CN109042320B - Oil peony callus culture method - Google Patents
Oil peony callus culture method Download PDFInfo
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- CN109042320B CN109042320B CN201810814581.0A CN201810814581A CN109042320B CN 109042320 B CN109042320 B CN 109042320B CN 201810814581 A CN201810814581 A CN 201810814581A CN 109042320 B CN109042320 B CN 109042320B
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- A—HUMAN NECESSITIES
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Abstract
The invention belongs to the technical field of plant callus culture, and particularly relates to a method for culturing oil peony callus. The method comprises collecting peony leaf for oil, cleaning, and sterilizing to obtain sterile leaf; then cutting the aseptic leaves along the main vein direction of the leaves, namely, dividing each leaf into two halves, and then carrying out induction culture and multiplication culture, wherein ginger juice is added into an induction culture medium and a multiplication culture medium respectively. The method of the invention takes the peony leaves for oil as the object, successfully cultivates the peony callus for oil, and lays a test foundation for the establishment of a peony for oil regeneration system.
Description
Technical Field
The invention belongs to the technical field of plant callus culture, and particularly relates to a method for culturing oil peony callus.
Background
At present, the wild peony varieties in China are mainly distributed in Henan, Shaanxi, Hubei, Gansu, Sichuan, Yunnan, Tibet and the like. According to incomplete statistics, more than 100 have the fruit setting capability. The oil peony is a peony variety with high seed yield, high oil content and high effective components. The content of unsaturated fatty acid in Paeonia ostii and Paeonia suffruticosa seed exceeds 90%, wherein the content of linolenic acid is about 40%. Linolenic acid is essential amino acid for human, has the effects of refreshing brain, enhancing memory, reducing blood fat, reducing blood pressure, preventing cardiovascular and cerebrovascular diseases and the like, and plays a key role in human health. Therefore, the mass culture of the oil peony has better ornamental effect and high medicinal value. The traditional planting method of the oil peonies mainly comprises seed propagation and plant division propagation, and the time consumption in the culture process is long.
The plant cultured by the tissue culture technology can grow strongly, leaves are dark green, the fruit quality can be effectively changed, the yield is improved, the propagation is fast, and the plant is not restricted by seasons. The common tissue culture technology is callus culture, for example, nanguo pears, oil peonies, carrots and the like can be used for callus culture, but because the variety of the oil peonies is few, the traditional plant callus induction and subculture hormone proportion are not suitable for callus culture of all plants, and a method for callus culture by using the oil peonies is not found at present.
Disclosure of Invention
The invention aims to provide a method for culturing oil peony callus, which takes oil peony leaves as an object to successfully culture the oil peony callus and lays a test foundation for establishing an oil peony regeneration system.
The invention provides a method for culturing oil peony callus, which comprises the following steps:
s1, taking peony leaves for oil, cleaning and sterilizing to obtain sterile leaves;
s2, inducing: cutting the aseptic leaves along the main pulse direction of the leaves, namely dividing each leaf into two halves, inoculating the cut 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 4.74g of MS solid culture medium and 20g of cane sugar into 1L of water, heating for dissolving, adjusting the pH value to 5.8-6.0, then adding 0.1mg of NAA, 10-20 ml of ginger juice and 6-8 g of agar, boiling until the agar is completely dissolved, and sterilizing;
s3, proliferation: taking out the leaf callus, cutting into blocks, placing in a proliferation culture medium, and culturing at 25 +/-2 ℃ until the white peony leaf callus for the pine oil is obtained;
the proliferation culture medium is prepared by the following method: adding 4.74g of MS solid culture medium and 30g of cane sugar into 1L of water, heating for dissolving, adjusting the pH value to 5.8-6.0, then adding 2mg of 6-BA, 10-20 ml of ginger juice and 6-8 g of agar, boiling until the agar is completely dissolved, and sterilizing;
the ginger juice in S2 and S3 is prepared by the following method: slicing ginger, mixing the sliced ginger with water according to the ratio of 20g to 200ml, boiling for 5-10 min, and filtering to collect filtrate as ginger juice.
Preferably, the method for culturing the peony callus for oil, in S1, the specific sterilization method is as follows: soaking cleaned oil in 75% ethanol solution, sterilizing, washing with sterile water, and adding HgCl 0.1g/100ml2Soaking in the solution for 8min for sterilization, taking out the leaves, washing with sterile water, and taking out to obtain sterile leaves.
Preferably, in the method for culturing oil-use peony callus, in S1, the length of the oil-washed peony leaf in the main vein direction is 2 to 6 cm.
Preferably, in the method for culturing the peony callus for oil, the thickness of the ginger slices in S2 and S3 in S1 is 2-4 mm.
