CN116784237A - Efficient fraxinus mandshurica regeneration method based on light quality regulation and control - Google Patents
Efficient fraxinus mandshurica regeneration method based on light quality regulation and control Download PDFInfo
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Abstract
The invention belongs to the technical field of biology, and particularly relates to a high-efficiency regeneration method of fraxinus mandshurica based on light quality regulation and control, which comprises the following steps: obtaining hypocotyl by dark culture of the water yeast Liu Gezi embryo for 5d, and inducing the generation of adventitious buds of the hypocotyl in a monochromatic red light source and a bud forming culture medium; in blue: red light = 3:2 light source, induction of adventitious bud elongation in elongation medium, and continuous induction of elongation with white light after 15 d; in blue: red = 2:3 or blue: red light = 1:4 light source, rooting medium. The treatment of different light qualities and combinations of the LED light sources advances the appearance time of the hypocotyl bud point of the fraxinus mandshurica to 3 days, shortens the cultivation time of the bud stage to about 14 days, the cultivation time of the elongation stage to about 15 days and advances the rooting time to about 5 days. Effectively shortens the period of induction regeneration and propagation, improves the induction efficiency and ensures the quality of tissue culture seedlings.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a high-efficiency regeneration method of fraxinus mandshurica based on light quality regulation.
Background
The fraxinus plants of the Oleaceae of the genus fraxinus, tall and big fallen tree, and walnut tree, cortex phellodendri and being called as northeast China 'Sanda hard broad', are the main afforestation tree species for precious material tree species and national reserve forest construction; the wood has high overall strength, good shock resistance and excellent wood, is commonly used for buildings, furniture and the like, and has important economic value. Along with the increase of world population and the rapid change of global climate, the demand of wood is rapidly increased, the growth and production efficiency of artificial forests can not meet the demands of national economy development and ecological environment, the serious overmuch of the total amount of wood seedlings and the insufficient structural supply are contradictory and outstanding, and the effective supply of wood gradually becomes one of the restriction factors affecting people in China to the best life. There are many limiting factors in the sexual propagation process of fraxinus mandshurica, such as dormancy of seeds, long propagation period, unstable setting rate, long breeding period, etc., and fraxinus mandshurica Liu Moxing propagation and fraxinus mandshurica tissue culture rapid propagation system established on asexual propagation are attracting more and more attention.
Light is one of the most important environmental factors for plant growth and development, and plays an important role in regulating plant morphogenesis, growth and development, substance metabolism and the like. The LED (Light-Emitting Diode) Light source is used as a cold Light source, has the advantages of high Light energy utilization rate, good spectral performance, low energy consumption, simplicity, portability, small heating value, long service life, strong controllability and the like, and is applied to daily production and life such as illumination, home furnishing and the like on a large scale. The optical quality, i.e. the spectrum of different wavelengths, is one of the main factors affecting plant growth, leaf morphology, flower morphology, biochemical properties and photosynthetic efficiency. The plant absorption light is concentrated in the visible light part with the wavelength of 380-760 nm, and novel LED-based Light Emitting Diode (LED)The light source is widely applied in plant cultivation production, wherein red light (wavelength 620-760 nm) and blue light (wavelength 400-500 nm) are plant photosynthetic CO 2 The assimilation is mainly energy source, and the influence on the growth and development of plants is greatest. The red light is mainly used for generating assimilates and accumulating biomass; blue light is a necessary condition for chlorophyll synthesis and chloroplast formation, affecting plant morphogenesis by controlling stomatal movement. Researches show that the adoption of the proper LED light source and light quality ratio can not only enhance the photosynthetic efficiency of plants and improve the contents of carotenoid, VC, total phenol and the like, but also influence the photomorphogenesis of the plants, and effectively promote the tissue culture of the plants such as phalaenopsis, ornamental dendrobium, fir, ornamental crabapple and anthurium andraeanum.
