CN108812316B - Strawberry leaf callus induction method - Google Patents
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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
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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
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Abstract
The invention belongs to the technical field of plant callus culture, and particularly relates to a strawberry leaf callus induction method. The method comprises picking mature leaves of strawberry, cleaning, soaking in ethanol solution for sterilization, washing with sterile water, and adding HgCl 0.1g/100ml2Soaking and sterilizing the solution for 6min, washing with sterile water, and sucking water to obtain sterile leaves; then, the aseptic lamina is scored along the main vein direction of the lamina, and is subjected to induction culture in an induction culture medium and proliferation culture in a proliferation culture medium. The invention takes the strawberry leaves as the research material of the experiment, the leaves are convenient to obtain, the source is wide, the operation is easy, and the establishment of the high-efficiency regeneration system of the in vitro leaves is convenient for the research of the improved variety of the biotechnology.
Description
Technical Field
The invention belongs to the technical field of plant callus culture, and particularly relates to a strawberry leaf callus induction method.
Background
Strawberry (Fragaria ananassa Duch), perennial herb of strawberry of Rosaceae, is rich in nutrients such as vitamin C, pectin, cellulose, iron, calcium, etc. Has high economic value, nutritive value and health promotion effect, and can protect eyesight and promote digestion. Therefore, strawberries have been widely planted. The traditional propagation method comprises the plant division propagation and the seed propagation, and the propagation coefficient is low, the seedling quality is poor, and diseases of roots, leaves and fruits, such as leaf spot, powdery mildew, gray mold, fruit deformity and the like, are often accompanied. The tissue culture propagation is carried out as soon as possible, the tissue culture technology is utilized, the cultured strawberry seedlings are free of viruses, strong in growth and dark green in leaves, the fruit quality can be effectively changed, the yield is improved, the propagation is fast, and the method is not limited by seasons.
At present, a great deal of research is carried out on strawberry tissue culture at home and abroad, and related varieties mainly comprise 'Jingyao', 'Fengxiang', 'Hongyan', 'Quanmingxing', and the like. Researches show that the optimal induction callus culture medium of 'Fengxiang' is MS +6-BA 2.0mg/L +2, 4-D0.1 mg/L; the most suitable induction callus culture medium of 'octopus' is MS +6-BA 2.0mg/L + NAA 0.25 mg/L; the most suitable induction callus culture medium of 'whole star' is MS +6-BA 2.0mg/L + NAA 0.2 mg/L; the 'wool fragrance' has vitrification phenomenon when no addition or low NAA concentration, but the plant grows normally when the NAA concentration is higher, and the callus tissue appears when the NAA concentration is 0.1 mg/L. It can be seen that different varieties of strawberries are suitable for different culture mediums, and can not be replaced randomly.
The 'gorgeous' is a relatively new variety of strawberries, and has strong plant growth potential, plant height of about 20cm, conical fruits, regular fruit shapes and strong glossiness. Yellow green seed, orange red pulp, medium size of medulla, orange red, and hollow. Hardness of fruit 2.73kg cm-2It is storage and transportation resistant, and has certain resistance to gray mold and leaf diseases. Compared with other material-taking parts, the blade has convenient material taking and easy operation, becomes a main research object of modern bioengineering technology research, and the establishment of a high-efficiency regeneration system of the in vitro blade is also a basic work for improving varieties by applying biotechnology. However, there is currently a lack of induction methods for callus of 'brilliant' strawberry leaves.
Disclosure of Invention
The invention aims to provide a strawberry leaf callus induction method which is clear in induction culture method and beneficial to establishment of a gorgeous strawberry regeneration system.
