hour dark, humidity is to cultivate 15 days-20 days under conditions of 50%-70%；
Described division culture medium cultivate be temperature be 20 DEG C-25 DEG C, light intensity 2000Lux-4000Lux, photoperiod be 16 hours
Illumination/8 hour are dark, humidity is to cultivate 15 days-20 days under conditions of 50%-70%；
Described root culture be temperature be 20 DEG C-25 DEG C, light intensity 2000Lux-4000Lux, photoperiod be 16 hours illumination/8
Hour dark, humidity is to cultivate 20 days-30 days under conditions of 50%-70%.
The outer implant of described largeflower-like honeysuckle flower is newborn tender leaf or the largeflower-like honeysuckle flower new life stem section of largeflower-like honeysuckle flower.
Division culture medium for obtaining largeflower-like honeysuckle flower regeneration plant provided by the present invention is with strong points, and the suitability is good.This
Bright the provided method obtaining largeflower-like honeysuckle flower regeneration plant by tissue culture, has not by area, and weather limited with season,
It is easy to large-scale production and there is the individual plant of merit.Largeflower-like honeysuckle flower, currently without feasible renovation process, identifies that ash felt hair is born
In winter, the approach of gene function arabidopsis to be depended on or Nicotiana tabacum L. isotype plant are carried out, and have largely limited to it
Molecular mechanism research.Largeflower-like honeysuckle flower tissue culture regeneration technology provides hope for solving this problem.
Accompanying drawing explanation
Fig. 1 is the regenerative process of largeflower-like honeysuckle flower, and wherein, A is that the fresh tender leaf of largeflower-like honeysuckle flower is induced through wound healing as outer implant
Cultivate the wound healing obtained；B is that largeflower-like honeysuckle flower wound healing starts differentiation；C is the clump that largeflower-like honeysuckle flower wound healing obtains through differentiation culture
Bud.
Fig. 2 is clump bud condition of rooting after root culture.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Embodiment 1, by tissue culture obtain largeflower-like honeysuckle flower regeneration plant
Method I
One, outer implant sterilization
(public can obtain from Institute of Botany, Chinese Academy of Sciences, and this material is quoted from Longhui County, Hunan Province and plants to take largeflower-like honeysuckle flower
In Chinese Academy of Sciences's Beijing Botanical Garden more than 3 years；The non-patent literature recording this material is: Tang Xiaorong, Li Wuping, Peng Xiao
Cutting edge of a knife or a sword (2005), the tissue culture and rapid proliferation of largeflower-like honeysuckle flower. Plant Physiology Communications 41 (5), 642-642.) newborn tender
Leaf is outer implant, after clear water is rinsed well, is soaked in that flowing water is outstanding rushes 1h, aseptically, with aseptic water washing 1 time, uses
Sterilize 4min with the liquor natrii hypochloritis of 2% again after the alcohol disinfecting 20s of 70%, then turn the liquor natrii hypochloritis in 1% and sterilize 4
Min, then uses aseptic water washing 3-5 time, cuts into 0.5cm3Lamellar is suck dry moisture on aseptic filter paper.
Two, wound healing inducing culture
By the blade inoculation after step one sterilizing on calli induction media, it is 25 DEG C in temperature, light intensity 2000Lux, light week
Phase is that 12 hours illumination/12 hour are dark, and humidity is to cultivate 20 days under conditions of 50%, induces wound healing (A in Fig. 1),
Centre can extremely form the wound healing (B in Fig. 1) expanded for 2-5 time by subculture.Callus has budlet point preferable to compact.
Experiment sets three repetitions, results averaged, and each repetition sets 10 outer implant.
Callus induction rate (%)=induced synthesis wound healing number/inoculation outer implant number × 100%.
Result: Callus induction rate (%) is 100%.
