CN105918125B - The method and its culture medium of largeflower-like honeysuckle flower regeneration plant are obtained by tissue cultures - Google Patents
The method and its culture medium of largeflower-like honeysuckle flower regeneration plant are obtained by tissue cultures Download PDFInfo
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses methods and its culture medium that largeflower-like honeysuckle flower regeneration plant is obtained by tissue cultures.This hair provides a kind of culture medium for obtaining largeflower-like honeysuckle flower regeneration plant, is made of the nutritional ingredient of following mass parts：A great number of elements is with NH4NO31650 parts of meter, 0.83 part in terms of KI of trace element, molysite are with FeSO4·7H230000 parts of 37.3 parts of O meters, 0.5 part in terms of niacin of organic principle, 0.1 part 1.5 parts of zeatin, 0.1 part 0.5 part of indolebutyric acid and sucrose.Differential medium provided by the present invention for obtaining largeflower-like honeysuckle flower regeneration plant is with strong points, and applicability is good.The method provided by the present invention for obtaining largeflower-like honeysuckle flower regeneration plant by tissue cultures has not by area, weather and season limit, convenient for single plant of the large-scale production with merit.
Description
Technical field
The invention belongs to field of plant tissue culture technique, are related to a kind of by tissue cultures acquisition largeflower-like honeysuckle flower regeneration
The method and its culture medium of plant.
Technical background
Largeflower-like honeysuckle flower (Lonicera macranthoides Hand.-Mazz.) is that Caprifoliaceae Lonicera is perennial often
Green winding or upright undershrub plant.《Chinese Pharmacopoeia》Regulation largeflower-like honeysuckle flower be Honeysuckle flower medicinal material main plant source it
One, the chlorogenic acid content of bud is very high, to commonly use medicinal part.
Using a series of kinds that largeflower-like honeysuckle flower is formed as introduces a collection, bud is large number of, bud aggregates into umbrella or group
Locusta, corolla are neat, developmental stage is consistent and the florescence concentrates, flower is hardly open, can disposably pick, can in production
Plucking time and labour cost are greatlyd save, yield is significantly higher than the plant origin of honeysuckle, i.e., with the kind of honeysuckle origin, because
This, with the continuous extension developed and used to largeflower-like honeysuckle flower, the requirement to its quality and quantity also constantly enhances.
The breeding of largeflower-like honeysuckle flower at present is mostly based on vegetative propagation such as cutting propagation, and different cultivars tree characteristics makes a variation
It is larger, and cuttage survival rate is low, cannot meet the market demand；In addition, it is excellent to cultivate largeflower-like honeysuckle flower by molecular breeding means
Kind makes it be also a problem to be solved as the excellent substitute of honeysuckle (honeysuckle).And pass through the asexual of tissue cultures
Breeding can not only realize Industrialization of seeds and seedlings in a short time, and certain basis is also provided for transgenic technology.Ash felt at present
The imperfect regenerating system of hairy honeysuckle, these hamper the further research to largeflower-like honeysuckle flower, are given birth to especially by molecule
The means of object study its high chlorogenic acid content, spend more and mechanism study phenomena such as bud is kept closed.Therefore, mesh
The preceding regeneration techniques for needing to study and promote a set of efficient largeflower-like honeysuckle flower.
Invention content
It is an object of the present invention to provide a kind of culture mediums for obtaining largeflower-like honeysuckle flower regeneration plant.