The invention provides a strawberry leaf callus induction method, which comprises the following steps:
s1, picking mature leaves of strawberry, cleaning, soaking in ethanol solution for sterilization, washing with sterile water, and adding HgCl 0.1g/100ml2Soaking and sterilizing the solution for 6min, washing with sterile water, and sucking water to obtain sterile leaf;
s2, carrying out marking treatment on the sterile leaves along the main vein direction of the leaves, then inoculating the marked leaves into an induction culture medium, and culturing at 25 +/-2 ℃ until callus grows out to obtain leaf callus;
the induction culture medium is prepared by the following method: adding 6g of agar powder into 1L of water, boiling until the agar powder is completely dissolved, adding 4.74g of MS solid culture medium, adding 30g of sucrose, heating to dissolve, adjusting pH to 5.8-6.0, adding 2mg of TDZ and 0.1mg of NAA, and sterilizing;
s3, taking out the leaf callus, cutting into blocks, placing the blocks in a proliferation culture medium, and culturing at 25 +/-2 ℃ until white loose strawberry leaf callus is obtained;
the proliferation culture medium is prepared by the following method: adding 6g of agar powder into 1L of water, boiling until the agar powder is completely dissolved, adding 4.74g of MS solid culture medium, adding 30g of sucrose, heating to dissolve, adjusting pH to 5.8-6.0, adding 2mg of 6-BA and 0.1mg of NAA, and sterilizing.
Preferably, in the method for inducing the strawberry leaf callus, in S1, the strawberry variety is gorgeous, and the picking time of mature leaves of the strawberry is 3-4 months.
Preferably, in the method for inducing the strawberry leaf callus, in step S1, the mature strawberry leaves are washed by tap water, then soaked in 2g/L washing powder solution for 2 hours, and finally washed clean by tap water.
Preferably, in the method for inducing strawberry leaf callus, in S1, the ethanol solution has a volume fraction of 75%, and the ethanol solution has a disinfection time of 25S.
Preferably, in the method for inducing strawberry leaf callus, S2, the number of the scratch treatment per leaf is 3-4, and the length of each scratch is 1-4 mm.
Preferably, the strawberry leaf callus induction method is carried out under the same culture conditions in S2 and S3 as follows: the illumination time is 16h/d, and the light intensity is 1500-.
Compared with the prior art, the strawberry leaf callus induction method provided by the invention has the following beneficial effects:
although inducingThere are many growth regulators for forming plant callus, but different factors and concentrations have different effects on the induction of strawberry callus. Selection of strawberry leaves and HgCl2The solution treatment time has great effect on the induction of callus, and when the leaf selects mature leaf, HgCl2When the solution treatment time is 6min, the induction effect of the callus is better. The present invention can raise wound healing rate effectively, and the wound is stimulated with hormone to produce callus.
In addition, in order to promote the formation of callus, the MS culture medium is combined with lower concentration of auxin NAA, cytokinin 6-BA and TDZ to promote the induction of leaf callus. We also found that the induction effect of the strawberry leaves picked in different seasons is different, wherein the leaves picked in 3-4 months are more beneficial to the induction of the callus.
The invention takes 'gorgeous' strawberry leaves as the research materials of the experiment, the leaves are convenient to obtain, the source is wide, the operation is easy, the invention can be used as the main research object of the modern bioengineering technology research, the induction culture method is clear, and the invention is beneficial to the establishment of the 'gorgeous' strawberry regeneration system.
Drawings
FIG. 1 shows the curling of the leaves after 7d inoculation;
wherein FIG. 1A is young leaf, FIG. 1B is middle-aged leaf, and FIG. 1C is old leaf;
FIG. 2 is a diagram showing callus formation induced by different induction media;
wherein FIG. 2A is MS1L + TDZ 1.0mg/L + NAA 0.1mg/L, FIG. 2B is MS1L + TDZ 1.5mg/L + NAA 0.1mg/L, and FIG. 2C is MS1L + TDZ 2.0mg/L + NAA 0.1 mg/L.
