Slice analysis method for rapidly identifying columnar apples

Abstract

The invention discloses a slice analysis method for rapidly identifying columnar apples, and belongs to the technical field of apple breeding. The invention provides a method for rapidly identifying the column type apple by utilizing the ratio (L / R) of the distance L from the xylem vascular cells of the phloroglucinol dyed section of the stem tissue of the apple to the epidermis to the transecting radius R for the first time, and testing the section dyeing analysis L / R by taking 89 parts of materials taken from 20 parts of apple germplasm as test materials. And the accuracy of the method is verified through a molecular marker identification method. Experimental results show that the identification accuracy of the section staining analysis L / R method provided by the invention is 98.17%, a feasible and effective tool is provided for rapidly identifying column type apple materials in the field and breeding and popularizing column type apples, the identification method is low in cost, rapid, efficient and low in error rate, and the production efficiency is greatly improved.

Description

Technical Field

[0001] The invention relates to the technical field of apple breeding, and in particular to a slice analysis method for rapidly identifying columnar apples. Background Art

[0002] Tree structure is an important agronomic trait that affects shaping, pruning, orchard management, and fruit yield. Dwarfing and dense planting is the development trend of China's modern apple production. The promotion of tree types suitable for light simplification and mechanized management will meet the future development needs of the apple industry. The columnar apple (Colmnar apple) is derived from a mutant of the common apple. It has the characteristics of compact tree body, short internodes, axillary buds germinating to form a large number of short branches, and high photosynthetic efficiency. This tree type does not require branch pulling, and the amount of pruning is extremely light. It is suitable for mechanized and standardized management and is an important germplasm resource for the selection and breeding of lightly simplified apple cultivars (Tobutt, 1985; Zhang & Dai, 2011; Petersen & Krost, 2013; Liu Wei et al., 2016; Liang Meixia et al., 2017). The promotion and application of columnar apple varieties will greatly save production costs such as labor and help labor-saving apple cultivation. Therefore, how to accurately identify and screen columnar apple varieties is the focus of current research.

[0003] The columnar trait of apple is a quality trait controlled by the dominant single gene Co (Zhao Yujun et al., 1998; Gao Hua et al., 2004; Tobutt, 1994). MdCoL is a key candidate gene that regulates the formation of apple columnar traits (Wolters et al., 2013; Okada et al., 2016; Petersen et al., 2015; Okada et al., 2020). At present, molecular markers for identifying columnar apples have been developed based on the apple Co gene, but the instruments and reagents required for molecular marker identification are cumbersome and time-consuming, and the operator needs to have a certain molecular biology foundation, which is not suitable for rapid identification of columnar apple materials in the field. Therefore, it is urgent to develop a simple method for rapid identification of columnar apple materials based on the morphological characteristics of columnar apples to assist in the selection and promotion of columnar apple materials. Summary of the invention

[0004] The purpose of the present invention is to provide a slice analysis method for quickly identifying columnar apples to solve the problems existing in the above-mentioned prior art. The slice analysis method of the present invention can quickly, efficiently, low-cost and accurately identify columnar apples by calculating the ratio of the distance between the xylem vascular cells of the apple stem tissue and the epidermis and the cross-sectional radius.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] The present invention provides a method for identifying columnar apples, which is characterized by comprising the following steps:

[0007] Sampling the stem tissue of the apple to be tested, slicing it, and obtaining a tissue slice;

[0008] staining the tissue slice to obtain a stained section;

[0009] Observe the stained sections, determine the tissue center, measure the distance between the xylem vascular cells and the epidermis, record it as L, measure the cross-sectional radius, record it as R, and calculate the ratio of L to R;

[0010] On the same stained section, L and R of xylem vascular cells at different positions are measured, and the average value of the ratio of L to R is calculated and recorded as L / R to identify whether the apple to be tested is a columnar apple.

