CN108849534A - A kind of application of eucalyptus leaves anthocyanidin in Banana Tissue culture medium - Google Patents
A kind of application of eucalyptus leaves anthocyanidin in Banana Tissue culture medium Download PDFInfo
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- CN108849534A CN108849534A CN201811154866.2A CN201811154866A CN108849534A CN 108849534 A CN108849534 A CN 108849534A CN 201811154866 A CN201811154866 A CN 201811154866A CN 108849534 A CN108849534 A CN 108849534A
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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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Abstract
The present invention relates to Banana Tissue culture technique fields, in particular to application of a kind of eucalyptus leaves anthocyanidin in Banana Tissue culture medium, the Banana Tissue culture medium of the application is made of garlic P.E, tapioca, potato juice, chitosan, banana stem extracting solution, agar, aloe polysaccharide, eucalyptus leaves anthocyanidin and heteroauxin, it is its reasonable composition, simple, it can be suitably used for the Explants In Banana of culture banana stem preparation, explant can be promoted to take root, enhance the survival ability of explant.
Description
【Technical field】
The present invention relates to Banana Tissue culture technique field, in particular to a kind of eucalyptus leaves anthocyanidin is in Banana Tissue culture
The application of base.
【Background technique】
Banana belongs to Musaceae Musa to be one of main fresh fruit type in the world originating from Tropical Asian area for many years.
Banana, which belongs to triploid, height sterility, and fruit does not have seed, can only be passed on by vegetative propagation, and common breeding practice is
Tissue cultures, common explant is usually that banana inhales bud in existing quick-breeding method, is divided into two culture using bud stem apex is inhaled
Method, first generation bud ratio is generally 2-3, and breeding radix is very low, so that subculture algebra be made to increase, increases variating seedling hair
Raw risk, and it is time-consuming very long, from suction bud is taken, to differentiation, emergence of taking root lasts at least nine moon；Currently, rarely about using
Banana stem is that the report of the method for tissue culture of explant is deposited this is because banana stem is not high as its differentiation degree of explant
Motility rate is low, and is mostly chemical raw material in conventional use of culture medium, and ingredient more or less can all influence Explants In Banana
Survival rate is exclusively used in culture banana stem explant for this reason, it is necessary to study a kind of safe and effective culture medium prescription.
Anthocyanidin is widely present in various plants body, is considered to have anti-oxidant, anti-aging, anticancer, anti-inflammatory, anti-prominent
Become, protect the multi-efficiencies such as cardiovascular system.The research of anthocyanidin focuses mostly in medical domain at present, and answers about tissue cultures
Relatively fewer with reporting, in eucalyptus anthocyanidin other than containing Anthocyanins, there are also the objects such as many terpenes, eucalyptus oil
Matter, these ingredients can play good bacteriostasis, if can be good bacteriostatic agent, can be used for preparing training using proper
Support base.
【Summary of the invention】
In view of above content, it is necessary to provide a kind of safe and effective culture medium prescription, be exclusively used in culture banana stem explant
Body.
In order to achieve the above objectives, the technical scheme adopted by the invention is that：
A kind of Banana Tissue culture medium, garlic P.E, 50g/L-60g/L of the culture medium by 2.0g/L-5.0g/L
Tapioca, the potato juice of 100g/L-200g/L, 1.0g/L-3.0g/L chitosan, 100g/L-200g/L banana stem
Extracting solution, the agar of 10g/L-20g/L, the aloe polysaccharide of 1.0g/L-3.0g/L, 3.0g/L-5.0g/L eucalyptus leaves anthocyanidin
It is formed with the heteroauxin of 0.5mg/L-1mg/L.
Further, allicin content is 156mg/g-178mg/g in the garlic P.E.
Further, the cyanide content of the tapioca is 2%-5%.
Further, the preparation method of the potato juice is：Potato is cooked, mashed potato is pressed into, is cooled to room
The yeast powder of mashed potato quality 3%-5% is added after temperature ferment at constant temperature 4-6 days in 37 DEG C of insulating box, Ma Ling is then added
The distilled water of 2-3 times of mass parts of mashed potatoes, is sufficiently stirred, and is placed in 50 DEG C -60 DEG C of insulating box and extracts 3-4h, filters to take filter
Liquid is up to potato juice；The living bacteria count of the yeast powder is 2.0 × 109cfu/g-3.0×109cfu/g。
Further, the preparation method of the banana stem extracting solution is：New fresh bananas stem is shredded, banana stem is then added
The biological enzyme solutions of 2%-4% are isothermal reaction -26h for 24 hours in 37 DEG C of insulating box in temperature, after being heated to 50 DEG C -60 DEG C plus
Enter the ethyl ester solution that 3-5 times of mass parts percentage by volume is 20%-25%, stir evenly, the reaction was continued 3h-4h is cooled to room
Temperature filters, filtrate stratification is taken to remove supernatant up to banana extracting solution；The biology enzyme solutions by enzyme-activity unit are
The cellulase of 1000U/g-1500U/g.
