CN110278875A - A kind of method of orchardgrass children fringe in vitro culture regeneration plant - Google Patents
A kind of method of orchardgrass children fringe in vitro culture regeneration plant Download PDFInfo
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- CN110278875A CN110278875A CN201910690019.6A CN201910690019A CN110278875A CN 110278875 A CN110278875 A CN 110278875A CN 201910690019 A CN201910690019 A CN 201910690019A CN 110278875 A CN110278875 A CN 110278875A
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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
- A01H4/001—Culture apparatus for tissue culture
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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
- A01H4/008—Methods for regeneration to complete plants
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Abstract
It is fast the invention discloses a kind of callus induction speed and inductivity is high, the method for orchardgrass children's fringe in vitro culture regeneration plant of differentiation rate and high survival rate.The present invention is using orchardgrass children fringe as explant, orchardgrass children's fringe is taken to carry out excised cotyledon, callus can be formed by being inoculated in induced medium 5~7 days after young fringe disinfection, 15~20d of culture in subculture medium is transferred to after 15~30d of Fiber differentiation, then it is transferred to 15~25d of culture in differential medium, then it is transferred to 7~15d of culture in root media, finally when seedling grows to 5cm or more and with sturdy, it is transplanted after carrying out hardening treatment to seedling, the present invention establishes regenerating system using orchardgrass children's fringe as explant, for orchardgrass genetic transformation and cultivates improved seeds and provide technical support.It is suitble to promote the use in field of biotechnology.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of method of orchardgrass children fringe in vitro culture regeneration plant.
Background technique
Orchardgrass (Dactylis glomerata L.) also known as halymenia dentata, orchard grass belong to grass family perennial herb and dredge clump type
Plant, originating from Europe, north African and Asia temperate zone, after be introduced into Temperate Region in China, planted extensively as excellent Cool-Season Forage Species
Training.China is also one of area of origin of orchardgrass, and Wild ornamental resources are abundant, but are distributed mainly on the Mountain Ranges of Tian Shan Mountains forest in Xinjiang
Edge zone, the Mount Emei in Sichuan, Erlongshan Mountains, Qionglai mountain range, the Liangshan Mountain and Mount Min mountain range 1000~3100m height above sea level forest side
Edge, shrubbery and patana, and sporadically appear in Southeast Daxinganling hillside fields, meanwhile, have on Chongqing, Yunnan, Guizhou, Hubei and other places
Distribution.
With the development of biotechnology, domestic and international forage plants breeding method is biotechnology breeding from traditional breeding method development,
The breeding of new variety wherein carried out based on Plant Tissue Breeding and genetic transformation application are efficiently and extensive, therefore, establish high
The tissue culturing system of effect can provide stable technical support for the breeding of good forage.So far, a variety of herbages have become
Function establishes plant regeneration system, such as English ryegrass, siberian wildrye, Elymus nutans, alfalfa.
The induction of callus is the key that establish plant regeneration system, by explant species, hormone kind and proportion
And many-sided influence such as nutritional condition.Its applicable explant is more single as graminous pasture for orchardgrass, such as seed, plasm
Body, leaf mesophyll cells, the tender flower spike of children, stipes etc..It can be used as explant evoked callus with mature seed, although overcoming
The season limit of explant supply, but its cell differentiation speed is slow, and embryo callus subculture inductivity is low.Select plant cell, plasm
Body is big for its extraction difficulty of explant, operates more multiple；Though and young fringe cannot be drawn materials for a long time by seasonal effect, its cell differentiation
Speed is fast, tissue internal hormone content is high, can induce and forms callus, the later period can subculture embryo callus subculture can be obtained.Currently, grass
Section herbage such as Elymus nutans, siberian wildrye, sudangrass, rye grass etc. establishes regenerating system with children's fringe, and orchardgrass conduct
The main graminous pasture in southwest is rarely reported using the research that its children's fringe establishes regenerating system as explant, therefore, the present invention
Regenerating system is established using orchardgrass children's fringe as explant, is laid a good foundation for the verifying of later period orchardgrass gene function
Summary of the invention
It is fast technical problem to be solved by the invention is to provide a kind of callus induction speed and inductivity is high, differentiation rate and at
The method of the high orchardgrass children's fringe in vitro culture regeneration plant of motility rate.
