CN108812316A - A kind of Strawberry Leaves callus induction method - Google Patents
A kind of Strawberry Leaves callus induction method Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- 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
The invention belongs to plant callus Cultivating techniques fields, and in particular to a kind of Strawberry Leaves callus induction method.This method wins Strawberry ripening blade, cleans, is sterilized, aseptic water washing with alcohol solution dipping, place into the HgCl of 0.1g/100ml2Solution soaking disinfection 6min, aseptic water washing, suck dry moisture obtain aseptic blade；Then aseptic blade is carried out carving scar processing along blade master pulse direction, carries out Fiber differentiation in induced medium, Multiplying culture is carried out in proliferated culture medium.For the present invention using Strawberry Leaves as the research material of experiment, blade materials are convenient, and from a wealth of sources, operation is easy, and the high efficient regeneration system for establishing excised leaf is convenient for the research of bio-technology improvement kind.
Description
Technical field
The invention belongs to plant callus Cultivating techniques fields, and in particular to a kind of Strawberry Leaves callus induction side
Method.
Background technique
Strawberry (Fragaria ananassa Duch), the herbaceos perennial of rosaceae Fragaria is rich in
The nutriments such as vitamin C, pectin, cellulose, iron, calcium.There are higher economic value, nutritive value and health-care efficacy, can protect
Eyesight is protected, it is aid digestion.Therefore, strawberry is widely cultivated always.Traditional propagation method has division propagation and seminal propagation, they
It is not high that there is breeding coefficients, seedling poor quality, is often accompanied by root, leaf, and the illness on fruit occurs, such as leaf spot, powdery mildew, ash
Mildew, deformed fruit etc..Tissue culture propagation just comes into being, it utilizes tissue culture technique, and the Strawberry Seedlings of culture are virus-free, growth
Stalwartness, leaf dark green can effectively change fruit quality, improve yield, breeding is fast, and is not constrained by season.
Currently, carried out a large amount of research to Strawberry tissue culture both at home and abroad, the kind being related to mainly have ' brilliant precious jade ',
' Feng Xiang ', ' beauty ', ' all-star ' etc..The study found that the most suitable evoked callus culture medium of ' Feng Xiang ' is MS+6-BA
2.0mg/L+2,4-D 0.1mg/L；The most suitable evoked callus culture medium of ' a chapter Ji ' is MS+6-BA 2.0mg/L+NAA
0.25mg/L；The most suitable evoked callus culture medium of ' all-star ' is MS+6-BA 2.0mg/L+NAA 0.2mg/L；' Saga
Faint scent ' occur vitrification phenomenon when not adding or NAA concentration is low, but its plant normal growth when NAA concentration is higher, when
There is callus when being 0.1mg/L in NAA concentration.It can be seen that different cultivars strawberry be suitable for different culture mediums, different cultivars it
Between cannot arbitrarily replace.
' gorgeous ' is the newer kind of strawberry, its plant strain growth gesture is strong, plant height about 20cm, fruit cone, fruit shape
Rectify, glossiness is strong.Seed yellow green, pulp is orange red, medulla median size, orange red, there is cavity.The hardness of fruit
2.73kg·cm-2, storage tolerance, botrytis resistant and leaf diseases have certain resistance to powdery mildew.Blade compares other materials portions
Position materials are convenient, and operation is easy, and become the main study subject of modern biotechnology research, and establish excised leaf
High efficient regeneration system is also the basic work that Applied Biotechnology is improved the breed.But lacks be directed to ' gorgeous ' at present
The abductive approach of Strawberry Leaves callus.
Summary of the invention
The object of the present invention is to provide a kind of Strawberry Leaves callus induction methods, and method for inducing and cultivating is clear, favorably
In the foundation of ' gorgeous ' strawberry regenerating system.
A kind of Strawberry Leaves callus induction method provided by the invention, includes the following steps：
S1 wins Strawberry ripening blade, cleans, is sterilized with alcohol solution dipping, aseptic water washing places into 0.1g/
The HgCl of 100ml2Solution soaking disinfection 6min, aseptic water washing, suck dry moisture obtain aseptic blade, spare；
S2 carries out aseptic blade along blade master pulse direction to carve scar processing, then the blade inoculation after quarter scar exists
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：6g agar powder is added in 1L water, boils to agar powder and is completely dissolved,
Then 4.74g MS solid medium is added, 30g sucrose is added and dissolves by heating, adjusts pH to 5.8-6.0, is then added 2mg's
The NAA of TDZ and 0.1mg, sterilizing；
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 strawberry Callus of Leaf；
The proliferated culture medium is prepared by the following method：6g agar powder is added in 1L water, boils to agar powder and is completely dissolved,
Then 4.74g MS solid medium is added, 30g sucrose is added and dissolves by heating, adjusts pH to 5.8-6.0, is then added 2mg's
The NAA of 6-BA and 0.1mg, sterilizing.