It is provided by the present invention for obtaining the culture medium of largeflower-like honeysuckle flower regeneration plant, be as differential medium, by
The nutritional ingredient of following mass parts forms：
A great number of elements is with NH4NO31650 parts of meter, 0.83 part in terms of KI of trace element, molysite are with FeSO4·7H2O meters 37.3
Part, 0.5 part in terms of niacin of organic principle, 0.1 part -1.5 parts of zeatin, 0.1 part -0.5 part of indolebutyric acid and 30000 parts of sucrose；
The a great number of elements by following mass parts material composition：
NH4NO31650 parts, KNO31900 parts, KH2PO4170 parts, CaCl2·2H2440 parts of O and MgSO4·7H2O
370 parts；
The micro- material composition by following mass parts：
0.83 part of KI, Na2MoO4·2H20.25 part of O, H3BO36.2 parts, CuSO4·5H20.025 part of O, MnSO4·
H216.9 parts of O, CoCl2·6H20.025 part of O and ZnSO4·7H28.6 parts of O；
The molysite by following mass parts material composition：
Na2EDTA·2H227.8 parts of O and FeSO4·7H237.3 parts of O；
The organic principle by following mass parts material composition：
2.0 parts of 0.5 part of niacin, 100 parts of inositol, 0.1 part of thiamine hydrochloride, 0.5 part of puridoxine hydrochloride and glycine.
The culture medium consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·
5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, hydrochloric acid
Thiamine, puridoxine hydrochloride, glycine, zeatin, indolebutyric acid, sucrose and water；
Above ingredient concentration in the culture medium is respectively：
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
0.025mg/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, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride
0.1mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, zeatin 0.1mg/L-1.5mg/L, indolebutyric acid 0.1mg/
L-0.5mg/L and sucrose 30g/L.
It is a further object to provide a kind of solid mediums for obtaining largeflower-like honeysuckle flower regeneration plant.
It is provided by the present invention for obtaining the solid medium of largeflower-like honeysuckle flower regeneration plant, be by coagulator and above-mentioned
The solid medium that culture medium for obtaining largeflower-like honeysuckle flower regeneration plant is made into.
A concentration of 6g/L-8g/L of the coagulator in the solid medium；
The coagulator is agar.
The pH value of the 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 provided by the present invention for obtaining largeflower-like honeysuckle flower regeneration plant, includes the following steps：
Largeflower-like honeysuckle flower callus is inoculated into the solid medium for obtaining largeflower-like honeysuckle flower regeneration plant
On carry out differentiation culture, obtain largeflower-like honeysuckle flower regeneration bud；The largeflower-like honeysuckle flower regeneration bud is inoculated on root media
Culture of rootage is carried out to get to largeflower-like honeysuckle flower regeneration plant.
The largeflower-like honeysuckle flower callus is obtained by the method included the following steps：
Largeflower-like honeysuckle flower explant is inoculated on calli induction media and carries out Fiber differentiation, largeflower-like honeysuckle flower is obtained and is cured
Injured tissue；
The calli induction media consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·
5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, hydrochloric acid
Thiamine, puridoxine hydrochloride, glycine, 6-benzyladenine, kinetin, indolebutyric acid, sucrose, agar and water；
Above ingredient concentration in the calli induction media is respectively：
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
0.025mg/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, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride
0.1mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, 6-benzyladenine 0mg/L-3.0mg/L, kinetin 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 the inducing culture is 5.8-6.2.
The root media consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·
5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, hydrochloric acid
Thiamine, puridoxine hydrochloride, glycine, indolebutyric acid, sucrose, agar and water；
Above ingredient concentration in the root media is respectively：
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 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride
0.1mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, indolebutyric acid 0.2mg/L-1.0mg/L, sucrose 10g/L and fine jade
Fat 5g/L-8g/L；
The pH value of the root media is 5.8-6.2.
The Fiber differentiation is temperature is 20 DEG C -25 DEG C, light intensity 2000Lux-4000Lux, photoperiod were 12 small time
It is cultivated -20 days 15 days under conditions of being 50%-70% according to/12 hours dark, humidity；
The differential medium culture be temperature be 20 DEG C -25 DEG C, light intensity 2000Lux-4000Lux, the photoperiod 16
Hour illumination/8 hour are dark, humidity is cultivated -20 days 15 days under conditions of being 50%-70%；
The culture of rootage is temperature is 20 DEG C -25 DEG C, light intensity 2000Lux-4000Lux, photoperiod were 16 small time
It is cultivated -30 days 20 days under conditions of being 50%-70% according to/8 hours dark, humidity.