Detailed Description
The following detailed description of specific embodiments of the invention is provided, but it should be understood that the scope of the invention is not limited to the specific embodiments. Test methods in which specific conditions are not specified in the following examples are generally carried out under conventional conditions or under conditions recommended by the respective manufacturers. In addition, various reagents such as MS solid medium used in the following examples of the present invention are commercially available.
The invention provides a strawberry leaf callus induction method, which comprises the following steps:
s1, picking mature leaves of strawberry, cleaning, soaking in ethanol solution for sterilization, washing with sterile water, and adding HgCl 0.1g/100ml2Soaking and sterilizing the solution for 6min, washing with sterile water, and sucking water to obtain sterile leaf;
s2, carrying out marking treatment on the sterile leaves along the main vein direction of the leaves, then inoculating the marked leaves into an induction culture medium, and culturing at 25 +/-2 ℃ until callus grows out to obtain leaf callus;
the induction culture medium is prepared by the following method: adding 6g of agar powder into 1L of water, boiling until the agar powder is completely dissolved, adding 4.74g of MS solid culture medium, adding 30g of sucrose, heating to dissolve, adjusting pH to 5.8-6.0, adding 2mg of TDZ (thidiazuron) and 0.1mg of NAA (naphthylacetic acid), and sterilizing;
s3, taking out the leaf callus, cutting into blocks, placing the blocks in a proliferation culture medium, and culturing at 25 +/-2 ℃ until white loose strawberry leaf callus is obtained;
the proliferation culture medium is prepared by the following method: adding 6g of agar powder into 1L of water, boiling until the agar powder is completely dissolved, adding 4.74g of MS solid culture medium, adding 30g of sucrose, heating to dissolve, adjusting pH to 5.8-6.0, adding 2mg of 6-BA (6-benzylaminopurine) and 0.1mg of NAA, and sterilizing.
Preferably, the method for inducing the callus of the strawberry leaf comprises the following examples. In the following examples, the test material was a "gorgeous" plant of the strawberry variety, which was obtained from the strawberry greenhouse of the university academy of Shangqiu, and suitable strawberry leaves were selected as needed, inoculated on the day of sampling, and callus was induced.
Example 1
A strawberry leaf callus induction method comprises the following steps:
s1, picking mature leaves of strawberry (i.e. leaves which are unfolded and grow to the maximum size) in 3 months of winter and spring crossing season, cleaning, soaking and sterilizing with 75% ethanol solution by volume fraction for 25S on a super clean bench, washing with sterile water for 5 times, and adding HgCl of 0.1g/100ml2Soaking and sterilizing the solution for 6min, washing with sterile water for 5 times, and drying with sterile filter paper to obtain sterile leaf.
The method for cleaning the mature leaves of the strawberries comprises the steps of firstly washing the mature leaves of the strawberries with tap water, then soaking the mature leaves of the strawberries in 2g/L washing powder solution for 2 hours, and finally washing the mature leaves of the strawberries with tap water.
S2, placing the sterile leaf on a sterile culture dish, carrying out 3 scratch (not penetrating the leaf) treatments on the sterile leaf along the main vein direction of the leaf by using an inoculating knife, wherein the length of the scratch is 4mm, then inoculating the scratched leaf in an induction culture medium, and culturing at 25 +/-2 ℃, wherein the culture conditions are as follows: the illumination time is 16h/d, the light intensity is 2000lx, until 1cm multiplied by 1cm callus grows out, and the leaf callus is obtained after 45 days of culture; a total of 20 bottles were inoculated, and the contamination rate A and the recovery rate at this stage were counted.