Preferably, above-mentioned Strawberry Leaves callus induction method, in S1, strawberry cultivars are gorgeous kind, Strawberry ripening
The time of winning of blade is the 3-4 month.
Preferably, above-mentioned Strawberry Leaves callus induction method, in S1, the method for cleaning Strawberry ripening blade is, first
It is rinsed with tap water, then impregnates 2h with 2g/L washing powder solution, finally rinsed well with tap water.
Preferably, above-mentioned Strawberry Leaves callus induction method, in S1, the volume fraction of the ethanol solution is
75%, the disinfecting time of ethanol solution is 25s.
Preferably, above-mentioned Strawberry Leaves callus induction method, in S2, the number that each blade carves scar processing is 3-
4, the length of each scar is 1-4mm.
Preferably, above-mentioned Strawberry Leaves callus induction method, condition of culture is identical in S2 and S3, as follows：When illumination
Between 16h/d, light intensity 1500-2000lx.
Compared with prior art, Strawberry Leaves callus induction method provided by the invention, has the advantages that：
Although the growth regulator of induced synthesis plant callus has very much, different factors, various concentration are to grass
The induction of certain kind of berries callus has different influences.The selection of Strawberry Leaves and HgCl2Solution handles the time to callus
Induction play the role of it is very big, when blade select mature leaf, HgCl2When the solution processing time is 6min, the induction of callus
Effect is preferable.The present invention carves injury reason on blade can effectively improve healing rate, and wound receives irritating for hormone and is conducive to
The generation of callus.
In addition, in order to promote calli induction, we are by the auxin NAA and cell of MS culture medium and low concentration
Mitogen 6-BA, TDZ are combined, and promote the induction of Callus of Leaf.We are also the study found that the strawberry leaves that Various Seasonal is won
The inducing effect of piece is also different, and the blade that wherein 3-4 month wins is more advantageous to the induction of callus.
Research material of the present invention by " gorgeous " Strawberry Leaves as experiment, blade materials are convenient, from a wealth of sources, and operation is held
Easily, it can be used as the main study subject of modern biotechnology research, method for inducing and cultivating is clear, is conducive to ' gorgeous ' strawberry
The foundation of regenerating system, the invention discloses the most suitable culture medium of ' gorgeous ' strawberry and most suitable blade states, suitable disinfection
The time is handled, the test for the foundation of strawberry regenerating system lays the foundation, and establishes the high efficient regeneration system of excised leaf just
In the research for carrying out bio-technology improvement kind.
Detailed description of the invention
Fig. 1 is that inoculation 7d rear blade crimp occurs；
Wherein Figure 1A is young age blade, and Figure 1B is middle age blade, and Fig. 1 C is aged blade；
The case where Fig. 2 is different induced medium induced synthesis callus figure；
It is MS1L+TDZ 1.5mg/L+NAA that wherein Fig. 2A, which is MS1L+TDZ 1.0mg/L+NAA 0.1mg/L, Fig. 2 B,
0.1mg/L, Fig. 2 C are MS1L+TDZ 2.0mg/L+NAA 0.1mg/L.
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 various reagents such as following MS solid mediums used in the examples of the present invention
It is commercially available.
The present invention provides a kind of Strawberry Leaves callus induction methods, include the following steps：
S1 wins Strawberry ripening blade, cleans, is sterilized with alcohol solution dipping, aseptic water washing places into 0.1g/
The HgCl of 100ml2Solution soaking disinfection 6min, aseptic water washing, suck dry moisture obtain aseptic blade, spare；
S2 carries out aseptic blade along blade master pulse direction to carve scar processing, then the blade inoculation after quarter scar exists
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：6g agar powder is added in 1L water, boils to agar powder and is completely dissolved,
Then 4.74g MS solid medium is added, 30g sucrose is added and dissolves by heating, adjusts pH to 5.8-6.0, is then added 2mg's
The NAA (methyl α-naphthyl acetate) of TDZ (thiadiazole phenylurea) and 0.1mg, sterilizing；
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 strawberry Callus of Leaf；
The proliferated culture medium is prepared by the following method：6g agar powder is added in 1L water, boils to agar powder and is completely dissolved,
Then 4.74g MS solid medium is added, 30g sucrose is added and dissolves by heating, adjusts pH to 5.8-6.0, is then added 2mg's
The NAA of 6-BA (6-benzyl aminopurine) and 0.1mg, sterilizing.