[0011] Preferably, if the L / R of the apple stained section is greater than 0.40, the apple to be tested is identified as a columnar apple;

[0012] If the L / R of the apple stained section is not greater than 0.40, the apple to be tested is identified as a non-columnar apple.

[0013] Preferably, the sampling includes: counting from top to bottom, marking the first leaf of the normally growing branch of the apple to be tested with a length greater than 0.5 cm as the first leaf, and taking the stem tissue located between the fifth and sixth leaves.

[0014] Preferably, the slices are cross-sections, and slices with a thickness of 1 mm are selected to obtain the tissue slices.

[0015] Preferably, the staining is phloroglucinol staining, comprising:

[0016] A phloroglucinol solution was dripped onto the tissue slice until the tissue slice was completely covered, and the staining was continued for 2 minutes. The liquid on the tissue slice was blotted dry, and a drop of concentrated hydrochloric acid was dripped onto the center of the tissue slice, and the reaction was continued for 1 minute.

[0017] Preferably, each 10 mL of the phloroglucinol solution comprises 0.1 g of phloroglucinol, 8 mL of 95% anhydrous ethanol and 2 mL of concentrated hydrochloric acid.

[0018] Preferably, the determining the tissue center comprises: determining the tissue center of the stained section through two diameters of the stained section.

[0019] Preferably, the measuring of the distance between the xylem vascular cells and the epidermis comprises: selecting xylem vascular cells that are obviously stained, connecting the xylem vascular cells with the tissue center using a line segment and extending the line segment to the epidermis, and measuring the length of the line segment from the xylem vascular cells to the epidermis.

[0020] Preferably, the measuring of the cross-sectional radius comprises: selecting obviously stained xylem vascular cells, connecting the xylem vascular cells with the tissue center using a line segment, and extending the line segment to the epidermis, and measuring the length of the line segment from the tissue center to the epidermis.

[0021] Preferably, the number of different positions is 3-4.

[0022] The present invention discloses the following technical effects:

[0023] The present invention provides a new method for analyzing the position of xylem vascular cells and quickly identifying columnar apples by using the structural characteristics of the stem of columnar apples through freehand slicing combined with phloroglucinol staining. The present invention proposes for the first time to identify columnar apples by using the ratio (L / R) of the distance L between the xylem vascular cells and the epidermis of apple stem tissue slices and the cross-sectional radius R, and uses 89 materials taken from 20 apple germplasms as test materials to experiment with the above-mentioned slice staining and L / R analysis method, and verifies the accuracy of the method by molecular marker identification. The experimental results show that the identification accuracy of the slice staining and L / R analysis method provided by the present invention is 98.17%, which provides a feasible and effective tool for rapid identification of columnar apple materials and breeding and promotion of columnar apples in the field. The identification method is low-cost, fast and efficient, and has a low error rate, which will greatly improve production efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0025] Figure 1 Schematic diagram of the numerical measurement and analysis method after section staining; the full length of the red line segment represents the cross-section radius R, and the red line segment between the two short black lines represents the distance L between the lignified vascular cells passing through the segment and the epidermis;

[0026] Figure 2 Diagram of changes in stem tissue structure of poplar and apple overexpressing MdCoL; A is the stem tissue of poplar, WT represents wild-type 84K poplar, #1, #4 and #8 are three MdCoL transgenic lines, co+ph represents stem cortex plus phloem, xy represents xylem; B is the stem tissue of apple, WT represents wild-type GL-3 apple, OE-48 represents MdCoL transgenic line, L represents the distance between lignified vascular cells and epidermis; the scale bar in the figure is 500μm;

[0027] Figure 3 The results of staining analysis of slices of different apple varieties; C indicates that the sample is a columnar apple material, and N indicates that the sample is a non-columnar apple material; L / R indicates the ratio of the distance between the xylem vascular cells and the epidermis to the cross-section radius; the scale in the figure is 500μm;

[0028] Figure 4 This is a statistical chart of the results of staining analysis of slices of different apple varieties;

