CN111543324B - Banana tissue culture method suitable for DNA material requirements of BioNano technology - Google Patents

Abstract

The invention belongs to the technical field of plant tissue culture, in particular to a banana tissue culture method suitable for the technical material requirements of BioNano (optical atlas system). By improving the rooting medium, the invention uses MS liquid medium instead of agar, uses absorbent cotton to fix it, reduces the content of sucrose, cultivates in a shaking table, and quickly obtains strong and tender rooting shoots through tissue culture. The rooted seedlings cultivated by the method of the present invention can meet the requirements of BioNano technology for material DNA, and solve the technical problem that the rooted seedlings cultivated by the existing tissue culture method cannot meet the requirements of BioNano technology for material DNA; and the obtained banana The tissue culture seedlings are more tender, and the genetic traits of the seedlings are stable, which can better maintain the characteristics and characteristics of the female parent.

Description

Banana tissue culture method suitable for DNA material requirements of BioNano technology

Technical Field

The invention belongs to the technical field of plant tissue culture, particularly relates to a banana tissue culture method, and particularly relates to a banana tissue culture method suitable for requirements of BioNano technical materials.

Background

The BioNano optical mapping system is based on a single-molecule optical mapping technology, and enables complete single DNA molecules to be arranged in parallel in a nano channel through a special chip technology, and the complete single DNA molecules are photographed and imaged to display a more complete genome map. And a macro framework support of genome is provided through a monomolecular optical map, and the accuracy and the authenticity of a splicing and assembling result are ensured. In the region of the repeated sequence, the number of copies of the repeated fragment can be accurately determined. Through Illumina or PacBio sequencing and the combination of a BioNano map, Scaffolding is carried out, so that the assembly index is obviously improved. The BioNano optical mapping system has outstanding advantages: the accuracy of a sequencing assembly result can be verified, sequencing and directional positioning can be performed on sequencing assembly fragments, and potential error assembly in sequencing assembly can be identified; DNA molecules do not need to be broken into short fragments and amplified, the longest read length can reach Mb level, and genome information is truly displayed; the genome assembly effect can be improved, the length of the Scaffold N50 can be greatly improved, and the quantity of the Scaffold can be reduced; gap sizes between adjacent sequence fragments can be assessed.

BioNano optical mapping systems are very widely used. Currently, BioNano technology, such as goat, spinach, mustard, etc., is incorporated into some published assemblies of high quality genomes (Yang et al, 2016; Bickhart et al, 2017; Xu et al, 2017; Belser et al, 2018).

However, the BioNano technology has high requirements on DNA materials, and for plants, the DNA material with high yield and good purity is mainly derived from tender plants or etiolated seedlings. That is, DNA extraction is performed using young plants or etiolated seedlings. The requirements of the BioNano technology are as follows: for the database building grade A: the DNA solution is colorless, clear and transparent, the concentration is 35-100 ng/. mu.L, the CV is less than 0.25, and the main band is more than 300kb (PFGE); b: the DNA solution is colorless, has visible precipitation but is not obvious, the concentration is 35-100 ng/mu L, the CV is less than 0.25, and the main band is more than 300kb (PFGE); c: the DNA solution is colorless, has obvious visible precipitate, has better viscosity and lower concentration, CV is more than 0.25 and less than 0.5, and the main band is more than 300kb (PFGE); d: the DNA solution was colored, turbid, poorly viscous, low (<30 ng/. mu.L), CV >0.25, and there was degradation of the sample. A, B, C satisfies the requirement of BioNano technology on DNA material, D does not.

For the cultivation of triploid bananas, tissue culture technology is generally adopted in the prior art for propagation and seed preservation, and no seeds exist. The plant tissue culture is to culture plant organs, tissues, cells, protoplasts and the like by using an artificial culture medium under in vitro conditions to form a complete plant. The plant tissue culture technology plays a key role in agricultural production. The technology can be effectively used for plant detoxification, prevents variety degradation, rapidly propagates and grows seedlings, improves quality, is not limited by natural conditions and the like (Chenhaiwei, 2007; Shaoyyu and the like, 2019), has a particularly important effect in haploid, polyploid and mutant breeding, and has long breeding time and high hybridization difficulty if the traditional triploid plants are obtained by sexual hybridization of diploids and tetraploids, and the tissue culture technology effectively solves the breeding difficulty of the triploid. Under the aseptic condition, a small part of meristem of stem tip, flower, bud and other organs of banana is cultured in an artificially controlled environment to regenerate to form a complete plant. The tissue culture technology has the advantages that disease-free high-quality strong seedlings can be propagated in a large amount in a short time, and the excellent properties of the original variety can be maintained.