Preferably, a kind of Strawberry Leaves callus induction method of the present invention, including following embodiment.Following embodiments
In, material to be tested is strawberry cultivars " gorgeous " plant, is derived from Shangqiu Normal University's strawberry greenhouse, chooses suitable grass as needed
Certain kind of berries blade, is inoculated on the day of sampling, evoked callus.
Embodiment 1
A kind of Strawberry Leaves callus induction method, includes the following steps：
S1 hands over the March in season in Winter-Spring, wins Strawberry ripening blade (be unfolded and grow to maximum sized blade), wash
Only, 25s is sterilized with 75% alcohol solution dipping of volume fraction on superclean bench, aseptic water washing 5 times, places into 0.1g/
The HgCl of 100ml2Solution soaking disinfection 6min aseptic water washing 5 times, with aseptic filter paper suck dry moisture, obtains aseptic blade, standby
With.
The method for wherein cleaning Strawberry ripening blade is first to be rinsed with tap water, then impregnate 2h with 2g/L washing powder solution,
Finally rinsed well with tap water.
Aseptic blade is placed in sterile petri dish by S2, carries out 3 along blade master pulse direction to aseptic blade with vaccinating lancet
Scar (not penetrating blade) processing is carved, the length of scar is 4mm, then by the blade inoculation after quarter scar in induced medium
In, 25 ± 2 DEG C are cultivated, and condition of culture is as follows：Light application time 16h/d, light intensity 2000lx, until growing being cured for 1cm × 1cm size
Injured tissue has been cultivated 45 days, has obtained Callus of Leaf；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%；
The induced medium is prepared by the following method：6g agar powder is added in 1L water, boils to agar powder and is completely dissolved,
Then 4.74g MS solid medium (commercially available MS solid medium, without agar and sucrose) is added, 30g sucrose is added and heats
Then the NAA of the TDZ and 0.1mg of 2mg is added with the NaOH tune pH to 6.0 of 1mol/L in dissolution；High pressure sterilization at 121 DEG C
20min, it is ready-to-use；
S3 takes out the Callus of Leaf to grow fine with tweezers, is cut into 2mm × 2mm × 2mm in superclean bench
Specification fritter, is placed in proliferated culture medium, 25 ± 2 DEG C of cultures, and condition of culture is as follows：Light application time 16h/d, light intensity 2000lx,
The Strawberry Leaves callus of white loose is obtained after 50d.It is inoculated with 20 bottles altogether, counts the survival rate and dirt of the stage callus
Dye rate B.
Survival rate=current callus total bottle number × 100% of bottle number/callus
Pollution rate B=pollution bottle number/total bottle number × 100% of callus
The proliferated culture medium is prepared by the following method：6g agar powder is added in 1L water, boils to agar powder and is completely dissolved,
Then 4.74g MS solid medium (commercially available MS solid medium, without agar and sucrose) is added, 30g sucrose is added and heats
Then the 6-BA of the NAA and 2mg of 0.1mg is added with the NaOH tune pH to 6.0 of 1mol/L in dissolution；High pressure sterilization at 121 DEG C
20min, it is ready-to-use.
Embodiment 2
A kind of Strawberry Leaves callus induction method, includes the following steps：
S1 hands over the April in season in Winter-Spring, wins Strawberry ripening blade (be unfolded and grow to maximum sized blade), wash
Only, 25s is sterilized with 75% alcohol solution dipping of volume fraction on superclean bench, aseptic water washing 5 times, places into 0.1g/
The HgCl of 100ml2Solution soaking disinfection 6min aseptic water washing 5 times, with aseptic filter paper suck dry moisture, obtains aseptic blade, standby
With.
The method for wherein cleaning Strawberry ripening blade is first to be rinsed with tap water, then impregnate 2h with 2g/L washing powder solution,
Finally rinsed well with tap water.