Further, the eucalyptus leaves anthocyanidin by eucalyptus leaves by press leaf pretreatment, blue light processing, anthocyanidin extract and
Cyanine is plain to be proposed several steps and is prepared；Specially：
(1) leaf pretreatment is pressed：It is 1 that leaf and pretreatment fluid, which will be pressed, according to mass ratio:1-3 is mixed, and is then placed in
- 26h for 24 hours is heated in the water-bath that temperature is 40 DEG C -50 DEG C, filtrate is taken to obtain by leaf leachable after filtering；Eucalyptus leaves are carried out
Stifling 40min-50min, then mixes with biological enzyme solutions, and constant temperature handles 12h-14h in 36 DEG C -38 DEG C of insulating box；So
The centrifugal treating 5min-15min in the centrifuge that revolving speed is 1000r/min-1500r/min afterwards, takes supernatant to complete eucalyptus leaves
Pretreatment；
(2) blue light is handled：The eucalyptus leaves clear liquid that step (1) has pre-processed is put into blue light lamp box and carries out blue light illumination,
A length of 48h-50h when irradiation；The blue light intensity of illumination of irradiation is 1000Lx-1500Lx；
(3) anthocyanidin extracts：By after step (2) blue light illumination eucalyptus leaves clear liquid and organic solvent according to mass ratio be 1:
3-5 is mixed, and is then placed in supersonic extractors and is carried out ultrasonic extraction；Rotation is concentrated by evaporation, dries after extracting 12h-14h
To anthocyanidin coarse powder；
(4) cyanine is plain mentions：It according to mass ratio is 1 by the anthocyanidin coarse powder of step (3) and distilled water:10-15 is mixed
To anthocyanidin solution, then adsorbed with macroporous resin column；When absorption, column flow rate is 0.5-1 times of cylinder on anthocyanidin solution
Product/hour, upper column temperature are 30-40 DEG C, are eluted after the completion of absorption with the ethanol solution that percentage by volume is 75%, then
Be concentrated in vacuo, be freeze-dried to obtain it is described by leaf anthocyanidin；The upper column flow rate of ethanol solution is 1-1.5 when the elution
Times column volume/hour.
Pretreatment fluid is molten by tartaric acid solution, citric acid solution and malic acid in the step of prepared by eucalyptus leaves anthocyanidin (1)
Liquid is 1 according to mass ratio:1-3:2-4 composition；The mass concentration of tartaric acid solution mesotartaric acid is 3-7mg/L；Citric acid solution
The mass concentration of middle citric acid is 10-15mg/L；The mass concentration of malic acid is 5-15mg/L in malic acid solution.
Fumigation temperature is 105 DEG C -110 DEG C in the step of prepared by eucalyptus leaves anthocyanidin (1).
Biological enzyme is according to mass ratio by protease, tannase and lipase in the step of prepared by eucalyptus leaves anthocyanidin (1)
1:3-5:4-6 is mixed to prepare；The enzyme activity of the protease is 800u/g-1000u/g, and the enzyme activity of tannase is 800u/g-
1000u/g, the enzyme activity of lipase are 1000u/g-1200u/g.
Organic solvent is 5- according to mass ratio by ethyl alcohol, methanol and n-butanol in the step of prepared by eucalyptus leaves anthocyanidin (3)
8：2-4:1 composition；The percentage by volume of the ethyl alcohol is 95%；The percentage by volume of the methanol is 25%；The n-butanol
Percentage by volume be 15%.
The extraction power of ultrasonic extraction is 500w-1000w in the step of prepared by eucalyptus leaves anthocyanidin (3).
Further, the culture medium of the application is used for the initial Fiber differentiation of Banana Tissue culture.
The present invention has the advantages that：
1, the Banana Tissue culture medium of the application is by garlic P.E, tapioca, potato juice, chitosan, banana stem
Extracting solution, agar, aloe polysaccharide, eucalyptus leaves anthocyanidin and heteroauxin form, and contain allicin in garlic P.E, are very
Good antipathogenic composition, but its additive amount can excessively destroy the institutional framework of Explants In Banana, to cause explant growth slow
It is slow, for this purpose, applicants have found that good fungistatic effect, cyanine can not only be played if it cooperates eucalyptus anthocyanidin to use
Element can also promote cell differentiation, further promote the growth of Explants In Banana, take root, explant is made to have stronger survival ability,
In the eucalyptus anthocyanidin of the application other than containing Anthocyanins, there are also the substances such as many terpenes, eucalyptus oil, these ingredients
Good bacteriostasis can be played；Also contain in culture medium：Potato juice, potato after slight fermentation process, wherein
Aminoacid ingredient be fully dissolved out, Explants In Banana can be given to provide nitrogen source abundant；Polysaccharide rich in banana stem,
Carbon source abundant can be provided for explant, but since its sugar is lower, applicant also study be added to aloe polysaccharide and
Chitosan, these polysaccharide components can promote the dispersion of nutriment, and each ingredient is uniformly mixed, moreover it is possible to play water conservation and make
With can improve the enrichment of effective component, and form one layer of very thin protective film in media surface, guarantee to be formed inside culture medium
One relatively sterile nontoxic growing environment will not generate antagonism, externally moreover, mentioned component derives from plant source
Implant causes to encroach on.