[0029] Figure 5 The results of staining slices of different apple varieties; C indicates that the sample is a columnar apple material, and N indicates that the sample is a non-columnar apple material; L / R indicates the ratio of the distance between the xylem vascular cells and the epidermis to the cross-section radius; the scale in the figure is 500μm;

[0030] Figure 6 This is the electrophoresis result diagram of molecular marker identification of different apple varieties; among them, C means that the sample is determined to be a columnar apple material, and N means that the sample is determined to be a non-columnar apple material. DETAILED DESCRIPTION

[0031] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] The apple varieties used in the present invention include non-columnar apple varieties Gala, Golden Crown, Chunyang, Fuli, Saijin, New Red Star and Xu, as well as columnar apple varieties Weisexu, Telamon, Tuscan, Guifei, Ruga No. 5 and Fulei, as well as Fuji, Fuzaohong and Ruga No. 6 as hybrid parents used in verification experiments.

[0037] Among them, 5 varieties, namely Gala, Golden Crown, Chunyang, Saijin and Fuji, have been disclosed in the document "Xu Meng, Guo Jiangshan, Yang Tianzi, et al. Evaluation of apple non-concentrated reduced juice processing suitability and screening of suitable varieties [J]. Journal of Qingdao Agricultural University (Natural Science Edition), 2021, 38(03): 164-175."; 9 varieties, namely Fuli, Xinhongxing, Xu, Weisekexu, Telamon, Taskan, Guifei, Ruga 5 and Ruga 6, have been disclosed in the document "Liu Gengsen. Development of apple SSR and SNP markers and their application in genetic map construction and variety identification [D]. Hunan Agricultural University, 2018."; 2 varieties, namely Fulei and Fuzaohong, have been disclosed in the document "Lei Mingshan, Zhou Xiaokang, Zhang Shoujiang. Investigation on rust resistance of major apple germplasm resources in Tianshui [J]. Forestry Science and Technology Newsletter, 2022, (03): 89-91.". The applicant promises to distribute the above biological materials to the public within 20 years from the date of application of this invention.

[0038] Phloroglucinol staining is a common method for observing plant lignified cells and is the simplest microscopic chemical method for detecting lignin in plant cell walls. Phloroglucinol can dye lignin red, and plant lignin is mainly concentrated in the xylem vessels. Its staining results can accurately reflect the location of xylem vascular cells.

[0039] The phloroglucinol staining method in the embodiment of the present invention is:

[0040] Reagents: 1% phloroglucinol staining solution (0.1 g phloroglucinol + 8 mL 95% anhydrous ethanol + 2 mL concentrated hydrochloric acid);

[0041] Method: Add 1% phloroglucinol to a 1 mm tissue slice until the entire slice is covered. Stain for 2 minutes. Dry the surface stain with a paper towel. Add a drop of concentrated hydrochloric acid to the center of the slice. Allow to react for 1 minute. Observe the slice under a stereo microscope or by other visual methods.

[0042] The inventors' team found in previous studies that transgenic poplars heterologously expressing the apple column gene MdCoL had reduced plant height, shortened internodes, compact growth, and an increased proportion of stem cortex (co) plus phloem (ph), while a decreased proportion of xy (xy) (e.g. Figure 2 ); the transgenic apples overexpressing MdCoL not only grew compactly, but also had an increased ratio of cortex to phloem, and a larger distance L between the xylem vascular cells and the epidermis (as shown in Figure 2 In addition, the distance L between the xylem vascular cells and the epidermis of the original mutant material 'Weisekexu' of the columnar apple is also larger than that of the normal non-mutated material 'Xu' (as shown in Figure 3 This suggests that the increased distance L between xylem vascular cells and epidermis is part of the molecular function of the apple columnar gene MdCoL.