Compared with the prior art, the method for culturing the oil peony callus provided by the invention has the following beneficial effects:
the peony leaves for oil are used as materials, the callus cultured by the method has vigorous growth capability and high growth speed, and the callus can grow to the total volume of about 5-6 cm after about 25 days of enrichment culture3The color of the product is changed into dark yellow after 35 d. The callus cultured by adopting the comparative example is dark yellow at the initial stage of subculture, then gradually becomes tan and has harder texture and slow growth, and the total volume of the callus cultured by about 25 days of propagation culture is only 1-2 cm3And about 30d is aged and blackened.
The method of the invention lays a test foundation for establishing the regeneration system of the peony for oil and lays a foundation for researching the variety improvement of the peony for oil by utilizing the regeneration system technology.
Drawings
FIG. 1 is a photograph of callus obtained after proliferation in example 1;
FIG. 2 is a photograph of callus obtained after proliferation in comparative example 1.
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 (not containing sucrose) used in the following examples of the present invention are commercially available.
The invention provides a method for culturing oil peony callus, which comprises the following steps:
s1, taking peony leaves for oil, cleaning and sterilizing to obtain sterile leaves;
s2, cutting the aseptic leaves along the main pulse direction of the leaves, namely dividing each leaf into two halves, inoculating the cut 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 4.74g of MS solid culture medium and 20g of cane sugar into 1L of water, heating for dissolving, adjusting the pH value to 5.8-6.0, then adding 0.1mg of NAA, 10-20 ml of ginger juice and 6-8 g of agar, boiling until the agar is completely dissolved, and sterilizing;
s3, taking out the leaf callus, cutting into blocks, putting into a proliferation culture medium, and culturing at 25 +/-2 ℃ until the white peony leaf callus for the pine oil is obtained;
the proliferation culture medium is prepared by the following method: adding 4.74g of MS solid culture medium and 30g of cane sugar into 1L of water, heating for dissolving, adjusting the pH value to 5.8-6.0, then adding 2mg of 6-BA, 10-20 ml of ginger juice and 6-8 g of agar, boiling until the agar is completely dissolved, and sterilizing;
the ginger juice in S2 and S3 is prepared by the following method: slicing ginger, mixing the sliced ginger with water according to the ratio of 20g to 200ml, boiling for 5-10 min, and filtering to collect filtrate as ginger juice.
Preferably, the invention relates to a method for culturing oil peony callus, which comprises the following examples. In the following examples, the test material is a Paeonia ostii variety "Paeonia ostii" plant, and proper Paeonia ostii leaves are selected according to needs and inoculated on the day of sampling to induce callus.
Example 1
A method for culturing oil peony callus comprises the following steps:
s1, taking peony leaves for oil, cleaning and sterilizing the peony leaves for oil with the length of 6cm along the main vein direction to obtain sterile leaves; the specific mode of disinfection is as follows: soaking cleaned oil in 75% ethanol solution, sterilizing, washing with sterile water, and adding HgCl 0.1g/100ml2Soaking in the solution for 8min for sterilization, taking out the leaves, washing with sterile water, and taking out to obtain sterile leaves.
S2, inducing: cutting sterile leaves along the main pulse direction of the leaves, namely, dividing each leaf into two halves, inoculating the cut leaves into an induction culture medium, culturing at 25 +/-2 ℃, wherein the illumination time is 16h/d, the light intensity is 2000lx, culturing for 20 days, and growing 1cm multiplied by 1cm callus to obtain leaf callus;
the induction culture medium is prepared by the following method: adding 4.74g MS solid culture medium and 20g sucrose into 1L water, heating to dissolve, adjusting pH to 5.8, adding 0.1mg NAA, 10ml rhizoma Zingiberis recens juice and 6g agar, boiling until agar is completely dissolved, and sterilizing;
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;
s3, proliferation: taking out leaf callus, cutting into 2mm × 2mm × 1mm pieces, placing in proliferation culture medium, culturing at 25 + -2 deg.C under illumination for 16h/d and light intensity of 2000lx for 25 days to obtain 4-5 cm3White pine oil with large and small sizes is used as peony leaf callus;
the proliferation culture medium is prepared by the following method: adding 4.74g MS solid culture medium and 30g sucrose into 1L water, heating to dissolve, adjusting pH to 5.8, adding 2mg 6-BA, 10ml rhizoma Zingiberis recens juice and 6g agar, boiling until agar is completely dissolved, and sterilizing;
and (4) inoculating 20 bottles of the callus, and counting the contamination rate B of the callus at the stage.
The contamination rate B is the number of contaminated bottles/total number of callus bottles × 100%.
The ginger juice in S2 and S3 is prepared by the following method: slicing rhizoma Zingiberis recens, mixing with water at a ratio of 20g to 200ml, boiling for 5min to obtain rhizoma Zingiberis recens juice, and filtering with rapid qualitative filter paper.