The existing tissue culture regeneration technical means of the fraxinus mandshurica still has the problems of long adventitious bud induction time period, low bud yield and the like, and the tissue culture regeneration efficiency of the fraxinus mandshurica can be further improved by applying different light combinations to the tissue culture regeneration culture of the fraxinus mandshurica, so that the bottleneck of rapid propagation of the fraxinus mandshurica is broken through.
Disclosure of Invention
In view of the above, the invention provides a high-efficiency regeneration method of fraxinus mandshurica based on light quality regulation. The method adopts different artificial light sources and combinations to carry out irradiation treatment at different stages of tissue culture regeneration of the fraxinus mandshurica, realizes rapid induction of budding, elongation and rooting of the fraxinus mandshurica hypocotyl, shortens the rapid propagation period, and improves the induction efficiency and the quality of tissue culture seedlings.
In order to achieve the aim of the invention, the invention provides a fraxinus mandshurica efficient regeneration method based on light quality regulation, which comprises the following steps:
(1) Obtaining the hypocotyl of fraxinus mandshurica;
(2) Budding culture;
(3) Elongation culture;
(4) Rooting culture;
preferably, the step (2) uses monochromatic red light as a light source, and the step (3) uses blue light: red light=3:2 and white light as light sources, step (4) uses blue light: red = 2:3 or blue: red = 1:4 is a light source.
Preferably, the photo-quality control-based fraxinus mandshurica is highThe effective regeneration method uses an LED lamp as an artificial light source, and the illumination intensity is 12 mu mol/m 2 S, the power is 40W, the culture temperature is 23+/-2 ℃, and the photoperiod is 16h illumination/8 h darkness; the wavelength of the white light is 410-690nm; the wavelength of the red light is 600-900nm, and the peak is 612nm; the blue light wavelength is 410-540nm, and the peak is 435nm.
Further, in the stage of obtaining the hypocotyl of the fraxinus mandshurica, the specific steps include: soaking the fraxinus mandshurica seeds in tap water for 48 hours, removing seed coats, and washing for 24 hours; sterilizing with 75% (v/v) alcohol in an ultra clean bench for 2min, and cleaning with sterile water once; sterilizing with 10% sodium hypochlorite for 15min, repeating twice, and cleaning with sterile water for three to five times; the zygotic embryos are removed and inoculated in WPM+20g/L sucrose+7 g/L agar (pH=5.8-6.0) medium and cultured in darkness for 5d. The embryo and radicle are excised by a scalpel, the hypocotyl is obtained, and the ultrasonic treatment is carried out for 90s.
Further, in the budding culture stage, WPM+1.0mg/L TDZ+3.0mg/L6-BA+30 g/L sucrose+7 g/L agar (pH=5.8-6.0) is used as a budding culture medium for 14d.
Further, in the elongation culture stage, WPM+0.025mg/L TDZ+1.0mg/L GA 3 +30g/L sucrose+7 g/L agar (ph=5.8-6.0) as elongation medium, blue light: culturing under red light=3:2 for 15d, and then culturing under white light.
Further, in the rooting culture stage, WPM+1mg/L IAA+1mg/L IBA+20g/L sucrose+7 g/L agar (pH=5.8-6.0) is used as a culture medium.
The invention also provides an application of the fraxinus mandshurica efficient regeneration method based on light quality regulation in fraxinus mandshurica tissue culture rapid propagation and genetic transformation.
Compared with the prior art, the invention has the following beneficial effects:
the high-efficiency fraxinus mandshurica regeneration method based on light quality regulation, provided by the invention, applies light quality treatment to the fraxinus mandshurica tissue culture rapid propagation process, and gives different light sources and combinations aiming at different stages of fraxinus mandshurica tissue culture bud regeneration, so that the method is strong in pertinence, and can be used for rapidly and effectively inducing fraxinus mandshurica hypocotyl to bud, elongate and root, thereby shortening the induction regeneration propagation period, improving the induction efficiency and ensuring the quality of tissue culture seedlings.