2, it includes pressing leaf pretreatment, blue light processing, anthocyanidin extraction and cyanine that the eucalyptus leaves anthocyanidin of the application, which extracts,
Plain to propose four steps, in preprocessing process, pretreatment fluid is had by tartaric acid solution, citric acid solution and malic acid solution etc.
Machine acid composition, these Determination of Organic Acids can corrode eucalyptus leaves in acidity well, carry out after heating with the active material in blade
Reaction, can effectively remove eucalyptus vein after filtering (ingredient is mainly lignin)；After acid processing heating, it can contain in filtrate higher
The tannin and essential oil substance of ingredient；Tannin and essential oil will be effectively removed after stifling, in reaction solution at this time still containing compared with
The macromolecular substances, especially colloid such as colloid abundant, cellulose can form coating to influence the precipitation of anthocyanidin；Shen
Asking someone, addition biological enzyme will promote the decomposition of macromolecular substances at this time for discovery, carry out broken wall for the cell of eucalyptus leaves, accelerate
The precipitation of Dissolve things inside, to improve anthocyanidin yield；It is found in applicant's research process, if carried out at this time to extracting solution blue
Light processing will further be enriched with anthocyanidin, and concrete reason is also making further research at present, tentatively conclude it is because blue light promotees
Certain enzymatic reactions have been made to promote the synthesis of anthocyanidin intermediate；After blue light illumination, pass through organic solvent extraction and macroreticular resin
Absorption, can effectively promote the precipitation and purifying of anthocyanidin, can be effectively improved using the present processes production eucalyptus leaves anthocyanidin
The content and yield of anthocyanidin in extract, while tannin content in extract can also be effectively reduced.
【Specific embodiment】
All features disclosed in this specification or disclosed all methods or in the process the step of, in addition to mutually exclusive
Feature and/or step other than, can combine in any way.
Any feature disclosed in this specification (including any accessory claim, abstract), unless specifically stated, each
Feature is an example in a series of equivalent or similar characteristics.
Embodiment 1：
Present embodiments provide a kind of Banana Tissue culture medium, the culture medium is by the garlic P.E of 2.0g/L, 50g/L
Tapioca, the potato juice of 100g/L, 1.0g/L chitosan, 100g/L banana stem extracting solution, the agar of 10g/L, 1.0g/
The heteroauxin of the aloe polysaccharide of L, the eucalyptus leaves anthocyanidin of 3.0g/L and 0.5mg/L is mixed to prepare.
Allicin content is 156mg/g in garlic P.E in the present embodiment culture medium.
The cyanide content of tapioca is 2% in the present embodiment culture medium.
The preparation method of potato juice is in the present embodiment culture medium：Potato is cooked, mashed potato is pressed into, is cooled to
The yeast powder of addition mashed potato quality 3% ferment at constant temperature 4 days in 37 DEG C of insulating box, are then added mashed potato after room temperature
The distilled water of 2 times of mass parts, is sufficiently stirred, and is placed in 50 DEG C of insulating box and extracts 3h, filters to take filtrate up to potato
Juice；The living bacteria count of the yeast powder is 2.0 × 109cfu/g。
The preparation method of banana stem extracting solution is in the present embodiment culture medium：New fresh bananas stem is shredded, is then added fragrant
The biological enzyme solutions of any of several broadleaf plants stem 2%, for 24 hours, 3 times of quality are added in isothermal reaction in the insulating box that temperature is 37 DEG C after being heated to 50 DEG C
The ethyl ester solution that part percentage by volume is 20%, stirs evenly, the reaction was continued 3h, is cooled to room temperature, filters, filtrate is taken to stand point
Layer removes supernatant up to banana extracting solution；The cellulase that the biology enzyme solutions are 1000U/g by enzyme-activity unit.