Nevertheless, the banana rooted seedlings cultured by the tissue culture technology in the prior art can meet the requirements of molecular related experiments on materials, including PCR, fluorescent quantitative PCR, third generation sequencing (Pacbio sequence), high-throughput chromosome conformation capture technology (Hi-C), full-length transcription group and the like, but can not meet the requirements of the BioNano technology on DNA materials. The inventors dark-treated young rooted shoots for 4 days and used for DNA extraction of the BioNano-technology material. The DNA extraction process of the BioNano technology comprises the steps of taking a plant tender tissue sample, embedding agar, digesting a gel block by protease K, and carrying out quality inspection after sol and enzyme sol. The DNA detection result is D, which does not meet the library building requirement of the BioNano technology, namely the DNA solution is colored, turbid, poor in viscosity, low in concentration (<30 ng/mu L), CV >0.25 and degraded. The DNA extracted in several times is detected as D. For this reason, the present invention needs to improve the plant tissue culture method to meet the requirement of the BioNano technology on the DNA of the material.

However, the factors influencing the plant tissue culture are many, including the components of the culture medium, such as the culture medium, the components of the culture medium are many, various components have different influences on the tissue culture of each plant, the tissue culture results are influenced by the culture conditions, such as illumination, temperature, humidity and the like, and particularly, the tissue culture seedlings can meet the requirements of the BioNano technology on the DNA of the materials.

Reference documents:

Belser C,Istace B,Denis E,et al.Chromosome-scaleassemblies of plant genomes using nanopore long reads and optical maps.Nature Plants,2018,4:879-887.

Bickhart,DM,Rosen BD,Koren S,et al.Single-molecule sequencing and chromatin conformation capture enable de novo reference assembly of the domestic goat genome.Nature Genetics,2017,49(4):643-650.

Xu C,Jiao C,Sun H,et al.Draft genome of spinach and transcriptome diversity of 120 Spinacia accessions.Nature Communications,2017,8:15275.

Yang J,Liu D,Wang X,et al.The genome sequence of allopolyploid Brassica juncea and analysis of differential homoeolog gene expression influencing selection.Nature Genetics,2016,48(10):1225-1232.

chenhaiwei, progress of plant tissue culture research, Chifeng college of academic, 2007,23(6):16-17.

Shaoyin, lylurin, application progress of plant tissue culture technology in garden plant breeding, rural economy and science and technology 2019,30(9):56-57.

Disclosure of Invention

Aiming at the technical problem, the invention provides a tissue culture method of a fragrant coke, which is used for meeting the requirement of a BioNano technology on material DNA.

The technical scheme of the invention is as follows:

a banana tissue culture method suitable for DNA material requirements of BioNano technology comprises the following steps:

(1) selecting an explant:

selecting strong and well-growing plants without diseases and insect pests to sprout in sunny days;

(2) and (3) explant sterilization:

washing the bud in the step (1) under tap water, removing the outer old leaves, leaf sheaths and part of seedling growing points, and using 0.1% HgCl in a sterile workbench₂Soaking the solution for 30 minutes, then washing with sterile water for 3-4 times, and sucking the surface water with sterile filter paper;

(3) explant induction:

splitting the explant sterilized in the step (2) from the middle, longitudinally dividing the explant containing a growing point into two parts, then inoculating the two parts into an induction culture medium, and culturing for 40-50 days under the conditions of a culture temperature of 26-30 ℃, an illumination intensity of 1600-2000 lx and an illumination time of 12 hours/day, wherein the basal part of the explant generates an adventitious bud cluster; the induction culture medium is as follows: MS culture solution, 6-BA 4mg/L, NAA0.2 mg/L, sucrose 30g/L and agar 5-10g/L, and the pH value is 5.8-6.0;

(4) and (3) proliferation culture:

selecting adventitious bud clusters generated by explant induction in the step (3), cutting the adventitious bud clusters with the height of 2-3cm into small blocks containing 2-3 buds, inoculating the small blocks into a solid proliferation subculture medium, and culturing for 30-40 days under the conditions that the culture temperature is 26-30 ℃, the illumination intensity is 1600-2000 lx and the illumination time is 12 hours/day to obtain seedlings with the height of 3-5cm, wherein the solid proliferation subculture medium is as follows: MS culture solution, NAA0.15 mg/L, sucrose 30g/L, active carbon 0.5g/L, agar 5-10g/L and pH 5.8-6.0;