Described inducing culture consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, 6-benzyladenine, kinetins, indolebutyric acid, fine jade
Fat, sucrose and water；
Above composition concentration in described inducing culture is respectively as follows:
NH4NO3 1.65g/L、KNO3 1.90g/L、KH2PO4 0.17g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O
0.37g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、CuSO4·5H2O 0.025
mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L, hydrochloric acid
Thiamine 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, 6-benzyladenine 2.0mg/L, kinetins 1
Mg/L, indolebutyric acid 0.1mg/L, sucrose 30g/L and agar 7g/L；
The pH value of described inducing culture is 5.8.
Three, differentiation culture
Wound healing step 2 obtained turns and is inoculated in division culture medium, is 25 DEG C in temperature, light intensity 2000Lux, and the photoperiod is
16 hours illumination/8 hour are dark, and humidity is to cultivate 20 days under conditions of 50%, and subculture 2-3 time is to being differentiated to form bud length 3～5cm
Clump bud (C in Fig. 1).
Experiment sets three repetitions, results averaged, and each repetition sets 10 wound healing.
Differentiation rate (%)=be differentiated to form clump bud number/inoculation wound healing number × 100%.
Result: differentiation rate (%) is 100%.
Described division culture medium consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, zeatin, indolebutyric acid, agar, sucrose and water；
Above composition concentration in described division culture medium is respectively as follows:
NH4NO3 1.65g/L、KNO3 1.90g/L、KH2PO4 0.17g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O
0.37g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、CuSO4·5H2O 0.025
mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L, hydrochloric acid
Thiamine 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, zeatin 1.0mg/L, indolebutyric acid 0.1
Mg/L, sucrose 30g/L and agar 7g/L；
The pH value of described division culture medium is 5.8.
Four, root culture
Clump bud after above-mentioned steps three enrichment culture is inoculated in root media, is 25 DEG C in temperature, light intensity 2000Lux,
Photoperiod is that 16 hours illumination/8 hour are dark, and humidity is to cultivate 20 days, to growing 10～20 roots (figure under conditions of 50%
2)。
Experiment sets three repetitions, results averaged, and each repetition sets 10 clump buds.
The clump bud number of the clump bud number/inoculation of rooting rate (%)=take root.
Result: rooting rate is 100%.
Described root media consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, indolebutyric acid, sucrose, agar and water；
Above composition concentration in described root media is respectively as follows:
NH4NO3 0.825g/L、KNO3 0.95g/L、KH2PO4 0.085g/L、CaCl2·2H2O 0.22g/L、
MgSO4·7H2O 0.185g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、
CuSO4·5H2O 0.025mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6
mg/L、Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L,
Thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, indolebutyric acid 0.2mg/L, sucrose
10g/L and agar 5g/L；
The pH value of described root media is 5.8.
Method II
One, outer implant sterilization
Taking largeflower-like honeysuckle flower new life stem section is outer implant, after clear water is rinsed well, aseptically, with aseptic water washing 1 time,
Sterilize 8min with the liquor natrii hypochloritis of 2% again with after the alcohol disinfecting 30s of 70%, then with aseptic water washing 3-5 time, cut
It is cut into 1～2cm without stem section suck dry moisture on aseptic filter paper of axillalry bud.
Two, wound healing inducing culture
Newborn leaf stem section after step one sterilizing is seeded on inducing culture, is 20 DEG C in temperature, light intensity 3000Lux, light
Cycle is that 12 hours illumination/12 hour are dark, and humidity is to cultivate 15 days under conditions of 60%, centre can subculture 2-3 time to inducing
Go out wound healing.
Experiment sets three repetitions, results averaged, and each repetition sets 10 outer implant.
Callus induction rate (%)=induced synthesis wound healing number/inoculation outer implant number × 100%.
Result: Callus induction rate (%) is 100%.