The largeflower-like honeysuckle flower explant is the newborn tender leaf or largeflower-like honeysuckle flower new life stem section of largeflower-like honeysuckle flower.
Differential medium provided by the present invention for obtaining largeflower-like honeysuckle flower regeneration plant is with strong points, applicability
It is good.The method provided by the present invention for obtaining largeflower-like honeysuckle flower regeneration plant by tissue cultures has not by area, weather with
Season limit, convenient for single plant of the large-scale production with merit.Largeflower-like honeysuckle flower is currently without feasible regeneration method, mirror
Determining the approach of gene function in largeflower-like honeysuckle flower will often carry out dependent on arabidopsis or tobacco isotype plant, very great Cheng
Its molecular mechanism research is limited on degree.Largeflower-like honeysuckle flower tissue culture regeneration technology is solves the problems, such as that this provides hope.
Description of the drawings
Fig. 1 is the regenerative process of largeflower-like honeysuckle flower, wherein A is for the fresh tender leaf of largeflower-like honeysuckle flower as explant through callus
The callus that Fiber differentiation obtains；B is that largeflower-like honeysuckle flower callus starts to break up；C is that largeflower-like honeysuckle flower callus is obtained through differentiation culture
Clump bud.
Fig. 2 is condition of rooting of the clump bud after culture of rootage.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1 obtains largeflower-like honeysuckle flower regeneration plant by tissue cultures
Method I
One, explant sterilizes
Taking largeflower-like honeysuckle flower, (public can obtain from Institute of Botany, Chinese Academy of Sciences, and the material is quoted from Hunan Province Longhui County
And it plants in Chinese Academy of Sciences's Beijing Botanical Garden 3 years or more；Recording the non-patent literature of the material is：Tang Xiaorong, Li Wu
It is flat, Peng Xiaofeng (2005), the tissue culture and rapid proliferation Plant Physiology Communications 41 (5) of largeflower-like honeysuckle flower, 642-642.)
Newborn tender leaf is explant, after clear water is rinsed well, is soaked in that flowing water is outstanding to rush 1h, aseptically, with aseptic water washing 1
It is secondary, with 4min is sterilized with 2% liquor natrii hypochloritis again after 70% alcohol disinfecting 20s, then turn the liquor natrii hypochloritis in 1%
4min is sterilized, then uses aseptic water washing 3-5 times, cuts into 0.5cm3Sheet is in suck dry moisture on aseptic filter paper.
Two, callus Fiber differentiation
Blade inoculation after step 1 is sterilized is 25 DEG C, light intensity 2000Lux in temperature on calli induction media,
Photoperiod is 12 hours illumination/12 hour dark, and humidity is cultivated 20 days under conditions of being 50%, induces callus (A in Fig. 1),
It centre can 2-5 callus (B in Fig. 1) expanded to formation of subculture.Callus has small bud point preferable to compact.
Experiment is set to be repeated three times, and results are averaged, each to repeat to set 10 explants.
Callus induction rate (%)=induced synthesis callus number/inoculation explant number × 100%.
As a result：Callus induction rate (%) is 100%.
The inducing culture consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·
5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, hydrochloric acid
Thiamine, puridoxine hydrochloride, glycine, 6-benzyladenine, kinetin, indolebutyric acid, agar, sucrose and water；
Above ingredient concentration in the inducing culture is respectively：
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
0.025mg/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, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride
0.1mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, 6-benzyladenine 2.0mg/L, kinetin 1mg/L, indoles
Butyric acid 0.1mg/L, sucrose 30g/L and agar 7g/L；
The pH value of the inducing culture is 5.8.