(5) rooting culture:

cutting the plantlets with the height of 3-5cm obtained by culturing in the step (4), inoculating the cut plantlets into a tissue culture bottle with a rooting culture medium, fixing the cut banana plantlets by absorbent cotton, and culturing for 10-20 days under the conditions that the shaking table temperature is 26-30 ℃, the shaking table speed is 50-70 times/min, the illumination intensity is 1600-2000 lx and the illumination time is 8 hours/day, so as to obtain the rooting plantlets for DNA extraction of the BioNano technology, wherein the rooting culture medium is as follows: MS culture solution, NAA0.5mg/L, sucrose 10-15g/L and pH 5.6-5.8;

the basic components of the MS culture solutionIs NH₄NO₃ 190mg/L，KNO₃ 165mg/L，KH₂PO₃ 17mg/L，MgSO₄ 37mg/L，CaCl₂ 44mg/L，MnSO₄ 22.3μg/L，ZnSO₄ 8.6μg/L，NaMoO₄ 0.25μg/L，CuSO₄0.025μg/L，CoCl₂ 0.025μg/L，KI 0.83μg/L，H₃BO₃ 6.2μg/L，VB₁ 0.5-2.5μg/L，VB₆ 2.5μg/L，VB₃2.5 mu g/L, 10 mu g/L of amino acetic acid, 0.5mg/L of inositol, 37.25mg/L of disodium edetate and 27.85mg/L of ferrous sulfate.

Different from the conventional tissue culture of the fragrant coke, the conventional tissue culture of the fragrant coke aims at obtaining coarse seedlings, and has high survival rate and strong reproduction rate or germplasm resource preservation, but the invention is suitable for the BioNano technology, meets the requirements of the BioNano technology on material DNA, needs tender tissue culture seedlings, has stable genetic character and small coefficient of variation, and can better maintain the characteristics and the characteristics of female parents. Therefore, the invention improves the original tissue culture method to meet the requirements of the BioNano technology.

The rooting culture medium uses an MS culture medium without agar, is fixed by absorbent cotton, further reduces the content of sucrose, and is cultured in a liquid culture medium by using a shaking table during rooting culture, and the rooting seedlings are strong and tender after tissue culture. In addition, on one hand, the system is more uniform by shake culture in a shaking table, so that the components of the culture medium can be better and fully contacted with the culture, the components in the culture medium can better play a role, the difference among samples is small, and the variation coefficient of the DNA solution is small; meanwhile, the special liquid rooting culture medium without agar and with reduced sucrose content is adopted, so that less sucrose and agar are involved in the subsequent material DNA extraction process, the requirements of the BioNano technology on the material DNA are met, and the technical problem that the rooting seedlings cultured by the conventional tissue culture method cannot meet the requirements of the BioNano technology on the material DNA is solved.

Compared with the prior art, the invention has the beneficial effects that:

the rooting seedling cultured by the tissue culture method can meet the requirement of the BioNano technology on the DNA of the material, and the technical problem that the rooting seedling cultured by the conventional tissue culture method cannot meet the requirement of the BioNano technology on the DNA of the material is solved.

By adopting the tissue culture method, the obtained aromatic coke seedlings have stable hereditary characters and small variation, and can better maintain the characteristics and the characteristics of the female parent.

The culture medium disclosed by the invention is fast in induced differentiation and growth, and the rooted seedlings cultured by the tissues are tender, so that the culture medium is more suitable for the requirements of the BioNano technology on material DNA, and meanwhile, the time and the cost are saved.

The invention creatively adopts shake culture in a shaking table in the rooting culture stage, so that the system is more uniform, the components of the culture medium can be better and fully contacted with the culture, and the components in the culture medium can better play a role, thereby ensuring that the sample is more uniform and being beneficial to improving the quality of DNA; meanwhile, the special agar-free liquid rooting culture medium with reduced sucrose is adopted, so that the method is beneficial to less sucrose in the subsequent material DNA extraction process, reduces the influence of agar on the later-stage DNA extraction, and is beneficial to improving the quality of the total DNA of the bananas.