[0043] Example 1: Rapid identification of different columnar and non-columnar apple varieties by section staining analysis method

[0044] Based on the functional characteristics of the apple columnar gene MdCoL verified by the inventor's team in the early genetic transformation experiment, the present invention proposes a method for analyzing the characteristics of apple branch tissue structure and quickly identifying columnar apples and non-columnar apples in combination with freehand sectioning and phloroglucinol staining. In the cross section of the stem of columnar apples, the ratio of the distance L of the xylem vascular cells from the epidermis to the cross-section radius R is L / R>0.40, while the L / R of non-columnar apples is ≤0.40.

[0045] In order to quickly identify columnar apples and non-columnar apples using a slice staining analysis method, the present invention determines a detailed operation method, including slice position selection, staining method, and analysis and calculation method.

[0046] 1. Location Selection and Staining Method

[0047] In this embodiment, 75 branches of 13 varieties, including non-columnar apple varieties Gala, Golden Crown, Chunyang, Fuli, Saijin, New Red Star, and Xu, and columnar apple varieties Wesakexu, Telamon, Tuskan, Guifei, Ruga No. 5, and Fulei, were used as identification test materials.

[0048] Take a branch with normal growth, count the first leaf with a length greater than 0.5 cm as the first leaf, select the 5th internode (N5) between the 5th and 6th leaves, avoid the middle position of the leaf, and use a blade to make a freehand cross-section with a thickness of about 1 mm; add 1% phloroglucinol to cover the entire slice, stain for 2 minutes, dry the surface stain with a paper towel, add a drop of concentrated hydrochloric acid to the center of the slice, react for 1 minute, observe the slice under a stereo microscope, and the staining results are shown in Figure 3 .

[0049] 2. Measurement, calculation and analysis methods

[0050] like Figure 1 As shown, first, the tissue center O is determined by two diameters; then, four obviously stained xylem vascular cells are selected according to the staining results, and the distance L1 / 2 / 3 / 4 from the epidermis and the cross-sectional radius R1 / 2 / 3 / 4 passing through the xylem vascular cells are measured respectively, and L1 / R1, L2 / R2, L3 / R3 and L4 / R4 are calculated; and the average value L / R is calculated by taking the above four groups of data.

[0051] The analysis of the 75 groups of L / R average values ​​and 300 original L / R values ​​calculated by the above method showed that the L / R average value of columnar apples was 0.44, the L / R average value of non-columnar apples was 0.37, and at least 3 / 4 of the L / R ratios of columnar apples and non-columnar apples were distributed around 0.40 (e.g. Figure 4 Further research found that when L / R>0.40 and L / R≤0.40 were used as the dividing line, the accuracy of using a single ratio to analyze columnar apple varieties was 78.13%, the accuracy of non-columnar varieties was 81.98%, and the overall accuracy was 80.33%; the accuracy of using the average of the three ratios to analyze columnar apple varieties was 90.63%-96.88%, the accuracy of non-columnar apples was 90.70%-97.67%, and the overall accuracy was 92.00%-96.00%; the accuracy of using the average of the four ratios to analyze columnar apple varieties was 100%, the accuracy of non-columnar apple varieties was 97.67%, and the overall accuracy was 98.67% (as shown in Figure 2). Figure 4 As shown). It was determined that the average value of the four groups of L / R data was used as the evaluation content, and the dividing line of L / R between columnar apples and non-columnar apples was 0.40, that is, if L / R>0.40, the material was a columnar apple, and if L / R≤0.40, the sample was a non-columnar apple.

[0052] The results of the identification show that the above-mentioned section staining analysis method is effective in identifying columnar apple materials. The columnar and non-columnar apple materials can be clearly distinguished by L / R=0.40 as the dividing line, and the accuracy rate of identifying columnar materials can reach 100%, indicating that the above-mentioned method is feasible and effective. The technical method is low-cost, fast and efficient, and has a low error rate. It provides a simple and effective method for the breeding and promotion of columnar apples, which will greatly improve production efficiency.

[0053] Table 1 Statistics of identification results of different apple varieties by section staining analysis

[0054]

[0055]

[0056] Note: The "C" or "N" before the variety name in the variety column and in the result column indicates that the sample is a "column-shaped" or "non-column-shaped" apple material; the number after the variety name indicates the biological replicates of different trees; the L / R in the table is the average of 4 L / R data.