Drawings
FIG. 1 shows the effect of different light treatments on the budding of the hypocotyl of fraxinus mandshurica, the first, second and third rows being budding culture 3d, 5d and 11d respectively;
FIG. 2 shows the effect of different photoproduction on the gene expression associated with the regeneration of fraxinus mandshurica shoots;
FIG. 3 shows the effect of different light treatments on the elongation of the fraxinus mandshurica sprouts, the first, second and third rows being elongation cultures 3d, 9d and 12d respectively;
FIG. 4 is the effect of different photoproduction treatments on the expression of genes related to the elongation of the fraxinus mandshurica bud;
FIG. 5 shows the effect of different light treatments on the rooting of the fraxinus mandshurica, the first and second rows being rooting cultures 5d and 20d respectively;
FIG. 6 is the effect of different photoperiod treatments on the root number and root length of fraxinus mandshurica;
FIG. 7 shows the effect of different light treatments on the growth status of tissue culture seedlings of fraxinus mandshurica, the first and second rows being different light cultures 25d and 60d respectively; the method comprises the steps of carrying out a first treatment on the surface of the
FIG. 8 is the effect of different photoperiod treatments on the quality of tissue culture seedlings of fraxinus mandshurica, where a is leaf area, b is total chlorophyll content, c is chlorophyll a/b, d is carotenoid content, and e is plant height;
FIG. 9 is a culture flow chart of photo-based tissue culture regeneration of fraxinus mandshurica.
Detailed Description
For a better understanding of the present invention, reference will now be made in detail to the following examples and accompanying drawings, which are included to provide a further understanding of the invention, but it should be understood by those skilled in the art that the following examples are not intended to limit the scope of the invention and that any changes and modifications that would be made to the present invention are within the scope of the invention.
In the following examples, the experimental methods used are conventional methods unless otherwise specified.
Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.
Example 1: acquisition of hypocotyl of fraxinus mandshurica
The fraxinus mandshurica seeds are soaked in tap water for 48 hours, and are washed with tap water for 24 hours after the seed coats are removed. Sterilizing with 75% (v/v) alcohol in an ultra clean bench for 2min, and cleaning with sterile water once; sterilizing with 10% sodium hypochlorite for 15min, repeating twice, and cleaning with sterile water three to five times. The zygotic embryos are removed with sterile forceps and inoculated in WPM+20g/L sucrose+7 g/L agar (pH=5.8-6.0) medium and cultured in darkness for 5d. The embryo and radicle were excised with a sterile scalpel to obtain the hypocotyl, which was sonicated for 90s. Hypocotyls were inoculated in WPM medium (Table 1) of different ratios of TDZ and 6-BA, 30 hypocotyls were inoculated for each hormone combination, cultured under white light at 23.+ -. 2 ℃ with 16h light/8 h darkness, and adventitious bud induction rate was counted.
TABLE 1 type of adventitious bud regeneration induction Medium for fraxinus mandshurica
The results are shown in Table 2, the induction rate of adventitious buds is between 0.00% and 93.33%, wherein A1 cannot induce adventitious buds without any hormone; the highest induction rate of the adventitious buds of A5 and A8 is 93.33%, but the adventitious buds of A5 are easy to vitrify, and the adventitious buds of A8 have weaker growth vigor and are easy to brown; the inductances of A4 and A9 are 73.33%, but the A4 buds earlier and the growth condition in the subsequent culture is better. Therefore, the A4 combined culture medium is determined to be the optimal culture medium for adventitious bud regeneration of the fraxinus mandshurica.
TABLE 2 different media 7d budding Rate of the hypocotyl of fraxinus mandshurica
Example 2: influence of different lights on regeneration of adventitious buds of fraxinus mandshurica
Placing the hypocotyl of the fraxinus mandshurica in an A4 culture medium, wherein white light (W1), blue light (W2), red light (W3) and blue light are respectively obtained: red = 2:3 (W4), blue: red = 3:2 (W5), blue: red = 1:4 (W6) and blue: red = 4:1 (W7) seven different light quality combinations were incubated with different light quality parameters as shown in table 3. 30 hypocotyls were inoculated for each light source combination, the culture temperature was 23.+ -. 2 ℃, the photoperiod was 16h light/8 h dark, and the adventitious bud induction rate was counted.