The technical solution adopted by the present invention to solve the technical problems are as follows: the side of orchardgrass children's fringe in vitro culture regeneration plant
Method, comprising the following steps:
A, using orchardgrass children fringe as explant, 15 are cultivated by being inoculated into induced medium after orchardgrass children's fringe cleaning and sterilizing
~30d obtains callus, and the induced medium is by MS culture medium, 4mg/L2,4-D, 0.6mg/L copper sulphate, 0.5g/L dried meat
Propylhomoserin, 0.5g/L acid hydrolyzed casein, 30g/L sucrose, 3g/L gellan gum composition；
B, callus is transferred to 15~20d of culture in subculture medium, the subculture medium is by MS culture medium, 2mg/
L2,4-D, 0.4mg/LCPA, 30mol/L dicamba, 0.6mg/L copper sulphate, 0.5g/L proline, 0.5g/L sour water solution junket egg
White, 30g/L sucrose, 3g/L gellan gum composition；
C, 15~25d of culture in differential medium, the differentiation culture will be transferred to by the callus of step B processing
Base is made of MS culture medium, 2mg/LKT, 30g/L sucrose, 3g/L gellan gum；
D, 7~15d of culture, the culture of rootage in root media will be transferred to by the callus of step C processing
Base is made of 1/2MS culture medium, 0.2g/LNAA, 0.2g/LIBA, 30g/L sucrose, 3g/L gellan gum；
E, it when seedling grows to 5cm or more and with sturdy, is transplanted after carrying out hardening treatment to seedling.
Further, in step, the orchardgrass children fringe obtains in the following way: taking length is the duck of 2~5cm
Mao Yousui, clip orchardgrass section and top plant, incision are wrapped spare with preservative film.
Further, in step, it is as described below to the processing mode of orchardgrass children's fringe cleaning and sterilizing: firstly, by orchardgrass
Young fringe peels off outer blade, impregnates 1min with 75% alcohol in superclean bench, then uses distilled water flushing 3~5 times, then use
The sodium hypochlorite that concentration is 1.1% impregnates 1min, then uses distilled water flushing 3~5 times, is placed on aseptic filter paper and dries, with disappearing
The tweezers for crossing poison carefully strip young fringe, and the fringe section for being cut into about 0.2cm is spare.
Further, the MS culture medium contained in the induced medium, subculture medium, differential medium by
KNO3、NH4NO3、MgSO4·7H2O、KH2PO4、CaCl2·2H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、
NaMoO4·2H2O、CuSO4·5H2O、CoCl2·6H2O、Na2-EDTA、FeSO4·4H2O, glycine, thiamine hydrochloride, hydrochloric acid
Pyridoxol, niacin, inositol composition, the content of each component are as described below: KNO3For 1900mg/L, NH4NO3For 1650mg/L,
MgSO4·7H2O is 370mg/L, KH2PO4For 170mg/L, CaCl2·2H2O is 440mg/L, MnSO4·4H2O is 22.3mg/
L, ZnSO4·7H2O is 8.6mg/L, H3BO3For 6.2mg/L, KI 0.83mg/L, NaMoO4·2H2O is 0.25mg/L,
CuSO4·5H2O is 0.025mg/L, CoCl2·6H2O is 0.025mg/L, Na2- EDTA is 37.3mg/L, FeSO4·4H2O is
27.8mg/L, glycine 2.0mg/L, thiamine hydrochloride 0.1mg/L, puridoxine hydrochloride 0.5mg/L, niacin 0.5mg/
L, inositol 100mg/L.
