CN109042320A - A kind of oil tree peony callus cultural method - Google Patents
A kind of oil tree peony callus cultural method Download PDFInfo
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- CN109042320A CN109042320A CN201810814581.0A CN201810814581A CN109042320A CN 109042320 A CN109042320 A CN 109042320A CN 201810814581 A CN201810814581 A CN 201810814581A CN 109042320 A CN109042320 A CN 109042320A
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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Abstract
The invention belongs to plant callus Cultivating techniques fields, and in particular to a kind of oil tree peony callus cultural method.This method takes oil tree peony blade, cleans, and disinfection obtains aseptic blade；Then aseptic blade is cut along blade master pulse direction, i.e., each blade is divided equally into two, then carries out Fiber differentiation and Multiplying culture, is added to ginger juice in induced medium and proliferated culture medium respectively.Method of the invention uses tree peony leaf as object using oil, and successful incubation is fuel-displaced to use tree peony callus, has established experimental basis with the foundation of tree peony regenerating system for oil.
Description
Technical field
The invention belongs to plant callus Cultivating techniques fields, and in particular to a kind of oil tree peony callus culture side
Method.
Background technique
Wild tree peonies kind in China's is mainly distributed on the ground such as Henan, Shaanxi, Hubei, Gansu, Sichuan, Yunnan, Tibet at present.
According to incompletely statistics, there is ability about more than 100 kinds.Oil with tree peony refer to sub real yield is high, oil content is high, effectively at
Divide high Varieties of Peony type.Unsaturated fatty acid content is more than 90% in phoenix pellet tree peony and paeonia rockii seed, wherein linolenic acid
Content accounts for 40% or so.Linolenic acid is manpower essential amino acid, have refresh the mind, enhance memory, reducing blood lipid, blood pressure lowering, in advance
Anti- cardiovascular and cerebrovascular disease and other effects plays key effect to human health.Therefore oil with the mass propgation of tree peony not only have compared with
Good appreciation effect also has very high medical value.Traditional oils are mainly seminal propagation and plant division with the implantation methods of tree peony
It breeds, time-consuming in incubation.
The plant energy robust growth of tissue culture technique culture, leaf dark green, and can effectively change fruit quality, it improves and produces
Amount, breeding is fast, and is not constrained by season.Common tissue culture technique is callus tissue culture, for example pear cv nanguo, oil are with male
Pellet, carrot etc. are used equally for callus tissue culture, but due to the oil plant callus of less types, traditional of tree peony
Induction and subculture hormone combination are not particularly suited for the callus tissue cultures of all plants, at present not yet discovery using oil with tree peony into
The method of row callus tissue culture.
Summary of the invention
The object of the present invention is to provide a kind of oil tree peony callus cultural method, this method is with tree peony leaf with oil
Object, successful incubation is fuel-displaced to use tree peony callus, has established experimental basis with the foundation of tree peony regenerating system for oil.
A kind of oil tree peony callus cultural method provided by the invention, comprising the following steps:
S1, takes oil tree peony blade, cleans, and disinfection obtains aseptic blade；
Induction: S2 aseptic blade is cut along blade master pulse direction, i.e., each blade is split into two halves, after incision
For blade inoculation in induced medium, 25 ± 2 DEG C of cultures obtain Callus of Leaf until growing callus；
The induced medium is prepared by the following method: 4.74g MS solid medium, 20g sucrose being added in 1L water, adds
PH to 5.8~6.0 is adjusted in heat of solution, and NAA, 10~20ml ginger juice, the 6~8g agar of 0.1mg is then added, boils to agar
It is completely dissolved, sterilizes；
S3, proliferation: taking out Callus of Leaf, and stripping and slicing is placed in proliferated culture medium, 25 ± 2 DEG C of cultures, until obtaining white
The loose oil tree peony Callus of Leaf of color；
The proliferated culture medium is prepared by the following method: 4.74g MS solid medium, 30g sucrose being added in 1L water, adds
PH to 5.8~6.0 is adjusted in heat of solution, and 6-BA, 10~20ml ginger juice, the 6~8g agar of 2mg is then added, boils complete to agar
Fully dissolved, sterilizing；
Wherein, ginger juice described in S2 and S3 is prepared in accordance with the following methods: with water according to 20g after ginger slice:
The ratio of 200ml mixes, and boils 5~10min, and the filtrate collected after filtering is ginger juice.