Eucalyptus leaves anthocyanidin is by eucalyptus leaves by mentioning by leaf pretreatment, blue light processing, anthocyanidin in the present embodiment culture medium
It takes and cyanine is plain proposes several steps and be prepared；Specific step is as follows：
(1) leaf pretreatment is pressed：It is 1 that leaf and pretreatment fluid, which will be pressed, according to mass ratio:1 mixed (pretreatment fluid by
The apple that the citric acid solution and mass concentration that tartaric acid solution that mass concentration is 3mg/L, mass concentration are 10mg/L are 5mg/L
Tartaric acid solution is 1 according to mass ratio:1:2 are mixed to prepare), it is then placed in the water-bath that temperature is 40 DEG C and heats for 24 hours, taken after filtering
Filtrate obtains by leaf leachable；By eucalyptus leaves under conditions of temperature is 105 DEG C stifling 40min, then with biological enzyme solutions
(the tannic acid enzyme solutions and enzyme activity that protein enzyme solution that biological enzyme is 800u/g by enzyme activity, enzyme activity are 800u/g are for mixing
The lipase solution of 1000u/g is 1 according to mass ratio:3:4 are mixed to prepare), constant temperature handles 12h in 36 DEG C of insulating box；So
The centrifugal treating 5min in the centrifuge that revolving speed is 1000r/min afterwards takes supernatant to complete eucalyptus leaves pretreatment；
(2) blue light is handled：The eucalyptus leaves clear liquid that step (1) has pre-processed is put into blue light lamp box and carries out blue light illumination,
A length of 48h when irradiation；The blue light intensity of illumination of irradiation is 1000Lx；
(3) anthocyanidin extracts：By after step (2) blue light illumination eucalyptus leaves clear liquid and organic solvent according to mass ratio be 1:
3 mixed the (methanol and percentage by volume that ethyl alcohol that organic solvent is 95% by percentage by volume, percentage by volume are 25%
It according to mass ratio is 5 for 15% n-butanol：2:1 is mixed to prepare)；It is then placed in supersonic extractors and carries out ultrasonic extraction, surpass
The power that sound extracts is 500w；Rotation is concentrated by evaporation after extraction 12h, drying obtains anthocyanidin coarse powder；
(4) cyanine is plain mentions：It according to mass ratio is 1 by the anthocyanidin coarse powder of step (3) and distilled water:10 are mixed to get flower
Green element solution, is then adsorbed with macroporous resin column；When absorption, column flow rate is 0.5 times of column volume/small on anthocyanidin solution
When, upper column temperature is 30 DEG C, is eluted after the completion of absorption with the ethanol solution that percentage by volume is 75%, then carries out vacuum
Concentration, freeze-drying obtain described by leaf anthocyanidin；The upper column flow rate of ethanol solution is 1 times of column volume/small when the elution
When.
Embodiment 2：
Present embodiments provide a kind of Banana Tissue culture medium, the culture medium is by the garlic P.E of 5.0g/L, 60g/L
Tapioca, the potato juice of 200g/L, 3.0g/L chitosan, 200g/L banana stem extracting solution, the agar of 20g/L, 3.0g/
The heteroauxin of the aloe polysaccharide of L, the eucalyptus leaves anthocyanidin of 5.0g/L and 1mg/L is mixed to prepare.
Allicin content is 178mg/g in garlic P.E in the present embodiment culture medium.
The cyanide content of tapioca is 5% in the present embodiment culture medium.
The preparation method of potato juice is in the present embodiment culture medium：Potato is cooked, mashed potato is pressed into, is cooled to
The yeast powder of addition mashed potato quality 5% ferment at constant temperature 6 days in 37 DEG C of insulating box, are then added mashed potato after room temperature
The distilled water of 3 times of mass parts, is sufficiently stirred, and is placed in 60 DEG C of insulating box and extracts 4h, filters to take filtrate up to potato
Juice；The living bacteria count of the yeast powder is 3.0 × 109cfu/g。
The preparation method of banana stem extracting solution is in the present embodiment culture medium：New fresh bananas stem is shredded, is then added fragrant
3-5 times of matter is added in the biological enzyme solutions of any of several broadleaf plants stem 4%, the isothermal reaction 26h in the insulating box that temperature is 37 DEG C after being heated to 60 DEG C
The ethyl ester solution that part percentage by volume is 25% is measured, is stirred evenly, the reaction was continued 3h-4h is cooled to room temperature, filters, takes filtrate
Stratification removes supernatant up to banana extracting solution；The cellulase that the biology enzyme solutions are 1500U/g by enzyme-activity unit.