Further, the 1/2MS culture medium contained in the root media is by KNO3、NH4NO3、MgSO4·7H2O、
KH2PO4、CaCl2·2H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·5H2O、
CoCl2·6H2O、Na2-EDTA、FeSO4·4H2O, glycine, thiamine hydrochloride, puridoxine hydrochloride, niacin, inositol composition, respectively
The content of component is as described below: KNO3For 950mg/L, NH4NO3For 825mg/L, MgSO47H2O is 185mg/L, KH2PO4For
85mg/L, CaCl2·2H2O is 220mg/L, MnSO4·4H2O is 11.15mg/L, ZnSO4·7H2O is 4.3mg/L, H3BO3For
3.1mg/L, KI 0.415mg/L, NaMoO4·2H2O is 0.125mg/L, CuSO4·5H2O is 0.0125mg/L, CoCl2·
6H2O is 0.0125mg/L, Na2- EDTA is 18.65mg/L, FeSO4·4H2O is 13.9mg/L, glycine 1.0mg/L, salt
Allithiamine element is 0.05mg/L, puridoxine hydrochloride 0.25mg/L, niacin 0.25mg/L, inositol 50mg/L.
Further, in step, the environment temperature of the Fiber differentiation is 25 ± 2 DEG C, and training method is dark
Training.
Further, the process of the hardening treatment is as described below in step E: opening the lid of culture bottle, and fall
Enter appropriate distilled water, cultivates 3d.
The beneficial effects of the present invention are: the present invention takes orchardgrass children's fringe to carry out in vitro tissue using orchardgrass children fringe as explant
Culture, callus can be formed by being inoculated in induced medium 5~7 days after the disinfection of young fringe, by MS culture medium, 4mg/L2,4-D,
The induction of 0.6mg/L copper sulphate, 0.5g/L proline, 0.5g/L acid hydrolyzed casein, 30g/L sucrose, 3g/L gellan gum composition
Induced velocity and inductivity can be improved in culture medium, carries out orchardgrass children fringe callus induction, induction using the induced medium
Rate reaches 75% or more, and 15~20d of culture is transferred in subculture medium after 15~30d of Fiber differentiation, by MS, 2mg/L2,4-D,
0.4mg/LCPA, 30mol/L dicamba, 0.6mg/L copper sulphate, 0.5g/L proline, 0.5g/L acid hydrolyzed casein, 30g/L
The subculture medium that sucrose, 3g/L gellan gum form can promote callus by the concentration and addition CPA and dicamba of reduction 2,4-D
Callus callus quality and embryo callus subculture incidence can be improved in tissue augmentation, is then transferred in differential medium and is trained
15~25d is supported, differentiation can be improved by the differential medium that MS culture medium, 2mg/LKT, 30g/L sucrose, 3g/L gellan gum form
Efficiency, the differential medium evoked callus form green seedling, and the speed of growth is fast, and differentiation rate is up to 86%；Then it is transferred to
7~15d is cultivated in root media, by 1/2MS culture medium, 0.2g/LNAA, 0.2g/LIBA, 30g/L sucrose, 3g/L gellan gum
The root media of composition produces the root system of sturdy prosperity, can greatly improve its efficiency of taking root, can be at after acclimatization and transplants
It is living, and then improve its survival rate；Finally when seedling grows to 5cm or more and with sturdy, after carrying out hardening treatment to seedling
Transplanting, the present invention establish regenerating system using orchardgrass children's fringe as explant, mention for orchardgrass genetic transformation and cultivating improved seeds
Technical support is supplied.A kind of method of orchardgrass children fringe in vitro culture regeneration plant according to the present invention simultaneously is a whole set of culture
System, links are closely connected to form a whole, and the implementation of any step is crucial whole process, and should
Method is for designed by orchardgrass children fringe.