Three, differentiation culture
The callus that step 2 obtains is turned to be inoculated in differential medium, is 25 DEG C, light intensity 2000Lux in temperature, light week
Phase is 16 hours illumination/8 hour dark, and humidity cultivate 20 days under conditions of being 50%, and subculture 2-3 times grows 3 to bud is differentiated to form
The clump bud (C in Fig. 1) of~5cm.
Experiment is set to be repeated three times, and results are averaged, each to repeat to set 10 callus.
Differentiation rate (%)=be differentiated to form clump bud number/inoculation callus number × 100%.
As a result：Differentiation rate (%) is 100%.
The differential medium consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·
5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, hydrochloric acid
Thiamine, puridoxine hydrochloride, glycine, zeatin, indolebutyric acid, agar, sucrose and water；
Above ingredient concentration in the differential medium is respectively：
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
0.025mg/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, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride
0.1mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, zeatin 1.0mg/L, indolebutyric acid 0.1mg/L, sucrose
30g/L and agar 7g/L；
The pH value of the differential medium is 5.8.
Four, culture of rootage
Clump bud after three Multiplying culture of above-mentioned steps is inoculated into root media, is 25 DEG C in temperature, light intensity
2000Lux, photoperiod are 16 hours illumination/8 hour dark, and humidity is cultivated 20 days under conditions of being 50%, until growing 10~20
Item root (Fig. 2).
Experiment is set to be repeated three times, and results are averaged, each to repeat to set 10 clump buds.
The clump bud number of the clump bud number/inoculation for rooting rate (%)=take root.
As a result：Rooting rate is 100%.
The root media consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·
5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, hydrochloric acid
Thiamine, puridoxine hydrochloride, glycine, indolebutyric acid, sucrose, agar and water；
Above ingredient concentration in the root media is respectively：
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 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride
0.1mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, indolebutyric acid 0.2mg/L, sucrose 10g/L and agar 5g/L；
The pH value of the root media is 5.8.
Method II
One, explant sterilizes
It is explant to take largeflower-like honeysuckle flower new life stem section, after clear water is rinsed well, aseptically, uses aseptic water washing
1 time, with 8min is sterilized with 2% liquor natrii hypochloritis again after 70% alcohol disinfecting 30s, then use aseptic water washing 3-5 times,
1~2cm is cut into without the stem section of axillary bud in suck dry moisture on aseptic filter paper.
Two, callus Fiber differentiation
Newborn leaf stem section after step 1 is sterilized is seeded on inducing culture, is 20 DEG C in temperature, light intensity
3000Lux, photoperiod are 12 hours illumination/12 hour dark, and humidity is cultivated 15 days under conditions of being 60%, and centre can subculture 2-
3 times to inducing callus.
Experiment is set to be repeated three times, and results are averaged, each to repeat to set 10 explants.
Callus induction rate (%)=induced synthesis callus number/inoculation explant number × 100%.
As a result：Callus induction rate (%) is 100%.
The inducing culture consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·
5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, hydrochloric acid
Thiamine, puridoxine hydrochloride, glycine, 6-benzyladenine, kinetin, indolebutyric acid, sucrose and water；
Above ingredient concentration in the inducing culture is respectively：
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
0.025mg/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, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride
0.1mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, 6-benzyladenine 3.0mg/L, kinetin 1.0mg/L, Yin
Diindyl butyric acid 0.2mg/L, sucrose 30g/L and agar 7g/L；
The pH value of the inducing culture is 6.2.
Three, differentiation culture
The callus that step 2 obtains is turned to be inoculated in differential medium, is 20 DEG C, light intensity 3000Lux in temperature, light week
Phase is 16 hours illumination/8 hour dark, and humidity is cultivated 20 days under conditions of being 60%, and subculture 2-3 times is to being differentiated to form clump bud.
Experiment is set to be repeated three times, and results are averaged, each to repeat to set 10 callus.
Differentiation rate (%)=be differentiated to form clump bud number/inoculation callus number × 100%.
As a result：Differentiation rate (%) is 100%.