Described inducing culture consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, 6-benzyladenine, kinetins, indolebutyric acid, sugarcane
Sugar and water；
Above composition concentration in described inducing culture is respectively as follows:
NH4NO3 1.65g/L、KNO3 1.90g/L、KH2PO4 0.17g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O
0.37g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、CuSO4·5H2O 0.025
mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L, hydrochloric acid
Thiamine 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, 6-benzyladenine 3.0mg/L, excitement
Element 1.0mg/L, indolebutyric acid 0.2mg/L, sucrose 30g/L and agar 7g/L；
The pH value of described inducing culture is 6.2.
Three, differentiation culture
Wound healing step 2 obtained turns and is inoculated in division culture medium, is 20 DEG C in temperature, light intensity 3000Lux, and the photoperiod is
16 hours illumination/8 hour are dark, and humidity is to cultivate 20 days under conditions of 60%, and subculture 2-3 time is to being differentiated to form clump bud.
Experiment sets three repetitions, results averaged, and each repetition sets 10 wound healing.
Differentiation rate (%)=be differentiated to form clump bud number/inoculation wound healing number × 100%.
Result: differentiation rate (%) is 100%.
Described division culture medium consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, zeatin, indolebutyric acid, agar, sucrose and water；
Above composition concentration in described division culture medium is respectively as follows:
NH4NO3 1.65g/L、KNO3 1.90g/L、KH2PO4 0.17g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O
0.37g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、CuSO4·5H2O 0.025
mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L, hydrochloric acid
Thiamine 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, zeatin 0.1mg/L, indolebutyric acid 0.2mg/L,
Sucrose 30g/L and agar 6g/L；
The pH value of described division culture medium is 6.0.
Four, root culture
Clump bud after above-mentioned steps three differentiation culture is inoculated in root media, is 20 DEG C in temperature, light intensity 3000Lux,
Photoperiod is that 16 hours illumination/16 hour are dark, and humidity is to cultivate 25 days, to growing 10～20 roots under conditions of 60%.
Experiment sets three repetitions, results averaged, and each repetition sets 10 clump buds.
The clump bud number of the clump bud number/inoculation of rooting rate (%)=take root.
Result: rooting rate is 100%.
Described root media consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, indolebutyric acid, agar, sucrose and water.
Above composition concentration in described root media is respectively as follows:
NH4NO3 0.825g/L、KNO3 0.95g/L、KH2PO4 0.085g/L、CaCl2·2H2O 0.22g/L、
MgSO4·7H2O 0.185g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、
CuSO4·5H2O 0.025mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6
mg/L、Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L,
Thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, indolebutyric acid 0.5mg/L, sucrose
10g/L and agar 5g/L；
The pH value of described root media is 5.8.
Method III
One, outer implant sterilization
The newborn blade taking largeflower-like honeysuckle flower is outer implant, and immersion flowing water hangs after rushing 2h, aseptically, rushes with sterilized water
Wash the liquor natrii hypochloritis of 2 use 2% to sterilize 8min, then with aseptic water washing 5 times, cut into 0.5 × 0.5cm blade
Suck dry moisture on aseptic filter paper.
Two, wound healing inducing culture
By the blade inoculation after step one sterilizing on calli induction media, it is 22 DEG C in temperature, light intensity 4000Lux, light week
Phase is that 12 hours illumination/12 hour are dark, and humidity is to cultivate 20 days under conditions of 70%, centre can subculture 2-3 time to inducing
Wound healing.
Described inducing culture consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, zeatin, indolebutyric acid, agar, sucrose and water；
Above composition concentration in described inducing culture is respectively as follows:
NH4NO3 1.65g/L、KNO3 1.90g/L、KH2PO4 0.17g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O
0.37g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、CuSO4·5H2O 0.025
mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L, hydrochloric acid
Thiamine 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, zeatin 1.5mg/L, indolebutyric acid 0.5
Mg/L, sucrose 30g/L and agar 7g/L；
The pH value of described inducing culture is 6.2.