[0057] Example 2 Comparison of Section Staining Analysis Method and Molecular Marker Method in Identifying Different Columnar and Non-columnar Apple Lines

[0058] In order to test the accuracy of slice staining analysis method in identifying columnar apples, 89 materials from 20 germplasms in the hybrid superior line evaluation garden of Qingdao Agricultural University were randomly selected as test materials. The L / R ratio was measured and calculated by slice staining analysis method, and columnar and non-columnar materials were identified. DNA extraction and molecular marker identification were also performed.

[0059] The parents of the above 20 germplasms include: Fuji, Fuli, Fuzaohong, Lujia No. 5 and Lujia No. 6; they are planted in the Hybrid Superior Line Evaluation Garden of Qingdao Agricultural University. The applicant promises to distribute the above biological materials to the public within 20 years from the date of application of the present invention.

[0060] 1. Section staining analysis method

[0061] A 1mm-thick tissue slice was cut from the middle of the N5 internode of 89 test materials, and 1% phloroglucinol was added for staining for 2 minutes. The surface stain was then dried, and a drop of concentrated hydrochloric acid was added. After reacting for 1 minute, photos were taken for observation. After the tissue center O was determined, 4 lignified vascular cells with obvious staining were selected, and the distance L1-L4 from the epidermis and the cross-section radius R1-R4 passing through the cells were measured. The final ratio L / R was calculated and rounded to 2 decimal places. The staining and analysis results are shown in Figure 5 If L / R>0.40, the material is columnar apple C, and if L / R≤0.40, the sample is non-columnar apple N.

[0062] 2. Molecular marker identification method

[0063] A young leaf was taken from each of the 20 apple germplasms tested, and the sample DNA was extracted using a plant genomic DNA extraction kit (Extron). The quality of the DNA sample was tested using a UV imager after 1% agarose gel electrophoresis.

[0064] The DNA concentration should be 10-30 ng / μL. 260 / 280 =1.8-2.0, A 260 / 230 =1.8-2.0.

[0065] Primers used for molecular markers:

[0066] AF1: 5'-GGTTTTGCGTCAAGTCTTATGTTAG-3' (SEQ ID NO. 1);

[0067] AR1: 5'-CCCTCTAGCTAGTCCGTCATTTATC-3' (SEQ ID NO. 2);

[0068] AR2: 5'-AAGGTTTGGGCTACGTCCATACTA-3' (SEQ ID NO. 3), the above primers are from previous studies (Wang et al., 2022), and the final concentration of the primers is 10 μM.

[0069] The PCR amplification system contained 1.4 μL AF1, 0.8 μL AR1 and 0.8 μL AR2, 1 μL DNA sample, 10 μL 2× TaqMix and 6 μL sterile water.

[0070] The PCR reaction program was as follows: pre-denaturation stage at 95°C for 5 min; 30 cycles of denaturation (95°C for 30 s), annealing (56.5°C for 30 s) and extension (72°C for 30 s); finally, extension at 72°C for 2 min and storage at 4°C.

[0071] After 1.8% agarose gel electrophoresis, the molecular marker identification results were observed by UV imaging (such as Figure 6 shown).

[0072] 3. Experimental results

[0073] The statistical analysis of the average values ​​of L / R of 89 groups of 20 germplasms showed that the L / R of all 19 samples of 5 germplasms was greater than 0.40, and they were identified as columnar apples. The L / R of 68 samples of all 70 samples of the remaining 15 germplasms was less than or equal to 0.4, and they were identified as non-columnar apples (as shown in Table 2). Figure 6 As shown, the PCR products of 5 of the tested materials had two bands, the same as the columnar control materials "Guifei" and "Fulei", and were determined to be columnar apples; the PCR products of 15 of the tested materials had only one non-columnar band, which was consistent with the non-columnar control materials "Fuli" and "Xinhongxing", and were determined to be non-columnar apples. The result numbers were all consistent with the identification result numbers of the above-mentioned section staining analysis method.