Drawings

FIG. 1 is a photograph of tissue culture seedlings of examples 1, 2 and 3, wherein FIG. 1(A) is the tissue culture seedling of example 1, FIG. 1(B) is the tissue culture seedling of example 2, and FIG. 1(C) is the tissue culture seedling of example 3;

FIG. 2 is a graph showing electrophoretic detection of DNA PFGE extracted from the tissue culture seedlings of examples 1, 2 and 3, wherein FIG. 2(A) is a graph showing electrophoretic detection of DNA PFGE extracted from the tissue culture seedlings of example 1, FIG. 2(B) is a graph showing electrophoretic detection of DNA PFGE extracted from the tissue culture seedlings of example 2, and FIG. 2(C) is a graph showing electrophoretic detection of DNA PFGE extracted from the tissue culture seedlings of example 1. M1 is 23k Marker (. lamda.DNA/HindIII); m2 is 1M Marker (Lambda PFG Ladder); y1 is tissue culture seedling DNA.

Detailed Description

Example 1 tissue culture method of Xiangjiao

(1) Selecting an explant:

selecting strong and well-growing plants without diseases and insect pests to sprout in sunny days;

(2) and (3) explant sterilization:

washing the bud in the step (1) under tap water, removing the outer old leaves, leaf sheaths and part of seedling growing points, and using 0.1% HgCl in a sterile workbench₂Soaking the solution for 30 minutes, then washing with sterile water for 3-4 times, and sucking the surface water with sterile filter paper;

(3) explant induction:

splitting the explant sterilized in the step (2) from the middle, longitudinally dividing the explant containing a growing point into two parts, then inoculating the two parts into an induction culture medium, and culturing for 40-50 days under the conditions of a culture temperature of 26-30 ℃, an illumination intensity of 1600-2000 lx and an illumination time of 12 hours/day, wherein the basal part of the explant generates an adventitious bud cluster; the induction culture medium is as follows: MS culture solution, 6-BA 4mg/L, NAA0.2 mg/L, sucrose 30g/L and agar 5-10g/L, and the pH value is 5.8-6.0;

(4) and (3) proliferation culture:

selecting adventitious bud clusters generated by explant induction in the step (3), cutting the adventitious bud clusters with the height of 2-3cm into small blocks containing 2-3 buds, inoculating the small blocks into a solid proliferation subculture medium, and culturing for 30-40 days under the conditions of culture temperature of 26-30 ℃, illumination intensity of 1600-2000 lx and illumination time of 12 hours/day to obtain seedlings with the height of 3-5 cm; the proliferation culture medium comprises: MS culture solution, NAA0.15 mg/L, sucrose 30g/L, active carbon 0.5g/L, agar 5-10g/L and pH 5.8-6.0;

(5) rooting culture:

cutting and inoculating the plantlets with the height of 3-5cm obtained by culturing in the step (4) into a tissue culture bottle with a rooting culture medium, fixing the cut banana plantlets by absorbent cotton, and culturing for 10-20 days under the conditions that the shaking table temperature is 26-30 ℃, the shaking table speed is 50-70 times/min, the illumination intensity is 1600-2000 lx and the illumination time is 8 hours/day, so as to obtain rooted plantlets for DNA extraction of a BioNano technology, wherein the rooting culture medium is as follows: MS culture solution, NAA0.5mg/L, cane sugar 15g/L and pH 5.6-5.8;

the basic component of the MS culture solution is NH₄NO₃ 190mg/L，KNO₃ 165mg/L，KH₂PO₃ 17mg/L，MgSO₄ 37mg/L，CaCl₂ 44mg/L，MnSO₄ 22.3μg/L，ZnSO₄ 8.6μg/L，NaMoO₄ 0.25μg/L，CuSO₄0.025μg/L，CoCl₂ 0.025μg/L，KI 0.83μg/L，H₃BO₃ 6.2μg/L，VB₁ 0.5-2.5μg/L，VB₆ 2.5μg/L，VB₃2.5 mu g/L, 10 mu g/L of amino acetic acid, 0.5mg/L of inositol, 37.25mg/L of disodium edetate and 27.85mg/L of ferrous sulfate.

Example 2 tissue culture method of Xiangjiao

The difference from the embodiment 1 is that: the content of sucrose in the rooting culture medium is 10 g/L. The picture of the tissue culture seedling is shown in FIG. 1 (B).

Example 3 tissue culture method of Banana seedlings in the prior art

The difference from the embodiment 1 is that: the rooting culture medium is a solid culture medium, the MS culture medium, NAA0.5mg/L, sucrose 30g/L and agar 5-10g/L are cultured for 10-20 days under the conditions that the illumination intensity is 1600-2000 lx and the illumination time is 8 hours/day, and the picture of the tissue culture seedling is shown in figure 1 (C).