Specific embodiment
The present invention takes orchardgrass children's fringe to carry out excised cotyledon, is inoculated in after young fringe disinfection using orchardgrass children fringe as explant
Callus can be formed within induced medium 5~7 days, by MS culture medium, 4mg/L2,4-D, 0.6mg/L copper sulphate, 0.5g/L dried meat
Propylhomoserin, 0.5g/L acid hydrolyzed casein, 30g/L sucrose, 3g/L gellan gum composition induced medium can be improved induced velocity and
Inductivity carries out orchardgrass children fringe callus induction using the induced medium, and inductivity reaches 75% or more, Fiber differentiation
15~20d of culture in subculture medium is transferred to after 15~30d, by MS, 2mg/L2,4-D, 0.4mg/LCPA, 30mol/L wheat straw
Fear, 0.6mg/L copper sulphate, 0.5g/L proline, 0.5g/L acid hydrolyzed casein, 30g/L sucrose, 3g/L gellan gum composition after
Callus amplification can promote by the concentration and addition CPA and dicamba of reduction 2,4-D for culture medium, callus can be improved
Callus quality and embryo callus subculture incidence are then transferred in differential medium 15~25d of culture, by MS culture medium,
Differentiation efficiency can be improved in the differential medium that 2mg/LKT, 30g/L sucrose, 3g/L gellan gum form, differential medium induction
Callus forms green seedling, and the speed of growth is fast, and differentiation rate is up to 86%；Then be transferred in root media culture 7~
15d, can by the root media that 1/2MS culture medium, 0.2g/LNAA, 0.2g/LIBA, 30g/L sucrose, 3g/L gellan gum form
The root system for generating sturdy prosperity, can greatly improve its efficiency of taking root, viable after acclimatization and transplants, and then improves it and survive
Rate；Finally when seedling it is long to 5cm or more and with sturdy when, transplanted after carrying out hardening treatment to seedling, the present invention with
Orchardgrass children's fringe is that explant establishes regenerating system, for orchardgrass genetic transformation and cultivates improved seeds and provides technical support.Simultaneously
A kind of method of orchardgrass children fringe in vitro culture regeneration plant according to the present invention is a whole set of cultivating system, and links are close
It is connected to form a whole, the implementation of any step is crucial whole process, and this method is for orchardgrass children
Designed by fringe.
Specifically, the method for orchardgrass children fringe in vitro culture regeneration plant of the present invention, specifically includes the following steps:
A, using orchardgrass children fringe as explant, 15 are cultivated by being inoculated into induced medium after orchardgrass children's fringe cleaning and sterilizing
~30d obtains callus, and the induced medium is by MS culture medium, 4mg/L2,4-D, 0.6mg/L copper sulphate, 0.5g/L dried meat
Propylhomoserin, 0.5g/L acid hydrolyzed casein, 30g/L sucrose, 3g/L gellan gum composition；
B, callus is transferred to 15~20d of culture in subculture medium, the subculture medium is by MS culture medium, 2mg/
L2,4-D, 0.4mg/LCPA, 30mol/L dicamba, 0.6mg/L copper sulphate, 0.5g/L proline, 0.5g/L sour water solution junket egg
White, 30g/L sucrose, 3g/L gellan gum composition；
C, 15~25d of culture in differential medium, the differentiation culture will be transferred to by the callus of step B processing
Base is made of MS culture medium, 2mg/LKT, 30g/L sucrose, 3g/L gellan gum；
D, 7~15d of culture, the culture of rootage in root media will be transferred to by the callus of step C processing
Base is made of 1/2MS culture medium, 0.2g/LNAA, 0.2g/LIBA, 30g/L sucrose, 3g/L gellan gum；
E, it when seedling grows to 5cm or more and with sturdy, is transplanted after carrying out hardening treatment to seedling.
During the method for above-mentioned orchardgrass children fringe in vitro culture regeneration plant, the orchardgrass children fringe obtains in the following way
To: taking length is orchardgrass children's fringe of 2~5cm, and clip orchardgrass section and top plant, incision are wrapped spare with preservative film.In addition,
In step, various ways can be used to the processing mode of orchardgrass children's fringe cleaning and sterilizing, in order to make cleaning effect, disinfection effect
Fruit reaches most preferably, and the present invention is by the way of as described below: as described below to the processing mode of orchardgrass children's fringe cleaning and sterilizing: first
First, orchardgrass children's fringe is peelled off into outer blade, impregnates 1min with 75% alcohol in superclean bench, then use distilled water flushing 3
~5 times, then 1min is impregnated for 1.1% sodium hypochlorite with concentration, it then uses distilled water flushing 3~5 times, is placed on aseptic filter paper
It dries, carefully strips young fringe with sterilized tweezers, the fringe section for being cut into about 0.2cm is spare.