Eucalyptus leaves anthocyanidin is by eucalyptus leaves by mentioning by leaf pretreatment, blue light processing, anthocyanidin in the present embodiment culture medium
It takes and cyanine is plain proposes several steps and be prepared；Specific step is as follows：
(1) leaf pretreatment is pressed：It is 1 that leaf and pretreatment fluid, which will be pressed, according to mass ratio:3 mixed (pretreatment fluid by
The citric acid solution and mass concentration that tartaric acid solution that mass concentration is 7mg/L, mass concentration are 10-15mg/L are 15mg/L
Malic acid solution according to mass ratio be 1:3:4 are mixed to prepare), it is then placed in the water-bath that temperature is 50 DEG C and heats 26h, filter
After take filtrate to obtain by leaf leachable；By eucalyptus leaves under conditions of temperature is 110 DEG C stifling 50min, then with biological enzyme
Solution mixing (the tannic acid enzyme solutions and enzyme that protein enzyme solution that biological enzyme is 1000u/g by enzyme activity, enzyme activity are 1000u/g
The lipase solution that vigor is 1200u/g is 1 according to mass ratio:5:6 are mixed to prepare), constant temperature is handled in 38 DEG C of insulating box
14h；Then the centrifugal treating 15min in the centrifuge that revolving speed is 1500r/min takes supernatant to complete eucalyptus leaves pretreatment；
(2) blue light is handled：The eucalyptus leaves clear liquid that step (1) has pre-processed is put into blue light lamp box and carries out blue light illumination,
A length of 50h when irradiation；The blue light intensity of illumination of irradiation is 1500Lx；
(3) anthocyanidin extracts：By after step (2) blue light illumination eucalyptus leaves clear liquid and organic solvent according to mass ratio be 1:
5 mixed the (methanol and percentage by volume that ethyl alcohol that organic solvent is 95% by percentage by volume, percentage by volume are 25%
It according to mass ratio is 8 for 15% n-butanol：4:1 is mixed to prepare)；It is then placed in supersonic extractors and carries out ultrasonic extraction, surpass
The power that sound extracts is 1000w；Rotation is concentrated by evaporation after extraction 14h, drying obtains anthocyanidin coarse powder；
(4) cyanine is plain mentions：It according to mass ratio is 1 by the anthocyanidin coarse powder of step (3) and distilled water:15 are mixed to get flower
Green element solution, is then adsorbed with macroporous resin column；When absorption, column flow rate is 1 times of column volume/hour on anthocyanidin solution,
Upper column temperature is 40 DEG C, is eluted after the completion of absorption with the ethanol solution that percentage by volume is 75%, and it is dense then to carry out vacuum
Contracting, freeze-drying obtain described by leaf anthocyanidin；The upper column flow rate of ethanol solution is 1.5 times of column volumes/small when the elution
When.
Embodiment 3：
Present embodiments provide a kind of Banana Tissue culture medium, the culture medium is by the garlic P.E of 4.0g/L, 55g/L
Tapioca, the potato juice of 150g/L, 2.0g/L chitosan, 150g/L banana stem extracting solution, the agar of 15g/L, 2.0g/
The heteroauxin of the aloe polysaccharide of L, the eucalyptus leaves anthocyanidin of 4.0g/L and 0.8mg/L is mixed to prepare.
Allicin content is 168mg/g in garlic P.E in the present embodiment culture medium.
The cyanide content of tapioca is 4% in the present embodiment culture medium.
The preparation method of potato juice is in the present embodiment culture medium：Potato is cooked, mashed potato is pressed into, is cooled to
The yeast powder of addition mashed potato quality 4% ferment at constant temperature 5 days in 37 DEG C of insulating box, are then added mashed potato after room temperature
The distilled water of 2.5 times of mass parts, is sufficiently stirred, and is placed in 55 DEG C of insulating box and extracts 3.5h, filters to take filtrate up to horse
Bell potato juice；The living bacteria count of the yeast powder is 2.5 × 109cfu/g。
The preparation method of banana stem extracting solution is in the present embodiment culture medium：New fresh bananas stem is shredded, is then added fragrant
The biological enzyme solutions of any of several broadleaf plants stem 2%-4%, the isothermal reaction 25h in the insulating box that temperature is 37 DEG C are added 4 times after being heated to 55 DEG C
The ethyl ester solution that mass parts percentage by volume is 22%, stirs evenly, and the reaction was continued 3.5h is cooled to room temperature, filters, takes filtrate
Stratification removes supernatant up to banana extracting solution；The cellulase that the biology enzyme solutions are 1200U/g by enzyme-activity unit.