Preferably, above-mentioned oil tree peony callus cultural method, in S1, the concrete mode of disinfection is as follows: by what is cleaned
Oil is sterilized with 75% alcohol solution dipping of tree peony blade volume fraction, and aseptic water washing places into the HgCl of 0.1g/100ml2
Solution soaking disinfection 8min, take out blade and use aseptic water washing, suck dry moisture taking-up, obtain aseptic blade.
Preferably, above-mentioned oil tree peony callus cultural method, in S1, wash oil is with tree peony blade along master pulse direction
Length is 2~6cm.
Preferably, above-mentioned oil tree peony callus cultural method, in S1, ginger slice described in S2 and S3 with a thickness of 2
~4mm.
Compared with prior art, oil tree peony callus cultural method provided by the invention, has the advantages that
Use tree peony blade as material using oil, growth speed vigorous using the callus growth ability of the method for the present invention culture
Degree is fast, and Multiplying culture 25d or so can be grown to total volume about 5~6cm3, good dispersion, white loose shape and quality is softer, 35d
Color just becomes dark yellow.It is later gradually in sepia using the callus of comparative example culture at subculture initial stage in dark yellow
And quality is harder, growing way is slow, and Multiplying culture 25d or so total volume is only 1~2cm3, 30d or so can aging blackening.
Method of the invention has established experimental basis with the foundation of tree peony regenerating system for oil, to utilize regenerating system technology
Oil is carried out to be laid a good foundation with the research that Varieties of Peony is improved.
Detailed description of the invention
Fig. 1 is the callus picture obtained after embodiment 1 is proliferated；
Fig. 2 is the callus picture obtained after comparative example 1 is proliferated.
Specific embodiment
The specific embodiment of invention is described in detail below, it is to be understood that protection scope of the present invention not by
The limitation of specific embodiment.The test method of actual conditions is not specified in the following example, usually according to normal condition, or
According to condition proposed by each manufacturer.In addition, the following MS solid mediums (being free of sucrose) used in the examples of the present invention
Etc. various reagents be commercially available.
The present invention provides a kind of oil tree peony callus cultural methods, comprising the following steps:
S1, takes oil tree peony blade, cleans, and disinfection obtains aseptic blade；
Aseptic blade is cut along blade master pulse direction, i.e., each blade is split into two halves, the blade after incision is connect by S2
For kind in induced medium, 25 ± 2 DEG C of cultures obtain Callus of Leaf until growing callus；
The induced medium is prepared by the following method: 4.74g MS solid medium, 20g sucrose being added in 1L water, adds
PH to 5.8~6.0 is adjusted in heat of solution, and NAA, 10~20ml ginger juice, the 6~8g agar of 0.1mg is then added, boils to agar
It is completely dissolved, sterilizes；
S3 takes out Callus of Leaf, and stripping and slicing is placed in proliferated culture medium, and 25 ± 2 DEG C of cultures are dredged until obtaining white
Pine tar tree peony Callus of Leaf；
The proliferated culture medium is prepared by the following method: 4.74g MS solid medium, 30g sucrose being added in 1L water, adds
PH to 5.8~6.0 is adjusted in heat of solution, and 6-BA, 10~20ml ginger juice, the 6~8g agar of 2mg is then added, boils complete to agar
Fully dissolved, sterilizing；
Wherein, ginger juice described in S2 and S3 is prepared in accordance with the following methods: with water according to 20g after ginger slice:
The ratio of 200ml mixes, and boils 5~10min, and the filtrate collected after filtering is ginger juice.
Preferably, the present invention a kind of oil tree peony callus cultural method, including following embodiment.Following embodiments
In, material to be tested is oily Varieties of Peony " phoenix pellet tree peony " plant, chooses suitable oil tree peony blade as needed, is sampling
The same day is inoculated with, evoked callus.
Embodiment 1
A kind of oil tree peony callus cultural method, comprising the following steps:
S1 takes oil tree peony blade, and length of the oil with tree peony blade along master pulse direction is 6cm, is cleaned, and disinfection obtains nothing
Bacterium blade；The concrete mode of disinfection is as follows: clean oil 75% alcohol solution dipping of tree peony blade volume fraction sterilized,
Aseptic water washing places into the HgCl of 0.1g/100ml2Solution soaking disinfection 8min, take out blade and use aseptic water washing, suction
Solid carbon dioxide point takes out, and obtains aseptic blade.