The results show that: callus culture was performed by the method of example 1, and the contamination rate A was 4.7%, the recovery rate was 68.3%, and the contamination rate B was 18.9%. The picture of the callus obtained after proliferation of S3 is shown in FIG. 1, and the texture is white and loose.
Example 2
A method for culturing oil peony callus comprises the following steps:
s1, taking peony leaves for oil, cleaning and sterilizing the peony leaves for oil with the length of 2cm along the main vein direction to obtain sterile leaves; the specific mode of disinfection is as follows: soaking cleaned oil in 75% ethanol solution, sterilizing, washing with sterile water, and adding HgCl 0.1g/100ml2Soaking in the solution for 8min for sterilization, taking out the leaves, washing with sterile water, and taking out to obtain sterile leaves.
S2, inducing: cutting the sterile leaves along the main pulse direction of the leaves, namely dividing each leaf into two halves, inoculating the cut leaves into an induction culture medium, culturing at 25 +/-2 ℃, wherein the illumination time is 16h/d, the light intensity is 2000lx, culturing for 20 days, and growing 1cm multiplied by 1cm callus to obtain leaf callus;
the induction culture medium is prepared by the following method: adding 4.74g MS solid culture medium and 20g sucrose into 1L water, heating to dissolve, adjusting pH to 6.0, adding 0.1mg NAA, 20ml rhizoma Zingiberis recens juice and 8g agar, boiling until agar is completely dissolved, and sterilizing;
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;
s3, proliferation: taking out leaf callus, cutting into 2mm × 2mm × 1mm pieces, placing in proliferation culture medium, culturing at 25 + -2 deg.C under illumination for 16h/d and light intensity of 2000lx for 25 days to obtain 4-5 cm3White pine oil with large and small sizes is used as peony leaf callus;
the proliferation culture medium is prepared by the following method: adding 4.74g MS solid culture medium and 30g sucrose into 1L water, heating to dissolve, adjusting pH to 6.0, adding 2mg 6-BA, 20ml rhizoma Zingiberis recens juice and 8g agar, boiling until agar is completely dissolved, and sterilizing;
and (4) inoculating 20 bottles of the callus, and counting the contamination rate B of the callus at the stage.
The contamination rate B is the number of contaminated bottles/total number of callus bottles × 100%.
The ginger juice in S2 and S3 is prepared by the following method: slicing rhizoma Zingiberis recens, mixing with water at a ratio of 20g to 200ml, boiling for 10min to give rhizoma Zingiberis recens juice, and filtering with rapid qualitative filter paper to give filtrate.
The results show that: callus culture was performed by the method of example 2, and the contamination rate A was 5.1%, the recovery rate was 69.4%, and the contamination rate B was 18.7%.
Example 3
A method for culturing oil peony callus comprises the following steps:
s1, taking peony leaves for oil, cleaning and sterilizing the peony leaves for oil with the length of 4.2cm along the main vein direction to obtain sterile leaves; the rest of the operation of S1 is the same as in example 1.
S2, inducing: cutting the sterile leaves along the main pulse direction of the leaves, namely dividing each leaf into two halves, inoculating the cut leaves into an induction culture medium, culturing at 25 +/-2 ℃, wherein the illumination time is 16h/d, the light intensity is 2000lx, culturing for 20 days, and growing 1cm multiplied by 1cm callus to obtain leaf callus;
the induction culture medium is prepared by the following method: adding 4.74g MS solid culture medium and 20g sucrose into 1L water, heating to dissolve, adjusting pH to 5.8, adding 0.1mg NAA, 15ml rhizoma Zingiberis recens juice and 6g agar, boiling until agar is completely dissolved, and sterilizing;
a total of 20 bottles were inoculated, and the contamination rate A and the recovery rate at this stage were counted.
The contamination rate A and the recovery rate were calculated in the same manner as in example 1.
S3, proliferation: taking out leaf callus, cutting into 2mm × 2mm × 1mm pieces, placing in proliferation culture medium, culturing at 25 + -2 deg.C under illumination for 16h/d and light intensity of 2000lx for 25 days to obtain 4-5 cm3White pine oil with large and small sizes is used as peony leaf callus;
the proliferation culture medium is prepared by the following method: adding 4.74g MS solid culture medium and 30g sucrose into 1L water, heating to dissolve, adjusting pH to 5.8, adding 2mg 6-BA, 15ml rhizoma Zingiberis recens juice and 6g agar, boiling until agar is completely dissolved, and sterilizing;
20 bottles were inoculated, and the contamination rate B of the callus at this stage was counted and calculated in the same manner as in example 1.