The induced medium, subculture medium, the MS culture medium contained in differential medium are by KNO3、NH4NO3、
MgSO4·7H2O、KH2PO4、CaCl2·2H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、
CuSO4·5H2O、CoCl2·6H2O、Na2-EDTA、FeSO4·4H2O, glycine, thiamine hydrochloride, puridoxine hydrochloride, niacin,
Inositol composition, the content of each component are as described below: KNO3For 1900mg/L, NH4NO3For 1650mg/L, MgSO47H2O is
370mg/L, KH2PO4For 170mg/L, CaCl2·2H2O is 440mg/L, MnSO4·4H2O is 22.3mg/L, ZnSO4·7H2O is
8.6mg/L, H3BO3For 6.2mg/L, KI 0.83mg/L, NaMoO4·2H2O is 0.25mg/L, CuSO4·5H2O is
0.025mg/L, CoCl2·6H2O is 0.025mg/L, Na2- EDTA is 37.3mg/L, FeSO4·4H2O is 27.8mg/L, sweet ammonia
Acid is 2.0mg/L, thiamine hydrochloride 0.1mg/L, puridoxine hydrochloride 0.5mg/L, and niacin 0.5mg/L, inositol is
100mg/L。
The 1/2MS culture medium contained in the root media is by KNO3、NH4NO3、MgSO4·7H2O、KH2PO4、
CaCl2·2H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·5H2O、CoCl2·6H2O、
Na2-EDTA、FeSO4·4H2O, glycine, thiamine hydrochloride, puridoxine hydrochloride, niacin, inositol composition, the content of each component is such as
It is lower described: KNO3For 950mg/L, NH4NO3For 825mg/L, MgSO47H2O is 185mg/L, KH2PO4For 85mg/L,
CaCl2·2H2O is 220mg/L, MnSO4·4H2O is 11.15mg/L, ZnSO4·7H2O is 4.3mg/L, H3BO3For 3.1mg/L,
KI is 0.415mg/L, NaMoO4·2H2O is 0.125mg/L, CuSO4·5H2O is 0.0125mg/L, CoCl2·6H2O is
0.0125mg/L, Na2- EDTA is 18.65mg/L, FeSO4·4H2O is 13.9mg/L, glycine 1.0mg/L, thiamine hydrochloride
Element is 0.05mg/L, puridoxine hydrochloride 0.25mg/L, niacin 0.25mg/L, inositol 50mg/L.Above-mentioned culture medium is matched
Side is specific to the configuration of orchardgrass children's fringe, is more suitable for orchardgrass children fringe tissue cultures.
In order to which the effect of Fiber differentiation is more preferable, the environment temperature of the Fiber differentiation is 25 ± 2 DEG C, and training method is dark
Training.
Embodiment
A, taking length is orchardgrass children's fringe of 2~5cm, and clip orchardgrass section and top plant, incision are wrapped standby with preservative film
With, orchardgrass children's fringe is then peelled off into outer blade, in superclean bench with 75% alcohol impregnate 1min, then rushed with distilled water
It washes 3~5 times, then the sodium hypochlorite for being 1.1% with concentration impregnates 1min, then uses distilled water flushing 3~5 times, is placed on sterile filter
It is dried on paper, carefully strips young fringe with sterilized tweezers, the fringe section for being cut into about 0.2cm is spare, then by orchardgrass children's fringe
It is inoculated into culture 15d in induced medium and obtains callus, the environment temperature of the Fiber differentiation is 25 DEG C, and training method
Secretly to train the induced medium by MS culture medium, 4mg/L2,4-D, 0.6mg/L copper sulphate, 0.5g/L proline, 0.5g/L acid
Caseinhydrolysate acid hydrolyzed casein, 30g/L sucrose 3g/L gellan gum composition；The induced medium is suitable for orchardgrass children fringe callus
The induction of tissue, experimentation, which finds that 2,4-D concentration is too low, causes inductivity low；The generation of excessively high meeting induced bud.And it applies and is somebody's turn to do
Culture medium carries out orchardgrass children fringe callus induction, and 5~7d can form callus, significantly improve induced velocity；Together
When, inductivity is up to 75%.