The pollution rate A is equal to the number of the leaves of the polluted mixed bacteria/the total number of the inoculated leaves multiplied by 100 percent;
the callus growth rate is the number of the leaves forming the callus/(the total number of inoculated leaves-the number of the polluted leaves) multiplied by 100 percent;
the induction culture medium is prepared by the following method: adding 6g of agar powder into 1L of water, boiling until the agar powder is completely dissolved, then adding 4.74g of MS solid culture medium (commercially available MS solid culture medium without agar and sucrose), adding 30g of sucrose, heating to dissolve, adjusting pH to 6.0 with 1mol/L NaOH, and then adding 2mg of TDZ and 0.1mg of NAA; sterilizing at 121 deg.C under high pressure for 20min, and mixing;
s3, taking out well-growing leaf callus by using tweezers in a clean bench, cutting the leaf callus into small pieces with the specification of 2mm multiplied by 2mm, placing the small pieces in a multiplication culture medium, and culturing at 25 +/-2 ℃, wherein the culture conditions are as follows: the illumination time is 16h/d, the light intensity is 2000lx, and white loose strawberry leaf callus is obtained after 50 d. And (4) inoculating 20 bottles in total, and counting the survival rate and the pollution rate B of the callus at the stage.
The survival rate is equal to the current bottle number of the callus/total bottle number of the callus multiplied by 100 percent
The contamination rate B is the number of contaminated bottles/total number of callus bottles multiplied by 100%
The proliferation culture medium is prepared by the following method: adding 6g of agar powder into 1L of water, boiling until the agar powder is completely dissolved, then adding 4.74g of MS solid culture medium (commercially available MS solid culture medium without agar and sucrose), adding 30g of sucrose, heating to dissolve, adjusting pH to 6.0 with 1mol/L NaOH, and then adding 0.1mg of NAA and 2mg of 6-BA; sterilizing at 121 deg.C under high pressure for 20min, and making into preparation.
Example 2
A strawberry leaf callus induction method comprises the following steps:
s1, picking mature leaves of strawberry (i.e. leaves which are unfolded and grow to the maximum size) in 4 months of winter and spring crossing season, cleaning, soaking and sterilizing with 75% ethanol solution by volume fraction for 25S on a super clean bench, washing with sterile water for 5 times, and adding HgCl of 0.1g/100ml2Soaking and sterilizing the solution for 6min, washing with sterile water for 5 times, and drying with sterile filter paper to obtain sterile leaf.
The method for cleaning the mature leaves of the strawberries comprises the steps of firstly washing the mature leaves of the strawberries with tap water, then soaking the mature leaves of the strawberries in 2g/L washing powder solution for 2 hours, and finally washing the mature leaves of the strawberries with tap water.
S2, placing the sterile leaf on a sterile culture dish, carrying out 4 scratch (not penetrating the leaf) treatments on the sterile leaf along the main vein direction of the leaf by using an inoculating knife, wherein the length of the scratch is 1mm, then inoculating the scratched leaf in an induction culture medium, and culturing at 25 +/-2 ℃, wherein the culture conditions are as follows: the illumination time is 16h/d, the light intensity is 1500lx, until 1cm multiplied by 1cm callus grows out, and the leaf callus is obtained after 45 days of culture;
the induction culture medium is prepared by the following method: adding 6g of agar powder into 1L of water, boiling until the agar powder is completely dissolved, then adding 4.74g of MS solid culture medium (commercially available MS solid culture medium without agar and sucrose), adding 30g of sucrose, heating to dissolve, adjusting pH to 6.0 with 1mol/L of NaOH, and then adding 2mg of TDZ (thidiazuron) and 0.1mg of NAA; sterilizing at 121 deg.C under high pressure for 20min, and mixing;
s3, taking out well-growing leaf callus by using tweezers in a clean bench, cutting the leaf callus into small pieces with the specification of 2mm multiplied by 2mm, placing the small pieces in a multiplication culture medium, and culturing at 25 +/-2 ℃, wherein the culture conditions are as follows: the illumination time is 16h/d, the light intensity is 1500lx, and white loose strawberry leaf callus is obtained after 50 d;
the proliferation culture medium is prepared by the following method: adding 6g of agar powder into 1L of water, boiling until the agar powder is completely dissolved, then adding 4.74g of MS solid culture medium (commercially available MS solid culture medium without agar and sucrose), adding 30g of sucrose, heating to dissolve, adjusting pH to 6.0 with 1mol/L NaOH, and then adding 0.1mg of NAA and 2mg of 6-BA; sterilizing at 121 deg.C under high pressure for 20min, and making into preparation.