Aseptic blade is placed in sterile petri dish by S2, carries out 4 along blade master pulse direction to aseptic blade with vaccinating lancet
Scar (not penetrating blade) processing is carved, the length of scar is 1mm, then by the blade inoculation after quarter scar in induced medium
In, 25 ± 2 DEG C are cultivated, and condition of culture is as follows：Light application time 16h/d, light intensity 1500lx, until growing being cured for 1cm × 1cm size
Injured tissue has been cultivated 45 days, has obtained Callus of Leaf；
The induced medium is prepared by the following method：6g agar powder is added in 1L water, boils to agar powder and is completely dissolved,
Then 4.74g MS solid medium (commercially available MS solid medium, without agar and sucrose) is added, 30g sucrose is added and heats
Then the NAA of TDZ (the thiadiazole phenylurea) and 0.1mg of 2mg is added with the NaOH tune pH to 6.0 of 1mol/L in dissolution；121℃
Lower high pressure sterilization 20min, it is ready-to-use；
S3 takes out the Callus of Leaf to grow fine with tweezers, is cut into 2mm × 2mm × 2mm in superclean bench
Specification fritter, is placed in proliferated culture medium, 25 ± 2 DEG C of cultures, and condition of culture is as follows：Light application time 16h/d, light intensity 1500lx,
The Strawberry Leaves callus of white loose is obtained after 50d；
The proliferated culture medium is prepared by the following method：6g agar powder is added in 1L water, boils to agar powder and is completely dissolved,
Then 4.74g MS solid medium (commercially available MS solid medium, without agar and sucrose) is added, 30g sucrose is added and heats
Then the 6-BA of the NAA and 2mg of 0.1mg is added with the NaOH tune pH to 6.0 of 1mol/L in dissolution；High pressure sterilization at 121 DEG C
20min, it is ready-to-use.
Embodiment 3
A kind of Strawberry Leaves callus induction method, step is substantially same as Example 1, and difference is in S1, cleans
The method of Strawberry ripening blade is tap water cleaning.
In order to verify effect of the invention, We conducted following experiments, in order to guarantee the reliability of result, following experiments
In the process, all blades are cut into the identical blade of size from peripheral edge by us, in addition to specific variable, remaining use
Identical processing mode.
1, different variable (blade leaf ages, HgCl2Solution soaking time) induced synthesis callus the case where
The strawberry young age blade (blade not being fully deployed) of gorgeous kind is selected respectively, age blade has been (i.e. just in strawberry
The blade being just unfolded), Strawberry ripening blade (be unfolded and grow to maximum sized blade), be divided into three big groups, per big component
For five groups, the HgCl of 4min, 5min, 6min, 7min, 8min is respectively adopted2Solution soaking time, remaining induction operation reference
The method of embodiment 1 carries out, and referring to the method statistic pollution rate A and healing rate of embodiment 1.
Each group callus appearance, color observation result are as follows：The 4th day part of blade after inoculation starts to crimp
Phenomenon, after 7 days, leaf rolling phenomenon is obvious.Wherein peak green, HgCl is presented in young age leaf rolling part2Solution processing
Time is the apparent browning of appearance of 7min, 8min；The browning of middle age blade does not have young age blade obvious；Climax leaves
There is not browning substantially in piece, but leaf rolling degree does not have young age blade obvious.Fig. 1 is that inoculation 7d rear blade is rolled up
Qu Xianxiang, wherein Figure 1A is young age blade, and Figure 1B is middle age blade, and Fig. 1 C is aged blade.
30 days after ripening blades begin with callus appearance.It is found in experimentation：The callus of mature leaf is raw
Long situation is best, and it is in opaque shape that the state of presentation, which is loosely organized, and the speed of growth is very fast；Young age blade is not due to complete
It trails, it is slower that callus forms speed；The callus growth that middle age blade is formed is slower, and the state of presentation is that structure is tight
It is close, it is harder, it is transparent, the speed of growth is slower, and part callus occurs with the case where color browning.
Different HgCl2Influence such as 1 institute of table that the solution processing time forms the gorgeous Strawberry Leaves callus of different leaf ages
Show.
1 difference HgCl of table2The influence that the solution processing time forms the gorgeous Strawberry Leaves callus of different leaf ages
The selection of sterilization time and blade plays an important role to the induction of Strawberry Leaves callus.HgCl2Belong to weight
Metal, can make the denaturation of protein, to play the role of disinfection, surface sterilization can be carried out to plant, but mercuric chloride itself has again
There is severe toxicity, processing overlong time will lead to Plant death.