B, callus is transferred in subculture medium and cultivates 20d, the subculture medium by MS culture medium, 2mg/L2,
4-D, 0.4mg/LCPA, 30mol/L dicamba, 0.6mg/L copper sulphate, 0.5g/L proline, 0.5g/L acid hydrolyzed casein,
30g/L sucrose, 3g/L gellan gum composition；The subculture medium is by reducing the concentration of 2,4-D and adding CPA, dicamba and battalion
Substance is supported, so that callus gradually becomes faint yellow and subculture is not easy browning, callus quality can be improved and promote embryo
The formation of callus.
C, will be transferred in differential medium by the callus of step B processing and cultivate 15d, the differential medium by
MS culture medium, 2mg/LKT, 30g/L sucrose, 3g/L gellan gum composition；Callus through basal culture medium carry out differentiation culture 4~
6d has green plant regeneration to be formed, its differentiation rate is counted after 15d and reaches 86%.The culture medium can accelerate point of callus
Change speed and improves differentiation rate.
D, it will be transferred in root media and cultivate by the green seedling of step C processing, the root media is trained by 1/2MS
Support base, 0.2g/LNAA, 0.2g/LIBA, 30g/L sucrose, 3g/L gellan gum composition；The green Miao Jing root media culture
It takes root, and can get sturdy, flourishing root system, to improve transplanting survival rate.
E, it when seedling grows to 5cm or more and with sturdy, is transplanted after carrying out hardening treatment to seedling.
A kind of method of orchardgrass children fringe in vitro culture regeneration plant according to the present invention is a whole set of cultivating system, each
Link is closely connected to form a whole, and the implementation of any step is crucial whole process, and this method is needle
To designed by orchardgrass children's fringe, orchardgrass children fringe callus induction is carried out using the induced medium in this method, inductivity reaches
To 75%, the induced velocity of callus is accelerated.The subculture medium can promote callus amplification, improve callus
Quality promotes the formation of embryo callus.The differential medium evoked callus forms green seedling, and the speed of growth is fast, point
Rate is up to 86%.The green Miao Jing root media culture produces the root system of sturdy prosperity, viable after acclimatization and transplants.
Min indicates minute in the present invention, and d indicates number of days, and 2,4-D indicate that 2,4- dichlorphenoxyacetic acid, NAA indicate naphthalene second
Acid, CPA indicate that 3,4- dichloro phenylalanine, KT indicate that kinetin, IBA indicate indolebutyric acid.
Claims (7)
1. a kind of method of orchardgrass children fringe in vitro culture regeneration plant, it is characterised in that the following steps are included:
A, using orchardgrass children fringe as explant, will be inoculated into after orchardgrass children's fringe cleaning and sterilizing in induced medium culture 15~
30d obtains callus, and the induced medium is by MS culture medium, 4mg/L2,4-D, 0.6mg/L copper sulphate, 0.5g/L dried meat ammonia
Acid, 0.5g/L acid hydrolyzed casein, 30g/L sucrose, 3g/L gellan gum composition；
B, callus is transferred in subculture medium 15~20d of culture, the subculture medium by MS culture medium, 2mg/L2,
4-D, 0.4mg/LCPA, 30mol/L dicamba, 0.6mg/L copper sulphate, 0.5g/L proline, 0.5g/L acid hydrolyzed casein,
30g/L sucrose, 3g/L gellan gum composition；
C, will be transferred in differential medium 15~25d of culture by the callus of step B processing, the differential medium by
MS culture medium, 2mg/LKT, 30g/L sucrose, 3g/L gellan gum composition；
D, will be transferred in root media 7~15d of culture by the callus of step C processing, the root media by
1/2MS culture medium, 0.2g/LNAA, 0.2g/LIBA, 30g/L sucrose, 3g/L gellan gum composition；
E, it when seedling grows to 5cm or more and with sturdy, is transplanted after carrying out hardening treatment to seedling.
2. the method for orchardgrass children fringe in vitro culture regeneration plant as described in claim 1, it is characterised in that: in step, institute
State orchardgrass children's fringe to obtain in the following way: taking length is orchardgrass children's fringe of 2~5cm, clip orchardgrass section and top plant, notch
Wrapped with preservative film spare in place.