[0074] From the results of molecular marker identification, the overlap between the identification results of the slice staining analysis L / R method and the molecular marker identification results is as high as 97.75%, among which the overlap of the identification of columnar apples can reach 100%. Comprehensive analysis of the identification results of 75 test materials of 7 known non-columnar apple varieties and 6 known columnar apple varieties, as well as 89 test materials of 20 unknown germplasms, showed that the accuracy of identifying columnar materials and non-columnar materials using the slice staining analysis method of the present invention can reach 98.17%, among which the accuracy of identifying columnar materials is 100%. It is proved that the method of slice staining analysis L / R for rapid identification of columnar apples is effective.

[0075] Table 2 Statistics of identification results of different columnar and non-columnar apple materials in hybrid lines

[0076]

[0077]

[0078] Note: In the hybrid number column, the number after “-” indicates the biological replication of different trees; L / R in the table is the average of 4 L / R data; “C” or “N” in the “Molecular marker results” column indicates that the sample is a “columnar” or “non-columnar” apple material; “Y” in the “Are the results consistent” column indicates that the L / R identification results are consistent with the molecular marker results, and “N” indicates inconsistency.

[0079] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for identifying columnar apples, characterized in that: The following steps are involved: Sampling the stem tissue of the apple to be tested, slicing it, and obtaining a tissue slice; staining the tissue slice to obtain a stained section; Observe the stained sections, determine the tissue center, measure the distance between the xylem vascular cells and the epidermis, record it as L, measure the cross-sectional radius, record it as R, and calculate the ratio of L to R; On the same stained section, L and R of xylem vascular cells at different positions are measured, and the average value of the ratio of L to R is calculated and recorded as L / R to identify whether the apple to be tested is a columnar apple.

2. The method according to claim 1, characterized in that If the L / R of the apple stained section is greater than 0.40, the apple to be tested is identified as a columnar apple; If the L / R of the apple stained section is not greater than 0.40, the apple to be tested is identified as a non-columnar apple.

3. The method according to claim 1, characterized in that The sampling includes: counting from top to bottom, marking the first leaf of the normally growing branch of the apple to be tested with a length greater than 0.5 cm as the first leaf, and taking the stem tissue located between the fifth and sixth leaves.

4. The method according to claim 1, characterized in that The slices are cross-sections, and a slice with a thickness of 1 mm is selected to obtain the tissue slices.

5. The method according to claim 1, characterized in that The staining is phloroglucinol staining, comprising: A phloroglucinol solution was dripped onto the tissue slice until the tissue slice was completely covered, and the staining was performed for 2 minutes. The liquid on the tissue slice was blotted dry, and a drop of concentrated hydrochloric acid was dripped onto the center of the tissue slice, and the reaction was performed for 1 minute.

6. The method according to claim 5, characterized in that Each 10 mL of the phloroglucinol solution includes 0.1 g of phloroglucinol, 8 mL of 95% anhydrous ethanol and 2 mL of concentrated hydrochloric acid.

7. The method according to claim 1, characterized in that The determining of the tissue center comprises: determining the tissue center of the stained section through two diameters of the stained section.

8. The method according to claim 1, characterized in that The method of measuring the distance between the xylem vascular cells and the epidermis comprises: selecting xylem vascular cells with obvious staining, connecting the xylem vascular cells with the tissue center using a line segment, and extending the line segment to the epidermis, and measuring the length of the line segment from the xylem vascular cells to the epidermis.

9. The method according to claim 1, characterized in that The measuring of the cross-sectional radius includes: selecting obviously stained xylem vascular cells, connecting the xylem vascular cells with the tissue center using a line segment, and extending the line segment to the epidermis, and measuring the length of the line segment from the tissue center to the epidermis.

10. The method according to claim 1, characterized in that The number of different positions is 3-4.

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