As can be seen from fig. 1: FIG. 1(B) shows the cultivation method of example 2, the cultured rooted shoots are more tender and the seedlings are more robust and suitable for transplantation after hardening-off, as shown in FIG. 1 (A).

Example 4 DNA extraction and DNA Qubit detection and results

(1) DNA extraction:

and extracting the total DNA of the young banana leaves by using a plant genome DNA extraction kit based on a Tiangen paramagnetic particle method.

The operation steps are as follows:

culturing the rooted seedlings obtained in the embodiments 1, 2 and 3 in the dark for 2 days, taking about 100mg of fresh tender tissues, and adding liquid nitrogen for fully grinding; quickly transferring the ground powder into a centrifuge tube which is pre-filled with 400 mu L of buffer solution GPM and 5 mu L of RNase A, quickly reversing and uniformly mixing, putting the centrifuge tube into a water bath at 70 ℃ for 10 minutes, and reversing the centrifuge tube in the water bath process to mix the sample for a plurality of times; ③ centrifuging for 4 minutes at 12000 r/min, transferring 300 mu L of supernatant to a new centrifuge tube; adding 300 mu L of buffer solution GHB, 300 mu L of isopropanol and 15 mu L of magnetic bead suspension G, and uniformly mixing by oscillation; standing at room temperature for 5 minutes; placing the centrifugal tube on a magnetic frame and standing for 1 minute, and carefully removing liquid when the magnetic beads are completely adsorbed; taking down the centrifugal tube from the magnetic frame, adding 500 mu L of deproteinized liquid RD, and uniformly mixing for 30 seconds; placing the centrifugal tube on a magnetic frame and standing for 30 seconds, and carefully absorbing the liquid after the magnetic beads are completely adsorbed; seventhly, adding 600 mu L of rinsing liquid PWB (whether absolute ethyl alcohol is added before use) and uniformly mixing the solution for 30 seconds by oscillation; placing the centrifugal tube on a magnetic frame and standing for 30 seconds, and carefully absorbing liquid after the magnetic beads are completely adsorbed; eighthly, repeating the step seven times; placing the centrifuge tube on a magnetic frame, and airing for 10-15 minutes at room temperature; ninthly, taking the centrifugal tube down from the magnetic frame, adding 50-100 mu L of elution buffer TB, oscillating, mixing uniformly, and incubating for 3 minutes at 65 ℃; placing the centrifuge tube on a magnetic frame for standing for 1 minute, carefully transferring the DNA solution into a new centrifuge tube after the magnetic beads are completely adsorbed, and detecting and performing subsequent tests on the DNA.

(2) DNA Qubit detection and results:

the adopted Qubit^TMdsDNA HS Assay kit.

The basic operation is that firstly, a standard product is configured and measured, and an instrument automatically corrects and generates a standard curve; and secondly, configuring a detection sample according to requirements, and performing computer detection. The results of DNAQubit detection of the DNA solutions of the rooted shoots of examples 1, 2 and 3 are shown in Table 1.

TABLE 1 comparison table of DNA Qubit test results

(3) DNA PFGE electrophoresis detection and result

PFGE electrophoresis detection was performed using a standard SOP protocol.

The method comprises the following specific steps of: 0.7 percent; voltage: 80V; separation conditions are as follows: 40-450 kb; electrophoresis time: 16 h; 23kMarker (M1): lambda DNA/HindIII (23130, 9416, 6557, 4361, 2322, 2027, 564 bp); 1M Marker (M2): lambda PFG Ladder; the samples of example 1, example 2 and example 3 were loaded at 500ng for the Qubit concentration. The DNA PFGE electrophoretic detection is shown in FIG. 2.

The result of DNA PFGE electrophoresis detection shows that:

the sample DNA solution of example 1 is colorless, has good viscosity and low concentration, and after electrophoresis for 9 hours by using the procedure of separating 1M sample at maximum, the visible main band of the sample is clear and bright, the main band is distributed at 50-350K, the fragments are short, and in conclusion, the sample is judged to be a risk sample, and a subsequent marking experiment can be tried.

The sample DNA solution in example 2 is colorless and transparent, electrophoresis is performed for 9 hours by using a procedure of maximally separating 1M samples, the visible main band of the sample is clear and bright, the mobility is low, the size of the band meets the requirement, the A260/A280 value of Nanodrop detection is 1.82 normal, the A260/A230 value is 1.43 low, and impurity pollution is possible.