3. the method for orchardgrass children fringe in vitro culture regeneration plant as described in claim 1, it is characterised in that: in step, right
The processing mode of orchardgrass children's fringe cleaning and sterilizing is as described below: firstly, orchardgrass children's fringe is peelled off outer blade, in superclean bench
1min is impregnated with 75% alcohol, is then used distilled water flushing 3~5 times, then the sodium hypochlorite for being 1.1% with concentration impregnates 1min,
Then it uses distilled water flushing 3~5 times, is placed on aseptic filter paper and dries, carefully strip young fringe with sterilized tweezers, be cut into
The fringe section of about 0.2cm is spare.
4. the method for orchardgrass children fringe in vitro culture regeneration plant as claimed in claim 3, it is characterised in that: the Fiber differentiation
Base, subculture medium, the MS culture medium contained in differential medium are by KNO3、NH4NO3、MgSO4·7H2O、KH2PO4、
CaCl2·2H2O、MnSO4·4H2O、ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·5H2O、CoCl2·6H2O、
Na2-EDTA、FeSO4·4H2O, glycine, thiamine hydrochloride, puridoxine hydrochloride, niacin, inositol composition, the content of each component is such as
It is lower described: KNO3For 1900mg/L, NH4NO3For 1650mg/L, MgSO47H2O is 370mg/L, KH2PO4For 170mg/L,
CaCl2·2H2O is 440mg/L, MnSO4·4H2O is 22.3mg/L, ZnSO4·7H2O is 8.6mg/L, H3BO3For 6.2mg/L,
KI is 0.83mg/L, NaMoO4·2H2O is 0.25mg/L, CuSO4·5H2O is 0.025mg/L, CoCl2·6H2O is
0.025mg/L, Na2- EDTA is 37.3mg/L, FeSO4·4H2O is 27.8mg/L, glycine 2.0mg/L, thiamine hydrochloride
For 0.1mg/L, puridoxine hydrochloride 0.5mg/L, niacin 0.5mg/L, inositol 100mg/L.
5. the method for orchardgrass children fringe in vitro culture regeneration plant as described in claim 1, it is characterised in that: the culture of rootage
The 1/2MS culture medium contained in base is by KNO3、NH4NO3、MgSO4·7H2O、KH2PO4、CaCl2·2H2O、MnSO4·4H2O、
ZnSO4·7H2O、H3BO3、KI、NaMoO4·2H2O、CuSO4·5H2O、CoCl2·6H2O、Na2-EDTA、FeSO4·4H2O, sweet
Propylhomoserin, thiamine hydrochloride, puridoxine hydrochloride, niacin, inositol composition, the content of each component are as described below: KNO3For 950mg/L,
NH4NO3For 825mg/L, MgSO47H2O is 185mg/L, KH2PO4For 85mg/L, CaCl2·2H2O is 220mg/L, MnSO4·
4H2O is 11.15mg/L, ZnSO4·7H2O is 4.3mg/L, H3BO3For 3.1mg/L, KI 0.415mg/L, NaMoO4·2H2O
For 0.125mg/L, CuSO4·5H2O is 0.0125mg/L, CoCl2·6H2O is 0.0125mg/L, Na2- EDTA is 18.65mg/
L, FeSO4·4H2O is 13.9mg/L, glycine 1.0mg/L, thiamine hydrochloride 0.05mg/L, and puridoxine hydrochloride is
0.25mg/L, niacin 0.25mg/L, inositol 50mg/L.
6. the method for orchardgrass children fringe in vitro culture regeneration plant as described in claim 1, it is characterised in that: in step, institute
The environment temperature for stating Fiber differentiation is 25 ± 2 DEG C, and training method is dark training.
7. the method for orchardgrass children fringe in vitro culture regeneration plant as described in claim 1, it is characterised in that: in step E, institute
The process for stating hardening treatment is as described below: opening the lid of culture bottle, pours into appropriate distilled water, cultivates 3d.
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| CN115885850A (en) * | 2022-11-30 | 2023-04-04 | 中国科学院西北高原生物研究所 | Tissue culture medium for regeneration of old mango wheat and tissue culture method for regeneration of mature embryo of old mango wheat |
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| CN115885850A (en) * | 2022-11-30 | 2023-04-04 | 中国科学院西北高原生物研究所 | Tissue culture medium for regeneration of old mango wheat and tissue culture method for regeneration of mature embryo of old mango wheat |
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