For young leaflet tablet, the too long induced synthesis that will affect Callus of Leaf of sterilization time, as can be seen from Table 1, sterilizing
It is 4min, 5min that time, which is the healing rate respectively less than processing time of 7min, 8min,.And for aged blade, to environmental stimuli
Resistivity it is slightly strong, but HgCl2The time is handled no more than 8min.For young age blade, sterilization time is 4min's
Healing rate highest is higher by 18.35%~66.67% than other several groups of processing for 83.33%；When being most suitable for the sterilizing of young age blade
Between be 4min.For middle age blade, sterilization time is the healing rate highest of 6min, is 60.71%；It is brown in conjunction with healing rate
Rate and pollution rate show that the sterilization time for being most suitable for the blade being just unfolded is 6min.For aged blade, healing rate
It is highest be sterilization time be 5min；Pollution rate it is minimum be sterilization time be 6min；Melting brown rate it is minimum be that sterilization time is
4min,6min.It is shown by data, the sterilization time for being most suitable for the blade being sufficiently spread out is 6min.To sum up, blade selects most
Suitable is aged blade, and corresponding most suitable sterilization time is 6min, and corresponding healing rate is higher, is 64.56%, and pollution rate is lower,
It is 5.00%, and does not occur browning.Therefore, selection of the follow-up test to blade and the processing time to mercuric chloride are all with this
Premised on conclusion.
Thus, mature leaf and HgCl of the present invention2Solution processing the time combined effect be it is best,
Although young age blade has stronger growth ability, but be not the suitable sample of callus induction.HgCl2Solution processing
Not only disinfection can more influence leaf growth and calli induction.And the longer pollution rate of non-processing is lower
Conventional rule.
Therefore, in experimentation below, blade chooses young age blade HgCl2The solution processing time all uses the knot
By.
2, the case where different induced medium induced synthesis callus
We are respectively adopted 2 four groups of induced mediums of table and carry out induction training using gorgeous Strawberry ripening blade as research object
It supports, remaining operation is referring to embodiment 1.As the result is shown：In following each group induced medium, the third day after blade inoculation starts
Existing crimp, the 3rd, 4 group of induced medium has two bottles browning occur respectively after 7 days, after 25 days, starts callus occur
Tissue, after 45 days, substantially there is callus in the blade of all survivals.
The influence that the different induced mediums of table 2 form gorgeous Strawberry ripening Callus of Leaf
6-BA, TDZ, NAA are critically important plant growth regulator for the induced synthesis of Strawberry Leaves callus,
The prior art research shows that the suitable culture medium of kind strawberry is MS+6-BA1.0mg/L+NAA 0.1mg/L, but this hair
The effect of bright research discovery TDZ (1- phenyl -3- (1,2,3,-thiadiazoles -5- base) urea, Thidiazuron, thiadiazole phenylurea) is wanted
Effect than 6-BA is good, and most suitable TDZ concentration is in 1-2mg/L.Therefore, using the concentration of TDZ as gradient, NAA concentration it is constant into
Row experiment.As a result, it has been found that：6-BA, TDZ concentration are identical be 1mg/L in the case where, the healing rate of Strawberry Leaves is respectively
63.6%, 100%, effect of the effect than 6-BA for demonstrating TDZ is good.And we can be found that the concentration of TDZ either
1.0mg/L, 1.5mg/L or 2.0mg/L, the healing rate of Strawberry Leaves are 100%.During observing experiment,
It is found after the growth conditions to callus compare, although TDZ concentration is the callus group of 1.0mg/L and 1.5mg/L
It is slower to knit the speed of growth, but TDZ concentration is 2.0mg/L, the speed of growth for the callus that blade is formed is very fast, and blade
The color itself presented is green.The callus ratio MS+6-BA1.0mg/L+NAA 0.1mg/L effect of formation is more preferable, by me
This draw a conclusion, the optimum medium of Strawberry Leaves (especially gorgeous strawberry) callus induction is MS+TDZ 2.0mg/L
+NAA 0.1mg/L。
The case where Fig. 2 is different induced medium induced synthesis callus figure, wherein Fig. 2A is MS1L+TDZ 1.0mg/
L+NAA 0.1mg/L, Fig. 2 B is that MS1L+TDZ 1.5mg/L+NAA 0.1mg/L, Fig. 2 C is MS1L+TDZ 2.0mg/L+NAA
0.1mg/L, Fig. 2 are similar to the result of table 2, and the optimum medium of Strawberry Leaves (especially gorgeous strawberry) callus induction is
MS+TDZ 2.0mg/L+NAA 0.1mg/L。
3, the case where different proliferated culture medium induced synthesis callus
It is divided into several fritters after callus growth to 1cm × 1cm and carries out Multiplying culture.We are with 6-BA's
Concentration gradient is variable, and NAA concentration is constant to be tested.Remaining is operated with the method in embodiment 1, and referring to embodiment 1
Method statistic dirt survival rate and pollution rate B.The results are shown in Table 3,
The influence that the different proliferated culture mediums of table 3 form the gorgeous Strawberry Leaves callus of different leaf ages
From the result of table 3 and from Multiplying culture result known to：The callus that young age blade is formed is more fragile,
When 6-BA concentration is 0.5mg/L, callus growth state is preferable, but the speed of growth is excessively slow；It is 1.0mg/L in 6-BA concentration
When, callus growth fast speed, but growth conditions are bad, find lurid callus after meeting water by dissection
Become the graininess of very little；When 6-BA concentration is 2.0mg/L, callus growth out of order, is substantially at and does not grow shape
State.