Example 3
A method for inducing callus of strawberry leaves, which comprises the steps substantially the same as those of example 1, except that in S1, the mature leaves of strawberry are washed with tap water.
In order to verify the effect of the invention, the following experiment is carried out, and in the following experiment process, all the blades are cut into the blades with the same size from the peripheral edge, and the same processing mode is adopted except for definite variables.
1. Different variables (age of leaf, HgCl)2Solution soaking time) inducing callus formation
Selecting young leaves (not completely unfolded leaves) of bright strawberry, middle-aged leaves (just unfolded leaves) of strawberry and mature leaves (unfolded and grown to the maximum size) of strawberry, and dividing into three groups, wherein each group comprises five groups of HgCl of 4min, 5min, 6min, 7min and 8min2The solution soaking time and the rest of the induction operations were carried out according to the method of example 1, and the contamination rate A and the recovery rate were counted according to the method of example 1.
The results of observing the appearance and color of the callus of each group are as follows: leaf first after inoculationCurling begins to appear in the 4-day part, and after 7 days, the curling of the leaves is obvious. Wherein the young leaf curly part is light green and HgCl2Obvious browning phenomenon appears when the solution treatment time is 7min and 8 min; the browning phenomenon of the middle-aged leaves is not obvious as that of the young leaves; the mature leaves are not browned basically, but the curling degree of the leaves is not obvious as that of young leaves. FIG. 1 shows the curling of the leaves after 7 days of inoculation, wherein FIG. 1A shows young leaves, FIG. 1B shows middle-aged leaves, and FIG. 1C shows old leaves.
After 30 days callus began to appear on the mature leaves. The experimental process shows that: the callus of the mature leaf has the best growth condition, and the present state is loose in structure, opaque and high in growth speed; the callus formation speed is slow because young leaves are not fully extended; the callus formed by the medium-aged leaves grows slowly, the callus is in a state of compact structure, hardness, transparency and slow growth speed, and part of the callus is accompanied by the condition of browning.
Different HgCl2The effect of solution treatment time on callus formation of different leaf age brilliant strawberry leaves is shown in table 1.
TABLE 1 different HgCl2Influence of solution treatment time on formation of callus of flamboyant strawberry leaves with different leaf ages
The sterilization time and the selection of the leaves play an important role in the induction of the strawberry leaf callus. HgCl2The mercuric chloride belongs to heavy metals, can denature protein, has a disinfection effect, can disinfect the surface of plants, but is highly toxic, and can cause the death of the plants after being treated for a long time.
For young and tender leaves, the induction formation of the leaf callus is affected by too long sterilization time, and as shown in table 1, the healing rate of 7min and 8min sterilization time is less than that of 4min and 5min sterilization time. But has slightly stronger resistance to external stimulation for old leaves,but HgCl2The treatment time cannot exceed 8 min. For young leaves, the healing rate is the highest when the sterilization time is 4min, and is 83.33 percent, which is 18.35 to 66.67 percent higher than that of other groups of treatment; the sterilization time most suitable for young leaves is 4 min. For the middle-aged leaf blades, the cure rate is 60.71% with the sterilization time of 6 min; the optimal sterilization time for the just developed leaves is 6min according to the healing rate, the browning rate and the pollution rate. For old leaves, the highest cure rate is that the sterilization time is 5 min; the lowest pollution rate is that the sterilization time is 6 min; the browning rate is the lowest, and the sterilization time is 4min and 6 min. The data show that the sterilization time for the best suited fully developed leaf is 6 min. In conclusion, the most suitable leaf is selected to be the old leaf, the corresponding most suitable sterilization time is 6min, the corresponding healing rate is higher and is 64.56%, the pollution rate is lower and is 5.00%, and the browning phenomenon does not occur. Therefore, the subsequent test for the selection of the blade and the treatment time of the mercury lift are based on the conclusion.