Induction: S2 aseptic blade is cut along blade master pulse direction, i.e., each blade is divided equally into two, after incision
Blade inoculation is in induced medium, and 25 ± 2 DEG C of cultures, light application time 16h/d, light intensity 2000lx have been cultivated 20 days, grown
The callus of 1cm × 1cm size, obtains Callus of Leaf；
The induced medium is prepared by the following method: 4.74g MS solid medium, 20g sucrose being added in 1L water, adds
PH to 5.8 is adjusted in heat of solution, and NAA, 10ml ginger juice, the 6g agar of 0.1mg is then added, boils to agar and is completely dissolved, go out
Bacterium；
It is inoculated with 20 bottles altogether, and counts the pollution rate A in the stage, healing rate.
Blade number/inoculation blade total number × 100% of pollution rate A=pollution microbes；
Healing rate=formation callus blade number/(number of blade total number-pollution blade of inoculation) ×
100%；
S3, proliferation: taking out Callus of Leaf, and 2mm × 2mm × 1mm stripping and slicing is placed in proliferated culture medium, light application time
16h/d, light intensity 2000lx, 25 ± 2 DEG C of cultures obtain 4~5cm after 25 days3The white loose oil of size tree peony blade callus
Tissue；
The proliferated culture medium is prepared by the following method: 4.74g MS solid medium, 30g sucrose being added in 1L water, adds
PH to 5.8 is adjusted in heat of solution, and 6-BA, 10ml ginger juice, the 6g agar of 2mg is then added, boils to agar and is completely dissolved, and is sterilized；
It is inoculated with 20 bottles altogether, counts the pollution rate B of the stage callus.
Pollution rate B=pollution bottle number/total bottle number × 100% of callus.
Ginger juice described in S2 and S3 is prepared in accordance with the following methods: with water according to 20g:200ml's after ginger slice
Ratio mixing, ginger slice with a thickness of 2mm, boil 5min, the filtrate collected after the filtering of fast qualitative filter paper is made a living
Ginger juice.
As the result is shown: carrying out callus tissue culture using the method for embodiment 1, pollution rate A is 4.7%, and healing rate is
68.3%, pollution rate B are 18.9%.The callus picture obtained after S3 proliferation is as shown in Figure 1, quality white loose.
Embodiment 2
A kind of oil tree peony callus cultural method, comprising the following steps:
S1 takes oil tree peony blade, and length of the oil with tree peony blade along master pulse direction is 2cm, is cleaned, and disinfection obtains nothing
Bacterium blade；The concrete mode of disinfection is as follows: clean oil 75% alcohol solution dipping of tree peony blade volume fraction sterilized,
Aseptic water washing places into the HgCl of 0.1g/100ml2Solution soaking disinfection 8min, take out blade and use aseptic water washing, suction
Solid carbon dioxide point takes out, and obtains aseptic blade.
Induction: S2 aseptic blade is cut along blade master pulse direction, i.e., each blade is split into two halves, after incision
Blade inoculation is in induced medium, and 25 ± 2 DEG C of cultures, light application time 16h/d, light intensity 2000lx have been cultivated 20 days, grown
The callus of 1cm × 1cm size, obtains Callus of Leaf；
The induced medium is prepared by the following method: 4.74g MS solid medium, 20g sucrose being added in 1L water, adds
PH to 6.0 is adjusted in heat of solution, and NAA, 20ml ginger juice, the 8g agar of 0.1mg is then added, boils to agar and is completely dissolved, go out
Bacterium；
It is inoculated with 20 bottles altogether, and counts the pollution rate A in the stage, healing rate.
Blade number/inoculation blade total number × 100% of pollution rate A=pollution microbes；
Healing rate=formation callus blade number/(number of blade total number-pollution blade of inoculation) ×
100%；
S3, proliferation: taking out Callus of Leaf, and 2mm × 2mm × 1mm stripping and slicing is placed in proliferated culture medium, light application time
16h/d, light intensity 2000lx, 25 ± 2 DEG C of cultures obtain 4~5cm after 25 days3The white loose oil of size tree peony blade callus
Tissue；
The proliferated culture medium is prepared by the following method: 4.74g MS solid medium, 30g sucrose being added in 1L water, adds
PH to 6.0 is adjusted in heat of solution, and 6-BA, 20ml ginger juice, the 8g agar of 2mg is then added, boils to agar and is completely dissolved, and is sterilized；
It is inoculated with 20 bottles altogether, counts the pollution rate B of the stage callus.