The ginger juice in S2 and S3 is prepared by the following method: slicing rhizoma Zingiberis recens, mixing with water at a ratio of 20g to 200ml, boiling for 8min to obtain rhizoma Zingiberis recens juice, and filtering with rapid qualitative filter paper to obtain filtrate.
The results show that: callus culture was performed by the method of example 3, and the contamination rate A was 4.5%, the recovery rate was 66.4%, and the contamination rate B was 15.1%.
Comparative example 1
A method for culturing oil peony callus comprises the following steps:
s1, taking peony leaves for oil, cleaning and sterilizing to obtain sterile leaves; the specific mode of disinfection is as follows: soaking cleaned peony leaf in 75% ethanol solution for sterilization, washing with sterile water, and adding HgCl 0.1g/100ml2Soaking in the solution for 8min for sterilization, taking out the leaves, washing with sterile water, and taking out to obtain sterile leaves.
S2, inducing: cutting the sterile leaves along the main pulse direction of the leaves, namely dividing each leaf into two halves, inoculating the cut leaves into an induction culture medium, culturing at 25 +/-2 ℃, wherein the illumination time is 16h/d, the light intensity is 2000lx, culturing for 20 days, and growing callus with the size of 0.5cm multiplied by 0.5cm to obtain leaf callus;
the induction culture medium is prepared by the following method: adding 4.74g MS solid culture medium and 20g sucrose into 1L water, heating to dissolve, adjusting pH to 5.8, adding 0.1mg NAA and 6g agar, boiling until the agar is completely dissolved, and sterilizing;
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;
s3, proliferation: taking out leaf callus, cutting into 2mm × 2mm × 1mm pieces, placing in proliferation culture medium, culturing at 25 + -2 deg.C under illumination for 16h/d and light intensity of 2000lx for 25 days to obtain 1-2 cm3White pine oil with large and small sizes is used as peony leaf callus;
the proliferation culture medium is prepared by the following method: adding 4.74g MS solid culture medium and 30g sucrose into 1L water, heating to dissolve, adjusting pH to 5.8, adding 2mg 6-BA,
Boiling 6g of agar until the agar is completely dissolved, and sterilizing;
and (4) inoculating 20 bottles in total, and counting the survival rate and the pollution rate B of the callus at the stage.
The contamination rate B is the number of contaminated bottles/total number of callus bottles multiplied by 100%
The results show that: callus culture was performed by the method of example 1, and the contamination rate A was 36.8%, the recovery rate was 51.0%, and the contamination rate B was 41.2%. The picture of callus obtained after propagation of S3 is shown in FIG. 2, and it is tan and hard in texture.
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 (1)
1. The application of the ginger in the callus culture of the oil peony is characterized by comprising the following steps:
s1, taking peony leaves for oil, cleaning and sterilizing the peony leaves for oil with the length of 6cm along the main vein direction to obtain sterile leaves; the specific mode of disinfection is as follows: soaking cleaned oil in 75% ethanol solution, sterilizing, washing with sterile water, and adding HgCl 0.1g/100ml2Soaking and sterilizing with the solution for 8min, taking out the leaves, washing with sterile water, and taking out to obtain sterile leaves; the peony variety for oil is paeonia ostii;
s2, inducing: cutting the sterile leaves along the main pulse direction of the leaves, namely dividing each leaf into two halves, inoculating the cut leaves into an induction culture medium, culturing at 25 +/-2 ℃, and culturing for 20 days with illumination time of 16h/d and light intensity of 2000lx to obtain leaf callus;
the induction culture medium is prepared by the following method: adding 4.74g MS solid culture medium and 20g sucrose into 1L water, heating to dissolve, adjusting pH to 5.8, adding 0.1mg NAA, 10ml rhizoma Zingiberis recens juice and 6g agar, boiling until agar is completely dissolved, and sterilizing;
s3, proliferation: taking out the leaf callus, cutting into pieces of 2mm multiplied by 1mm, placing the pieces in a proliferation culture medium, and culturing for 25 days at 25 +/-2 ℃ with illumination time of 16h/d and light intensity of 2000lx to obtain white peony leaf callus for the pine oil;
the proliferation culture medium is prepared by the following method: adding 4.74g MS solid culture medium and 30g sucrose into 1L water, heating to dissolve, adjusting pH to 5.8, adding 2mg 6-BA, 10ml rhizoma Zingiberis recens juice and 6g agar, boiling until agar is completely dissolved, and sterilizing;
the ginger juice in S2 and S3 is prepared by the following method: slicing ginger, mixing the sliced ginger with water according to a ratio of 20g to 200ml, boiling for 5-10 min when the sliced ginger is 2mm thick, and filtering by using quick qualitative filter paper to collect filtrate as ginger juice.
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