Therefore, the mature leaf and HgCl used in the present invention2The combined effect of solution treatment time was best, and young leaves, although stronger in growth capacity, were not suitable samples for callus induction. HgCl2Solution treatment is not only a disinfection effect, but also can influence the growth of leaves and the formation of callus. It is not a general rule that the longer the treatment time, the lower the contamination rate.
Therefore, in the course of the following experiments, young leaf HgCl was selected as the leaf2This conclusion was used for solution treatment times.
2. The callus formation conditions induced by different induction media
We used mature leaves of the beautiful strawberry as the study object and used four groups of induction culture media shown in Table 2 to perform induction culture, and the rest of the procedures refer to example 1. The results show that: in the following groups of induction culture media, curling phenomenon appears on the third day after leaf inoculation, two bottles of induction culture media of the 3 rd and 4 th groups appear browning phenomenon after 7 days, callus appears after 25 days, and callus appears on all surviving leaves basically after 45 days.
TABLE 2 Effect of different induction media on calli formation from mature leaves of Fragaria strawberries
6-BA, TDZ and NAA are plant growth regulators which are important for the induction formation of strawberry leaf callus, and the research of the prior art shows that the culture medium suitable for the strawberry variety is MS +6-BA1.0mg/L + NAA 0.1mg/L, but the research of the invention finds that the effect of TDZ (1-phenyl-3- (1, 2, 3-thiadiazole-5-yl) urea, thidiazuron and thiadiazophenyl urea) is better than that of 6-BA, and the optimal TDZ concentration is 1-2 mg/L. Therefore, experiments were performed with the concentration of NAA unchanged, using the concentration of TDZ as a gradient. As a result, it was found that: under the condition that the concentrations of 6-BA and TDZ are the same and are both 1mg/L, the healing rate of strawberry leaves is 63.6 percent and 100 percent respectively, and the TDZ effect is verified to be better than that of 6-BA. Moreover, we can find that the healing rate of strawberry leaves is 100% no matter the concentration of TDZ is 1.0mg/L, 1.5mg/L or 2.0 mg/L. In the course of observing the experiment, after comparing the growth state of the callus, it was found that, although the growth rate of the callus was slow at the TDZ concentrations of 1.0mg/L and 1.5mg/L, the growth rate of the callus formed from the leaf was fast at the TDZ concentration of 2.0mg/L, and the color of the leaf itself was green. The callus formed was more effective than MS +6-BA1.0mg/L + NAA 0.1mg/L, and we concluded that the optimal medium for callus induction of strawberry leaf (especially brilliant strawberry) was MS + TDZ 2.0mg/L + NAA 0.1 mg/L.
FIG. 2 is a diagram showing callus formation induced by different induction media, in which FIG. 2A shows MS1L + TDZ 1.0mg/L + NAA 0.1mg/L, FIG. 2B shows MS1L + TDZ 1.5mg/L + NAA 0.1mg/L, FIG. 2C shows MS1L + TDZ 2.0mg/L + NAA 0.1mg/L, and FIG. 2 shows results similar to those in Table 2, and the optimal medium for callus induction of strawberry leaf (especially Fragaria strawberry) is MS + TDZ 2.0mg/L + NAA 0.1 mg/L.