Eucalyptus leaves anthocyanidin is by eucalyptus leaves by mentioning by leaf pretreatment, blue light processing, anthocyanidin in the present embodiment culture medium
It takes and cyanine is plain proposes several steps and be prepared；Specific step is as follows：
(1) leaf pretreatment is pressed：It is 1 that leaf and pretreatment fluid, which will be pressed, according to mass ratio:2 mixed (pretreatment fluid by
The apple that the citric acid solution and mass concentration that tartaric acid solution that mass concentration is 5mg/L, mass concentration are 12mg/L are 7mg/L
Tartaric acid solution is 1 according to mass ratio:2:3 are mixed to prepare), it is then placed in the water-bath that temperature is 45 DEG C and heats 25h, taken after filtering
Filtrate obtains by leaf leachable；By eucalyptus leaves under conditions of temperature is 108 DEG C stifling 45min, then with biological enzyme solutions
(the tannic acid enzyme solutions and enzyme activity that protein enzyme solution that biological enzyme is 900u/g by enzyme activity, enzyme activity are 900u/g are for mixing
The lipase solution of 1100u/g is 1 according to mass ratio:2:5 are mixed to prepare), constant temperature handles 12h- in 37 DEG C of insulating box
14h；Then the centrifugal treating 8min in the centrifuge that revolving speed is 1200r/min takes supernatant to complete eucalyptus leaves pretreatment；
(2) blue light is handled：The eucalyptus leaves clear liquid that step (1) has pre-processed is put into blue light lamp box and carries out blue light illumination,
A length of 49h when irradiation；The blue light intensity of illumination of irradiation is 1300Lx；
(3) anthocyanidin extracts：By after step (2) blue light illumination eucalyptus leaves clear liquid and organic solvent according to mass ratio be 1:
4 mixed the (methanol and percentage by volume that ethyl alcohol that organic solvent is 95% by percentage by volume, percentage by volume are 25%
It according to mass ratio is 6 for 15% n-butanol：3:1 is mixed to prepare)；It is then placed in supersonic extractors and carries out ultrasonic extraction, surpass
The power that sound extracts is 700w；Rotation is concentrated by evaporation after extraction 13h, drying obtains anthocyanidin coarse powder；
(4) cyanine is plain mentions：It according to mass ratio is 1 by the anthocyanidin coarse powder of step (3) and distilled water:12 are mixed to get flower
Green element solution, is then adsorbed with macroporous resin column；When absorption, column flow rate is 0.8 times of column volume/small on anthocyanidin solution
When, upper column temperature is 35 DEG C, is eluted after the completion of absorption with the ethanol solution that percentage by volume is 75%, then carries out vacuum
Concentration, freeze-drying obtain described by leaf anthocyanidin；When the elution the upper column flow rate of ethanol solution be 1.3 times of column volumes/
Hour.
One, the application anthocyanidin quality and Study on extraction are as follows：
1, eucalyptus leaves anthocyanidin content and purity analysis in eucalyptus leaves extraction process：
Control group 1：
This control group does not have to pretreatment fluid and handles by leaf, and other preparation methods are identical with embodiment 1.
Control group 2：
For this control group not to leaf progress suffocating treatment is pressed, other preparation methods are identical with embodiment 1.
Control group 3：
This control group is handled eucalyptus leaves without using biological enzyme, and other preparation methods are identical with embodiment 1.
Control group 4：
This control group is handled without using blue light, and other preparation methods are identical with embodiment 1.
Control group 5：
Ethyl alcohol is used only in this control group organic solvent, that is, in the extraction of step (3) anthocyanidin：By step (2) blue light illumination
The ethanol solution that eucalyptus leaves clear liquid and percentage by volume afterwards is 95% is 1 according to mass ratio:3 are mixed, other preparation sides
Method is identical with embodiment 1.
Yield and purity test：
Anthocyanidin, tannin content in embodiment 1-3 and control group 1-5 extract are counted and detected, and calculates anthocyanidin and obtains
Rate, yield are calculated by following formula and are obtained, and concrete condition is shown in Table 1：
Table 1
As seen from the above table, the anthocyanidin content of embodiment 1-3, yield are all apparently higher than control group 1-5, illustrate the application's
Pretreatment fluid processing, suffocating treatment, cellulase treatment, blue light illumination processing and organic solvent formula can improve this well
The eucalyptus leaves anthocyanidin content of application, and it is indispensable；The tannin content of embodiment 1-3 is significantly lower than control group 1-3, and compares
Group 4-5 is not much different, and illustrates that the pretreatment fluid processing of the application, suffocating treatment, cellulase treatment can be effectively reduced in extract
Tannin content, and it is indispensable.
2, influence test of the pretreatment fluid component to eucalyptus leaves tannin content in eucalyptus leaves extraction process：
Eucalyptus leaves are pre-processed according to following processing group, test pretreatment front and back eucalyptus leaves tannin content, processing group
It is as follows：
Processing 1：Eucalyptus leaves are pre-processed according to the method for the embodiment of the present application 1；
Processing 2：Tartaric acid solution is free of in pretreatment fluid, other processing modes are identical with embodiment 1, that is, pre-process
The malic acid solution that the citric acid solution and mass concentration that liquid is 10mg/L by mass concentration are 5mg/L is 1 according to mass ratio:2
It is mixed to prepare.