Pollution rate B=pollution bottle number/total bottle number × 100% of callus.
Ginger juice described in S2 and S3 is prepared in accordance with the following methods: with water according to 20g:200ml's after ginger slice
Ratio mixing, ginger slice with a thickness of 4mm, boil 10min, the filtrate collected after the filtering of fast qualitative filter paper is made a living
Ginger juice.
As the result is shown: carrying out callus tissue culture using the method for embodiment 2, pollution rate A is 5.1%, and healing rate is
69.4%, pollution rate B are 18.7%.
Embodiment 3
A kind of oil tree peony callus cultural method, comprising the following steps:
S1 takes oil tree peony blade, and length of the oil with tree peony blade along master pulse direction is 4.2cm, is cleaned, and disinfection obtains
Aseptic blade；Remaining operation of S1 is the same as embodiment 1.
Induction: S2 aseptic blade is cut along blade master pulse direction, i.e., each blade is split into two halves, after incision
Blade inoculation is in induced medium, and 25 ± 2 DEG C of cultures, light application time 16h/d, light intensity 2000lx have been cultivated 20 days, grown
The callus of 1cm × 1cm size, obtains Callus of Leaf；
The induced medium is prepared by the following method: 4.74g MS solid medium, 20g sucrose being added in 1L water, adds
PH to 5.8 is adjusted in heat of solution, and NAA, 15ml ginger juice, the 6g agar of 0.1mg is then added, boils to agar and is completely dissolved, go out
Bacterium；
It is inoculated with 20 bottles altogether, and counts the pollution rate A in the stage, healing rate.
The calculation method of pollution rate A and healing rate is the same as embodiment 1.
S3, proliferation: taking out Callus of Leaf, and 2mm × 2mm × 1mm stripping and slicing is placed in proliferated culture medium, light application time
16h/d, light intensity 2000lx, 25 ± 2 DEG C of cultures obtain 4~5cm after 25 days3The white loose oil of size tree peony blade callus
Tissue；
The proliferated culture medium is prepared by the following method: 4.74g MS solid medium, 30g sucrose being added in 1L water, adds
PH to 5.8 is adjusted in heat of solution, and 6-BA, 15ml ginger juice, the 6g agar of 2mg is then added, boils to agar and is completely dissolved, and is sterilized；
It is inoculated with 20 bottles altogether, counts the pollution rate B of the stage callus, calculation method is the same as embodiment 1.
Ginger juice described in S2 and S3 is prepared in accordance with the following methods: with water according to 20g:200ml's after ginger slice
Ratio mixing, ginger slice with a thickness of 2.6mm, boil 8min, the filtrate collected after the filtering of fast qualitative filter paper is
Ginger juice.
As the result is shown: carrying out callus tissue culture using the method for embodiment 3, pollution rate A is 4.5%, and healing rate is
66.4%, pollution rate B are 15.1%.
Comparative example 1
A kind of oil tree peony callus cultural method, comprising the following steps:
S1, takes oil tree peony blade, cleans, and disinfection obtains aseptic blade；The concrete mode of disinfection is as follows: by what is cleaned
Oil is used with tree peony blade and is sterilized with 75% alcohol solution dipping of volume fraction, and aseptic water washing places into 0.1g/100ml's
HgCl2Solution soaking disinfection 8min, take out blade and use aseptic water washing, suck dry moisture taking-up, obtain aseptic blade.
Induction: S2 aseptic blade is cut along blade master pulse direction, i.e., each blade is split into two halves, after incision
Blade inoculation is in induced medium, and 25 ± 2 DEG C of cultures, light application time 16h/d, light intensity 2000lx have been cultivated 20 days, grown
The callus of 0.5cm × 0.5cm size, obtains Callus of Leaf；
The induced medium is prepared by the following method: 4.74g MS solid medium, 20g sucrose being added in 1L water, adds
PH to 5.8 is adjusted in heat of solution, and NAA, 6g agar of 0.1mg is then added, boils to agar and is completely dissolved, and is sterilized；
It is inoculated with 20 bottles altogether, and counts the pollution rate A in the stage, healing rate.