The differential medium consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·
5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, hydrochloric acid
Thiamine, puridoxine hydrochloride, glycine, zeatin, indolebutyric acid, agar, sucrose and water；
Above ingredient concentration in the differential medium is respectively：
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
0.025mg/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, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride
0.1mg/L, puridoxine 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 the differential medium is 6.0.
Four, culture of rootage
Clump bud after the differentiation culture of above-mentioned steps three is inoculated into root media, is 20 DEG C in temperature, light intensity
3000Lux, photoperiod are 16 hours illumination/16 hour dark, and humidity is cultivated 25 days under conditions of being 60%, until growing 10~20
Item root.
Experiment is set to be repeated three times, and results are averaged, each to repeat to set 10 clump buds.
The clump bud number of the clump bud number/inoculation for rooting rate (%)=take root.
As a result：Rooting rate is 100%.
The root media consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·
5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, hydrochloric acid
Thiamine, puridoxine hydrochloride, glycine, indolebutyric acid, agar, sucrose and water.
Above ingredient concentration in the root media is respectively：
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 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride
0.1mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, indolebutyric acid 0.5mg/L, sucrose 10g/L and agar 5g/L；
The pH value of the root media is 5.8.
Method III
One, explant sterilizes
It is explant to take the newborn blade of largeflower-like honeysuckle flower, after impregnating that flowing water is outstanding and rushing 2h, aseptically, uses sterile water
The liquor natrii hypochloritis for rinsing 2 use 2% sterilizes 8min, then uses aseptic water washing 5 times, cut into 0.5 × 0.5cm blades in
Suck dry moisture on aseptic filter paper.
Two, callus Fiber differentiation
Blade inoculation after step 1 is sterilized is 22 DEG C, light intensity 4000Lux in temperature on calli induction media,
Photoperiod is 12 hours illumination/12 hour dark, and humidity is cultivated 20 days under conditions of being 70%, centre can subculture 2-3 times to luring
Export callus.
The inducing culture consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·
5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, hydrochloric acid
Thiamine, puridoxine hydrochloride, glycine, zeatin, indolebutyric acid, agar, sucrose and water；
Above ingredient concentration in the inducing culture is respectively：
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
0.025mg/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, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride
0.1mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, zeatin 1.5mg/L, indolebutyric acid 0.5mg/L, sucrose
30g/L and agar 7g/L；
The pH value of the inducing culture is 6.2.
Three, differentiation culture
The callus that step 2 obtains is inoculated in differential medium, is 20 DEG C, light intensity 4000Lux in temperature, the photoperiod
For 16 hours illumination/8 hour dark, humidity was cultivated 15 days under conditions of being 70%, and subculture 2-3 times is to being differentiated to form clump bud.
The differential medium consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·
5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, hydrochloric acid
Thiamine, puridoxine hydrochloride, glycine, zeatin, indolebutyric acid, agar, sucrose and water；
Above ingredient concentration in the differential medium is respectively：
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
0.025mg/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, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride
0.1mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, zeatin 1.5mg/L, indolebutyric acid 0.5mg/L, sucrose
30g/L and agar 8g/L；
The pH value of the differential medium is 6.2.
Four, culture of rootage
Clump bud after the differentiation culture of above-mentioned steps three is inoculated into root media, is 25 DEG C in temperature, light intensity
4000Lux, photoperiod are 16 hours illumination/8 hour dark, and humidity is cultivated 20-30 days under conditions of being 70%, until grow 10~
20 roots.
The root media consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·
5H2O、MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, hydrochloric acid
Thiamine, puridoxine hydrochloride, glycine, indolebutyric acid, agar, sucrose and water；
Above ingredient concentration in the root media is respectively：
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 8.6mg/L、
Na2EDTA·2H2O 27.8mg/L、FeSO4·7H2O 37.3mg/L, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride
0.1mg/L, puridoxine hydrochloride 0.5mg/L, glycine 2.0mg/L, indolebutyric acid 1.0mg/L, sucrose 10g/L and agar 7g/L；
The pH value of the root media is 6.2.