Processing 3：Citric acid solution is free of in pretreatment fluid, other processing modes are identical with embodiment 1, that is, pre-process
The malic acid solution that the tartaric acid solution and mass concentration that liquid is 3mg/L by mass concentration are 5mg/L is 1 according to mass ratio:2 is mixed
It closes and is made.
Processing 4：Tartaric acid solution is free of in pretreatment fluid, other processing modes are identical with embodiment 1, that is, pre-process
The citric acid solution that the tartaric acid solution and mass concentration that liquid is 3mg/L by mass concentration are 10mg/L is 1 according to mass ratio:1
It is mixed to prepare.
Test result is shown in Table 2：
Table 2
| Tannin content (mg/g) | Processing 1 | Processing 2 | Processing 3 | Processing 4 |
| Before processing | 24.35 | 25.13 | 24.08 | 25.19 |
| After processing | 4.03 | 12.31 | 13.26 | 15.33 |
As seen from the above table, the ability of 1 processing tannin of processing is most strong, and the ability of processing 2-4 processing tannin is not so good as processing 1, by
This illustrates that content, the component of organic acid can reduce tannin content well in the application pretreatment fluid component, and ingredient lacks one not
It can.
3, influence test of the biological enzyme solution component to eucalyptus leaves tannin content in eucalyptus leaves extraction process：
Eucalyptus leaves are pre-processed according to following processing group, test pretreatment front and back eucalyptus leaves tannin content, processing group
It is as follows：
Handle A：Eucalyptus leaves are pre-processed according to the method for the embodiment of the present application 1；
Handle B：Protein enzyme solution is free of in pretreatment fluid, other processing modes are identical with embodiment 1, i.e. biological enzyme
The lipase solution that the tannic acid enzyme solutions and enzyme activity that liquid is 800u/g by enzyme activity are 1000u/g is 3 according to mass ratio:4 is mixed
It closes and is made.
Handle C：Tannic acid enzyme solutions are free of in pretreatment fluid, other processing modes are identical with embodiment 1, i.e. biological enzyme
The lipase solution that the protein enzyme solution and enzyme activity that liquid is 800u/g by enzyme activity by biological enzyme are 1000u/g is according to mass ratio
It is 1:4 are mixed to prepare.
Handle D：Lipase solution is free of in pretreatment fluid, other processing modes are identical with embodiment 1, i.e. biological enzyme
The tannic acid enzyme solutions that the protein enzyme solution and enzyme activity that liquid is 800u/g by enzyme activity by biological enzyme are 800u/g are according to mass ratio
It is 1:3 are mixed to prepare.
Test result is shown in Table 3：
Table 3
| Tannin content (mg/g) | Handle A | Handle B | Handle C | Handle D |
| Before processing | 24.35 | 25.13 | 24.08 | 25.19 |
| After processing | 4.03 | 8.65 | 9.06 | 8.33 |
As seen from the above table, the ability of processing A processing tannin is most strong, and the ability of processing B-D processing tannin is not so good as processing A, by
This illustrates that content, the component of biological enzyme can reduce tannin content well in the application biology enzyme solution component, and ingredient lacks one not
It can.
Two, impact analysis of the culture medium of the application to Banana Tissue culture：
The culture medium prescription of following processing group is prepared into culture medium, equal number of Explants In Bananas after sterilizing are seeded in
Fiber differentiation is carried out in induced medium in the incubator, cultivation temperature is 27 DEG C, is carried out with the white light that intensity of illumination is 800Lx
Interval light is shone, and interval light is " 3h (illumination) -1h (dark reaction) -3h (illumination) " according to processing method, is spaced the total of lighting process
Shi Changwei 30h；Explants In Banana is selected from the banana stem of same strain banana, and each processing sets 3 repetitions, counts and calculate banana
The survival rate of explant, the average root long of every plant of explant, concrete outcome are shown in Table 4.
Handle a：Use the culture medium of the embodiment of the present application 1；
Handle b：Use the culture medium of the embodiment of the present application 2；
Handle c：Use the culture medium of the embodiment of the present application 3；
Handle d：Use the culture medium for being free of garlic P.E；I.e. culture medium is by the tapioca of 50g/L, the horse of 100g/L
Bell potato juice, 1.0g/L chitosan, 100g/L banana stem extracting solution, the agar of 10g/L, the aloe polysaccharide of 1.0g/L, 3.0g/L
Eucalyptus leaves anthocyanidin and 0.5mg/L heteroauxin composition；Other embodiment is identical with embodiment 1.
Handle e：Use the culture medium for being free of tapioca；I.e. culture medium is by the garlic P.E of 2.0g/L, 100g/L
Potato juice, 1.0g/L chitosan, 100g/L banana stem extracting solution, the agar of 10g/L, the aloe polysaccharide of 1.0g/L, 3.0g/
The eucalyptus leaves anthocyanidin of L and the heteroauxin composition of 0.5mg/L；Other embodiment is identical with embodiment 1.