Blade number/inoculation blade total number × 100% of pollution rate A=pollution microbes；
Healing rate=formation callus blade number/(number of blade total number-pollution blade of inoculation) ×
100%；
S3, proliferation: taking out Callus of Leaf, and 2mm × 2mm × 1mm stripping and slicing is placed in proliferated culture medium, light application time
16h/d, light intensity 2000lx, 25 ± 2 DEG C of cultures obtain 1~2cm after 25 days3The white loose oil of size tree peony blade callus
Tissue；
The proliferated culture medium is prepared by the following method: 4.74g MS solid medium, 30g sucrose being added in 1L water, adds
Heat of solution, adjust pH to 5.8, then be added 2mg 6-BA,
6g agar, boils to agar and is completely dissolved, sterilizing；
It is inoculated with 20 bottles altogether, counts the survival rate and pollution rate B of the stage callus.
Pollution rate B=pollution bottle number/total bottle number × 100% of callus
As the result is shown: carrying out callus tissue culture using the method for embodiment 1, pollution rate A is 36.8%, and healing rate is
51.0%, pollution rate B are 41.2%.Obtained callus picture after S3 proliferation is as shown in Fig. 2, sepia and quality is harder.
It should be noted that when being related to numberical range in the present invention, except non-present invention is otherwise noted, each numberical range
Two endpoints and two endpoints between any one numerical value can be selected.Although preferred implementation of the invention has been described
Example, once a person skilled in the art knows basic creative concepts, then other change can be made to these embodiments
More and modify.So it includes preferred embodiment and all changes for falling into the scope of the invention that the following claims are intended to be interpreted as
More and modify.
Obviously, various changes and modifications can be made to the invention without departing from essence of the invention by those skilled in the art
Mind and range.In this way, if these modifications and changes of the present invention belongs to the range of the claims in the present invention and its equivalent technologies
Within, then the present invention is also intended to include these modifications and variations.
Claims (4)
1. a kind of oil tree peony callus cultural method, which comprises the following steps:
S1, takes oil tree peony blade, cleans, and disinfection obtains aseptic blade；
Induction: S2 aseptic blade is cut along blade master pulse direction, i.e., each blade is split into two halves, by the blade after incision
It is seeded in induced medium, 25 ± 2 DEG C of cultures obtain Callus of Leaf until growing callus；
The induced medium is prepared by the following method: 4.74g MS solid medium, 20g sucrose being added in 1L water, heats molten
Solution adjusts pH to 5.8~6.0, and NAA, 10~20ml ginger juice, the 6~8g agar of 0.1mg is then added, boils complete to agar
Dissolution, sterilizing；
S3, proliferation: taking out Callus of Leaf, and stripping and slicing is placed in proliferated culture medium, and 25 ± 2 DEG C of cultures are dredged until obtaining white
Pine tar tree peony Callus of Leaf；
The proliferated culture medium is prepared by the following method: 4.74g MS solid medium, 30g sucrose being added in 1L water, heats molten
Solution adjusts pH to 5.8~6.0, and 6-BA, 10~20ml ginger juice, the 6~8g agar of 2mg is then added, boils completely molten to agar
Solution, sterilizing；
Wherein, ginger juice described in S2 and S3 is prepared in accordance with the following methods: with water according to 20g:200ml's after ginger slice
Ratio mixing, boils 5~10min, and the filtrate collected after filtering is ginger juice.
2. it is according to claim 1 oil use tree peony callus cultural method, which is characterized in that in S1, disinfection it is specific
Mode is as follows: clean oil 75% alcohol solution dipping of tree peony blade volume fraction being sterilized, aseptic water washing places into
The HgCl of 0.1g/100ml2Solution soaking disinfection 8min, take out blade and use aseptic water washing, suck dry moisture taking-up, obtain nothing
Bacterium blade.
3. oil tree peony callus cultural method according to claim 1, which is characterized in that in S1, oil peony leaves
Length of the piece along master pulse direction is 2~6cm.
4. oil tree peony callus cultural method according to claim 3, which is characterized in that in S1, institute in S2 and S3
State ginger slice with a thickness of 2~4mm.
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| CN113678732A (en) * | 2021-08-17 | 2021-11-23 | 菏泽学院 | Peony stem leaf callus induction method |
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