The sample DNA solution of example 3 was stratified, had many impurities and had a non-uniform concentration of viscosity difference, and when electrophoresis was performed for 9 hours using a procedure of separating 1M of the sample at the maximum, the main band of the sample was not clear, and in conclusion, the sample was judged to be defective and no subsequent labeling experiment was performed.

As can be seen from table 1 and fig. 2: the DNA solution quality of the sample of example 2 was the best, that of example 1 and that of example 3 was the worst. The quality of the DNA solution is shown to be related to the agar and sucrose contents in the rooting medium during tissue culture. It is demonstrated that the agar content of rooting medium for banana tissue culture affects the quality of DNA solution, and from examples 1 and 2, the sucrose content of rooting medium also affects the quality of DNA solution.

Example 5 BioNano technical results

Sequencing data was generated 404G on the BioNano platform, and the results of off-line evaluation of the data met the data quality requirements, as shown in table 2.

TABLE 2 BioNano technology sequencing data off-line evaluation results table

In conclusion, the rooting seedling cultured by the tissue culture method can meet the requirement of the BioNano technology on the DNA of the material, and the technical problem that the rooting seedling cultured by the conventional tissue culture method cannot meet the requirement of the BioNano technology on the DNA of the material is solved; the obtained tissue culture seedling with the fragrant scorch is more tender, the genetic character of the seedling is stable, the variation is small, and the characteristics of the female parent can be well maintained.

Claims (1)

1. A banana tissue culture method applicable to BioNano technology DNA material requirements, comprising the following steps: (1) Explant selection: On sunny days, choose robust, well-growing, disease-free, pest-free plants to suck buds; (2) Sterilization of explants: Rinse the sucking buds in step (1) under tap water, peel off the outer layer old leaves, leaf sheaths and some seedling growth points, soak in 0.1% HgCl solution for 30 minutes in a sterile workbench, and then rinse with sterile water for ₃ -4 times, dry the surface moisture with sterile filter paper; (3) Induction of explants: The explant sterilized in the step (2) is cut from the middle, the explant containing the growth point is divided into two longitudinally, and then inoculated into the induction medium, at a culture temperature of 26 to 30 ° C, and a light intensity of 1600 ～2000lx, cultured for 40-50 days under the condition of 12 hours/day of light time, adventitious buds were formed at the base of explants, and the induction medium was: MS culture medium+6-BA4 mg/L+NAA0.2 mg/ L+sucrose 30g/L+agar 5-10g/L, pH 5.8～6.0; (4) Proliferation culture: The adventitious bud clusters induced by explants in step (3) are selected to be cut into small pieces containing 2-3 buds with a height of 2-3 cm, and inoculated into the solid proliferation subculture medium, and the culture temperature is 26-30 ℃. ℃, the light intensity is 1600～2000lx, and the light time is 12 hours/day for 30～40 days to obtain seedlings with a height of 3-5cm, and the solid proliferation subculture medium is: MS culture medium+NAA0.15 mg /L+sucrose 30g/L+activated carbon 0.5g/L+agar 5-10g/L, pH 5.8～6.0; (5) Rooting culture: The seedlings with a height of 3-5cm obtained in step (4) are cut, inoculated into a tissue culture bottle with a rooting medium, and the cut banana seedlings are fixed with absorbent cotton at a shaking temperature of 26-30 ° C and a shaking speed of 50-70 ℃. Under the condition of times/min, the light intensity is 1600～2000lx, and the light time is 8 hours/day, the culture is cultivated for 10-20 days, and the rooted seedlings are obtained for DNA extraction by BioNano technology, and the rooting medium is: MS culture medium+ NAA0.5 mg/L+sucrose 10-15g/L, pH 5.6～5.8; The basic components of the MS culture medium are NH ₄ NO ₃ 190 mg/L, KNO ₃ 165 mg/L, KH ₂ PO ₃ 17 mg/L, MgSO ₄ 37 mg/L, CaCl ₂ 44 mg/L, MnSO ₄ 22.3μg/L, ZnSO ₄ 8.6μg/L, NaMoO ₄ 0.25μg/L, CuSO ₄ 0.025μg/L, CoCl ₂ 0.025μg/L, KI 0.83μg/L, H ₃ BO ₃ 6.2μg/L, VB ₁ 0.5-2.5μg/L, VB ₆ 2.5μg/L, VB ₃ 2.5μg/L, Aminoacetate 10μg/L, Inositol 0.5mg/L, EDTA 37.25mg/L, ferrous sulfate 27.85mg/L.

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