The callus growth state that middle age blade is formed is preferable, and when 6-BA concentration is 0.5mg/L, callus is
The speed of growth is slow；When 6-BA concentration is 1.0mg/L, callus growth speed ratio 6-BA concentration is fast when being 0.5mg/L；
When 6-BA concentration is 2.0mg/L, callus growth state is preferable, and speed is also than very fast.
The callus pollution rate that mature leaf is formed is relatively high, and the speed of growth is also than very fast.
It is found after comparison：It is 2.0mg/L for 6-BA concentration most suitable needed for Callus Culture of Strawberry Multiplying culture.
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 (6)
1. a kind of Strawberry Leaves callus induction method, which is characterized in that include the following steps：
S1 wins Strawberry ripening blade, cleans, is sterilized with alcohol solution dipping, aseptic water washing places into 0.1g/100ml's
HgCl2Solution soaking disinfection 6min, aseptic water washing, suck dry moisture obtain aseptic blade, spare；
S2 carries out aseptic blade along blade master pulse direction to carve scar processing, is then inducing the blade inoculation after quarter scar
In culture medium, 25 ± 2 DEG C of cultures obtain Callus of Leaf until growing callus；
The induced medium is prepared by the following method：6g agar powder is added in 1L water, boils to agar powder and is completely dissolved, then
Be added 4.74g MS solid medium, be added 30g sucrose simultaneously dissolve by heating, adjust pH to 5.8-6.0, then be added 2mg TDZ and
The NAA of 0.1mg, sterilizing；
S3 takes out Callus of Leaf, and stripping and slicing is placed in proliferated culture medium, 25 ± 2 DEG C of cultures, until obtaining white loose grass
Certain kind of berries Callus of Leaf；
The proliferated culture medium is prepared by the following method：6g agar powder is added in 1L water, boils to agar powder and is completely dissolved, then
4.74g MS solid medium is added, 30g sucrose is added and dissolves by heating, adjusts pH to 5.8-6.0, the 6-BA of 2mg is then added
With the NAA of 0.1mg, sterilizing.
2. Strawberry Leaves callus induction method according to claim 1, which is characterized in that in S1, strawberry cultivars are
The time of winning of gorgeous kind, Strawberry ripening blade is the 3-4 month.
3. Strawberry Leaves callus induction method according to claim 1, which is characterized in that in S1, clean strawberry at
The method of ripe blade is first to be rinsed with tap water, then impregnate 2h with 2g/L washing powder solution, is finally rinsed well with tap water.
4. Strawberry Leaves callus induction method according to claim 1, which is characterized in that in S1, the ethyl alcohol is molten
The volume fraction of liquid is 75%, and the disinfecting time of ethanol solution is 25s.
5. Strawberry Leaves callus induction method according to claim 1, which is characterized in that in S2, each blade is carved
The number of scar processing is 3-4, and the length of each scar is 1-4mm.
6. Strawberry Leaves callus induction method according to claim 1, which is characterized in that condition of culture in S2 and S3
It is identical, it is as follows：Light application time 16h/d, light intensity 1500-2000lx.
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