Three, differentiation culture
Wound healing step 2 obtained is inoculated in division culture medium, is 20 DEG C in temperature, light intensity 4000Lux, and the photoperiod is 16
Hour illumination/8 hour are dark, and humidity is to cultivate 15 days under conditions of 70%, and subculture 2-3 time is to being differentiated to form clump bud.
Described division culture medium consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, zeatin, indolebutyric acid, agar, sucrose and water；
Above composition concentration in described division culture medium is respectively as follows:
NH4NO3 1.65g/L、KNO3 1.90g/L、KH2PO4 0.17g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O
0.37g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、CuSO4·5H2O 0.025
mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L, hydrochloric acid
Thiamine 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, zeatin 1.5mg/L, indolebutyric acid 0.5
Mg/L, sucrose 30g/L and agar 8g/L；
The pH value of described division culture medium is 6.2.
Four, root culture
Clump bud after above-mentioned steps three differentiation culture is inoculated in root media, is 25 DEG C in temperature, light intensity 4000Lux,
Photoperiod is that 16 hours illumination/8 hour are dark, and humidity is to cultivate 20-30 days, to growing 10～20 roots under conditions of 70%.
Described root media consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, indolebutyric acid, agar, sucrose and water；
Above composition concentration in described root media is respectively as follows:
NH4NO3 0.825g/L、KNO3 0.95g/L、KH2PO4 0.085g/L、CaCl2·2H2O 0.22g/L、
MgSO4·7H2O 0.185g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、
CuSO4·5H2O 0.025mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6
mg/L、Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L,
Thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, indolebutyric acid 1.0mg/L, sucrose
10g/L and agar 7g/L；
The pH value of described root media is 6.2.
Claims (10)
1., for obtaining a culture medium for largeflower-like honeysuckle flower regeneration plant, it is made up of the nutritional labeling of following mass parts:
A great number of elements is with NH4NO3Count 1650 parts, trace element in terms of KI 0.83 part, iron salt is with FeSO4·7H2O meter 37.3
Part, organic principle in terms of nicotinic acid 0.5 part, zeatin 0.1 part-1.5 parts, indolebutyric acid 0.1 part-0.5 part and sucrose 30000 parts；
Described a great number of elements is made up of the material of following mass parts:
NH4NO31650 parts, KNO31900 parts, KH2PO4170 parts, CaCl2·2H2O 440 parts and MgSO4·7H2O
370 parts；
Described trace element is made up of the material of following mass parts:
KI 0.83 part, Na2MoO4·2H2O 0.25 part, H3BO36.2 parts, CuSO4·5H2O 0.025 part, MnSO4·H2O
16.9 parts, CoCl2·6H2O 0.025 part and ZnSO4·7H2O 8.6 parts；
Described iron salt is made up of the material of following mass parts:
Na2EDTA·2H2O 27.8 parts and FeSO4·7H2O 37.3 parts；
Described organic principle is made up of the material of following mass parts:
0.5 part of nicotinic acid, inositol 100 parts, thiamine hydrochloride 0.1 part, pyridoxine hydrochloride 0.5 part and glycine 2.0 parts.
Culture medium the most according to claim 1, it is characterised in that:
Described culture medium consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, zeatin, indolebutyric acid, sucrose and water；
Above composition concentration in described culture medium is respectively as follows:
NH4NO3 1.65g/L、KNO3 1.90g/L、KH2PO4 0.17g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O
0.37g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、CuSO4·5H2O 0.025
mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L, hydrochloric acid
Thiamine 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, zeatin 0.1mg/L-1.5mg/L, Yin
Diindyl butanoic acid 0.1mg/L-0.5mg/L and sucrose 30g/L.
3., for obtaining a solid medium for largeflower-like honeysuckle flower regeneration plant, it is by described in coagulator and claim 1 or 2
The solid medium that is made into of culture medium.
Solid medium the most according to claim 3, it is characterised in that:
Described coagulator concentration in described solid medium is 6g/L-8g/L；
Described coagulator is agar.