3. The callus formation conditions induced by different proliferation media
After the callus grows to 1cm multiplied by 1cm, the callus is divided into a plurality of small blocks for multiplication culture. We performed experiments with a concentration gradient of 6-BA as variable and a concentration of NAA unchanged. The procedure was otherwise the same as in example 1, and the fouling survival rate and fouling rate B were counted by referring to example 1. The results are shown in Table 3 below,
TABLE 3 Effect of different multiplication media on callus formation of leaves of gorgeous strawberries of different leaf ages
From the results in table 3 and the results of the growth culture observed, it is understood that: the callus formed by young leaves is fragile, and when the concentration of 6-BA is 0.5mg/L, the callus has a good growth state but a too slow growth speed; when the concentration of 6-BA is 1.0mg/L, the callus grows faster, but the growth state is not good, and the light yellow callus is found to be small particles after meeting water through dissection; when the concentration of 6-BA is 2.0mg/L, the callus growth state is not good, and the callus is basically in a non-growth state.
The callus formed by the middle-aged leaves has a good growth state, and when the concentration of 6-BA is 0.5mg/L, the callus only grows at a relatively slow speed; when the concentration of 6-BA is 1.0mg/L, the growth speed of the callus is faster than that when the concentration of 6-BA is 0.5 mg/L; when the concentration of 6-BA is 2.0mg/L, the callus growth state is better and the speed is faster.
The callus formed by the mature leaves has higher pollution rate and higher growth speed.
After comparison, it was found that: the optimal 6-BA concentration required for the proliferation culture of strawberry callus is 2.0 mg/L.
It should be noted that when ranges are recited herein, unless otherwise stated, each endpoint, and any value between the endpoints, of each range can be selected. While preferred embodiments of the present invention have been described, additional variations and modifications in those embodiments may occur to those skilled in the art once they learn of the basic inventive concepts. Therefore, it is intended that the appended claims be interpreted as including preferred embodiments and all such alterations and modifications as fall within the scope of the invention.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present invention without departing from the spirit and scope of the invention. Thus, if such modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include such modifications and variations.
Claims (4)
1. A strawberry leaf callus induction method is characterized by comprising the following steps:
s1, picking mature leaves of strawberry, cleaning, soaking in ethanol solution for sterilization, washing with sterile water, and adding HgCl 0.1g/100ml2Soaking and sterilizing the solution for 6min, washing with sterile water, and sucking water to obtain sterile leaf; wherein, the strawberry variety is gorgeous, and the picking time of mature leaves of the strawberry is 3-4 months;
s2, performing scratch treatment on the sterile leaves along the main vein direction of the leaves, wherein the number of the scratch treatment on each leaf is 3-4, and the length of each scratch is 1-4mm, then inoculating the scratched leaves into an induction culture medium, and culturing at 25 +/-2 ℃ until callus grows out to obtain leaf callus;
the induction culture medium is prepared by the following method: adding 6g of agar powder into 1L of water, boiling until the agar powder is completely dissolved, adding 4.74g of MS solid culture medium, adding 30g of sucrose, heating to dissolve, adjusting pH to 5.8-6.0, adding 2mg of TDZ and 0.1mg of NAA, and sterilizing;
s3, taking out the leaf callus, cutting into blocks, placing the blocks in a proliferation culture medium, and culturing at 25 +/-2 ℃ until white loose strawberry leaf callus is obtained;
the proliferation culture medium is prepared by the following method: adding 6g of agar powder into 1L of water, boiling until the agar powder is completely dissolved, adding 4.74g of MS solid culture medium, adding 30g of sucrose, heating to dissolve, adjusting pH to 5.8-6.0, adding 2mg of 6-BA and 0.1mg of NAA, and sterilizing.
2. The method for inducing strawberry leaf callus according to claim 1, wherein in S1, mature leaves of strawberries are washed by tap water, then soaked in 2g/L washing powder solution for 2h, and finally washed clean by tap water.
3. The method for inducing strawberry leaf callus according to claim 1, wherein in S1, the volume fraction of the ethanol solution is 75%, and the disinfection time of the ethanol solution is 25S.
4. The method for inducing strawberry leaf callus according to claim 1, wherein the culture conditions in S2 and S3 are the same as follows: the illumination time is 16h/d, and the light intensity is 1500-.
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