Handle f：Use the culture medium for being free of potato juice；I.e. culture medium is by the garlic P.E of 2.0g/L, the wood of 50g/L
Sweet potato starch, 1.0g/L chitosan, 100g/L banana stem extracting solution, the agar of 10g/L, the aloe polysaccharide of 1.0g/L, 3.0g/L
Eucalyptus leaves anthocyanidin and 0.5mg/L heteroauxin composition；Other embodiment is identical with embodiment 1.
Handle g：Use the culture medium for being free of banana stem extracting solution；That is garlic P.E, 50g/L of the culture medium by 2.0g/L
Tapioca, 100g/L potato juice, 1.0g/L chitosan, the agar of 10g/L, the aloe polysaccharide of 1.0g/L, 3.0g/L
Eucalyptus leaves anthocyanidin and 0.5mg/L heteroauxin composition；Other embodiment is identical with embodiment 1.
Handle h：Use the culture medium for being free of eucalyptus leaves anthocyanidin；That is garlic P.E, 50g/L of the culture medium by 2.0g/L
Tapioca, the potato juice of 100g/L, 1.0g/L chitosan, the banana stem extracting solution of 100g/L, 10g/L agar,
The aloe polysaccharide of 1.0g/L and the heteroauxin composition of 0.5mg/L；Other embodiment is identical with embodiment 1.
Handle i：Use MS culture medium.
Table 4
As seen from the above table, handle the Explants In Banana survival rate of a-c, average root long is above processing d-h, higher than handle
Thus i illustrates, in the culture medium prescription of the application, garlic P.E, tapioca, potato juice, banana stem extracting solution and eucalyptus
Leaf anthocyanidin is used in compounding the survival rate that can effectively improve Explants In Banana, and explant is promoted to take root, and is preferably applied to
In subsequent squamous subculture, the medium component of the application is indispensable.
In conclusion Explants In Banana growth can be promoted using culture medium of the present invention, taken root, while the ingredient will not also be made
It is safe and reliable at the residual of chemical substance.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitation of the scope of the invention therefore cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art,
Without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection model of the invention
It encloses.Therefore, protection scope of the present invention should be determined by the appended claims.
Claims (7)
1. a kind of Banana Tissue culture medium, which is characterized in that the culture medium by 2.0g/L-5.0g/L garlic P.E,
The tapioca of 50g/L-60g/L, the potato juice of 100g/L-200g/L, 1.0g/L-3.0g/L chitosan, 100g/L-
Banana stem extracting solution, the agar of 10g/L-20g/L, the aloe polysaccharide of 1.0g/L-3.0g/L, 3.0g/L-5.0g/L of 200g/L
Eucalyptus leaves anthocyanidin and 0.5mg/L-1mg/L heteroauxin composition.
2. culture medium according to claim 1, which is characterized in that allicin content is in the garlic P.E
156mg/g-178mg/g。
3. culture medium according to claim 1, which is characterized in that the cyanide content of the tapioca is 2%-5%.
4. culture medium according to claim 1, which is characterized in that the preparation method of the potato juice is：By potato
It cooks, is pressed into mashed potato, the yeast powder of mashed potato quality 3%-5% is added after being cooled to room temperature in 37 DEG C of insulating box
Ferment at constant temperature 4-6 days, the distilled water of 2-3 times of mass parts of mashed potato is then added, is sufficiently stirred, and be placed in 50 DEG C -60 DEG C
Insulating box in extract 3-4h, filter to take filtrate up to potato juice；The living bacteria count of the yeast powder be 2.0 ×
109cfu/g-3.0×109cfu/g。
5. culture medium according to claim 1, which is characterized in that the preparation method of the banana stem extracting solution is：It will be new
The chopping of fresh bananas stem, is then added the biological enzyme solutions of banana stem 2%-4%, the isothermal reaction in the insulating box that temperature is 37 DEG C
The ethyl ester solution that 3-5 times of mass parts percentage by volume is 20%-25%, stirring is added in -26h for 24 hours after being heated to 50 DEG C -60 DEG C
Uniformly, the reaction was continued 3h-4h is cooled to room temperature, filters, filtrate stratification is taken to remove supernatant up to banana extracting solution；Institute
State the cellulase that biological enzyme solutions are 1000U/g-1500U/g by enzyme-activity unit.
6. culture medium according to claim 1, which is characterized in that the eucalyptus leaves anthocyanidin is pre- by pressing leaf by eucalyptus leaves
Processing, blue light processing, anthocyanidin, which are extracted, and cyanine is plain proposes several steps and is prepared.
7. the application of culture medium as described in claim 1-6, which is characterized in that the culture medium is initial for Banana Tissue culture
Fiber differentiation.
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