5. according to described culture medium arbitrary in claim 1-4, it is characterised in that: the pH value of described culture medium is 5.8-6.2.
6. the method obtaining largeflower-like honeysuckle flower regeneration plant, comprises the steps:
Largeflower-like honeysuckle flower callus is inoculated in claim 3-5 and carries out differentiation culture in arbitrary described culture medium, obtain
Largeflower-like honeysuckle flower regeneration bud；Described largeflower-like honeysuckle flower regeneration bud is inoculated on root media and carries out root culture, i.e. obtain ash
Felt hairy honeysuckle regeneration plant.
Method the most according to claim 6, it is characterised in that:
Described largeflower-like honeysuckle flower callus is to be obtained by the method comprised the steps:
Outer for largeflower-like honeysuckle flower implant is inoculated on calli induction media and carries out inducing culture, obtain largeflower-like honeysuckle flower callus；
Described calli induction media consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, 6-benzyladenine, kinetins, indolebutyric acid, sugarcane
Sugar, agar and water；
Above composition concentration in described calli induction media is respectively as follows:
NH4NO3 1.65g/L、KNO3 1.90g/L、KH2PO4 0.17g/L、CaCl2·2H2O 0.44g/L、MgSO4·7H2O
0.37g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、CuSO4·5H2O 0.025
mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L, hydrochloric acid
Thiamine 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, 6-benzyladenine 0mg/L-3.0mg/L, swash
Therbligs 0mg/L-1.5mg/L, zeatin 0-1.5mg/L, indolebutyric acid 0.1mg/L-0.5mg/L, sucrose 30g/L and agar
6g/L-8g/L；
The pH value of described inducing culture is 5.8-6.2.
Method the most according to claim 6, it is characterised in that:
Described root media consists of the following composition:
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、
CuSO4·5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O、
Nicotinic acid, inositol, thiamine hydrochloride, pyridoxine hydrochloride, glycine, indolebutyric acid, sucrose, agar and water；
Above composition concentration in described root media is respectively as follows:
NH4NO3 0.825g/L、KNO3 0.95g/L、KH2PO4 0.085g/L、CaCl2·2H2O 0.22g/L、
MgSO4·7H2O 0.185g/L、KI 0.83mg/L、Na2MoO4·2H2O 0.25mg/L、H3BO3 6.2mg/L、
CuSO4·5H2O 0.025mg/L、MnSO4·H2O 16.9mg/L、CoCl2·6H2O 0.025mg/L、ZnSO4·7H2O 8.6
mg/L、Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, nicotinic acid 0.5mg/L, inositol 100mg/L,
Thiamine hydrochloride 0.1mg/L, pyridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, indolebutyric acid 0.2mg/L-1.0mg/L,
Sucrose 10g/L and agar 5g/L-8g/L；
The pH value of described root media is 5.8-6.2.
9. according to the method described in claim 6 or 7, it is characterised in that:
Described inducing culture be temperature be 20 DEG C-25 DEG C, light intensity 2000Lux-4000Lux, photoperiod be 12 hours illumination/12
Hour dark, humidity is to cultivate 15 days-20 days under conditions of 50%-70%；
Described division culture medium cultivate be temperature be 20 DEG C-25 DEG C, light intensity 2000Lux-4000Lux, photoperiod be 16 hours
Illumination/8 hour are dark, humidity is to cultivate 15 days-20 days under conditions of 50%-70%；
Described root culture be temperature be 20 DEG C-25 DEG C, light intensity 2000Lux-4000Lux, photoperiod be 16 hours illumination/8
Hour dark, humidity is to cultivate 20 days-30 days under conditions of 50%-70%.
Method the most according to claim 7, it is characterised in that: the outer implant of described largeflower-like honeysuckle flower is largeflower-like honeysuckle flower
Newborn tender leaf or largeflower-like honeysuckle flower new life stem section.
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