Claims (2)
1. a kind of method obtaining largeflower-like honeysuckle flower regeneration plant, includes the following steps：
Largeflower-like honeysuckle flower explant is inoculated on calli induction media and carries out Fiber differentiation, obtains largeflower-like honeysuckle flower callus group
It knits；Largeflower-like honeysuckle flower callus is inoculated on differential medium and carries out differentiation culture, obtains largeflower-like honeysuckle flower regeneration bud；It will
The largeflower-like honeysuckle flower regeneration bud, which is inoculated on root media, carries out culture of rootage to get to largeflower-like honeysuckle flower regeneration plant；
The largeflower-like honeysuckle flower explant is the newborn tender leaf or largeflower-like honeysuckle flower new life stem section of largeflower-like honeysuckle flower；
The calli induction media consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·5H2O、
MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, thiamine hydrochloride
Element, puridoxine hydrochloride, glycine, 6-benzyladenine, kinetin, indolebutyric acid, sucrose, agar and water；
Above ingredient concentration in the calli induction media is respectively：
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 0.025mg/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, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride 0.1mg/L, hydrochloric acid pyrrole
Tremble alcohol 0.5mg/L, glycine 2.0mg/L, 6-benzyladenine 3.0mg/L, kinetin 1.0mg/L, indolebutyric acid 0.2mg/L,
Sucrose 30g/L and agar 7g/L；
The pH value of the inducing culture is 6.0；
The differential medium consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·5H2O、
MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, thiamine hydrochloride
Element, puridoxine hydrochloride, glycine, zeatin, indolebutyric acid, sucrose, agar and water；
Above ingredient concentration in the differential medium is respectively：
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 0.025mg/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, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride 0.1mg/L, hydrochloric acid pyrrole
It trembles alcohol 0.5mg/L, glycine 2.0mg/L, zeatin 0.1mg/L-1.5mg/L, indolebutyric acid 0.1mg/L-0.5mg/L, sucrose
30g/L and agar 6g/L-8g/L；
The pH value of the differential medium is 5.8-6.2；
The root media consists of the following compositions：
NH4NO3、KNO3、KH2PO4、CaCl2·2H2O、MgSO4·7H2O、KI、Na2MoO4·2H2O、H3BO3、CuSO4·5H2O、
MnSO4·H2O、CoCl2·6H2O、ZnSO4·7H2O、Na2EDTA·2H2O、FeSO4·7H2O, niacin, inositol, thiamine hydrochloride
Element, puridoxine hydrochloride, glycine, indolebutyric acid, sucrose, agar and water；
Above ingredient concentration in the root media is respectively：
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 8.6mg/L、Na2EDTA·2H2O
27.8mg/L、FeSO4·7H2O 37.3mg/L, niacin 0.5mg/L, inositol 100mg/L, thiamine hydrochloride 0.1mg/L, hydrochloric acid pyrrole
Tremble alcohol 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 the root media is 5.8-6.2.
2. according to the method described in claim 1, it is characterized in that：
The Fiber differentiation is temperature is 20 DEG C -25 DEG C, light intensity 2000Lux-4000Lux, photoperiod are 12 hours illumination/12
Hour dark, humidity are cultivated -20 days 15 days under conditions of being 50%-70%；
Differentiation culture is temperature is 20 DEG C -25 DEG C, light intensity 2000Lux-4000Lux, photoperiod are 16 hours illumination/8
Hour dark, humidity are cultivated -20 days 15 days under conditions of being 50%-70%；
The culture of rootage is temperature is 20 DEG C -25 DEG C, light intensity 2000Lux-4000Lux, photoperiod are 16 hours illumination/8
Hour dark, humidity are cultivated -30 days 20 days under conditions of being 50%-70%.
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