TABLE 3 different light quality parameters
The induction of adventitious buds is shown in FIG. 1, table 4 and Table 5. 3d, the bud points of the groups W3, W4, W5 and W6 appear, and W1, W2 and W7 have no obvious change, which indicates that the red light has a promoting effect on the induction of the adventitious buds of the fraxinus mandshurica; 5d, bud points appear in W1-W7, the density of adventitious buds formed by W3, W4 and W6 is higher, the density of the adventitious buds of W1 is lowest, the length is shortest, the induction of high-density adventitious buds can be promoted by red light and blue light, and the induction effect of red light is better; part of adventitious buds of W2 and W7 turn red, and other groups of adventitious buds are all light green; the bud ratio of the W3 explant was 90.00% at 11d, the average bud number of each explant was 30.34, which is significantly higher than the other groups. Therefore, in the induction process of adventitious buds of the hypocotyl of the fraxinus mandshurica, the use of monochromatic red light as a light source helps to induce budding.
TABLE 4 hypocotyl induced budding status under different photoperiod
TABLE 5 hypocotyl induced budding rates with different photoperiod
Freezing and storing the samples at 3d, 5d, 7d and 9d respectively, extracting the water yeast Liu Cailiao RNA by using a CTAB method, synthesizing cDNA by using a treasured organism reverse transcription kit, diluting the cDNA 10 times, and then using the cDNA for fluorescence quantitative PCR (qRT-PCR), and designing specific quantitative primers aiming at the water yeast sprout regeneration genes FmWOX5, fmWUS and FmWIND1 (table 6), wherein FmTUB is the water yeast Liu Nacan gene. WUS maintains totipotency of cells in shoot apical meristems; WOX5 is specifically expressed in the tissue center of stem cells, and plays an important role in maintaining stem tip stem cell activity; the WIND1 gene is a key regulator of wound-induced cell reprogramming, and the transcriptional level expression level is shown in FIG. 2. FmWOX5, fmWUS and FmWIND1 are highest in expression level under the W3 treatment during sampling, wherein the expression level of WOX5 gene is highest under the 7d of the W3 treatment and is 9.88 times of that of the control; the WUS gene has the highest expression level in the 9d of W3 treatment and is 12.35 times of that of the control; the WIND1 gene was expressed in the highest amount at 9d of W3 treatment, 1.61 times that of the control. The results show that the expression quantity of the bud regeneration genes under each red and blue light quality is higher than that of white light, and the expression quantity under the irradiation of monochromatic red light is highest.
TABLE 6 real-time fluorescent quantitative primers for fraxinus mandshurica bud regeneration genes
Example 3: influence of different lights on the elongation of adventitious buds of fraxinus mandshurica
Transferring adventitious buds of fraxinus mandshurica obtained in the step of processing 14d with W3 to WPM+0.025mg/L TDZ+1.0mg/L GA 3 +30g/L sucrose+7 g/L agar (ph=5.8-6.0) elongation medium in white light (W1), blue light (W2), red light (W3), blue light, respectively: red = 2:3 (W4), blue: red = 3:2 (W5), blue: red = 1:4 (W6) and blue: red = 4:1 (W7) seven different light quality combinations. 15 explants were inoculated for each light source combination, and the culture temperature was 23.+ -. 2 ℃ and the photoperiod was 16h light/8 h dark, and the adventitious bud elongation and survival rate were counted (FIG. 3, table 7 and Table 8).
TABLE 7 elongation of adventitious buds under different light quality
TABLE 8 elongation of adventitious buds under different light quality
The results showed that the survival rate of the explants under W3 was 80% at the highest, but the elongation of adventitious buds was 13.33% lower, and more calli were formed. The adventitious buds under W1 and W5 have higher elongation, and the adventitious buds of W5 have faster elongation at the initial stage of light quality treatment, but have slower growth at the later stage; the initial elongation speed of the W1 adventitious bud is slower, but the adventitious bud grows at a constant speed. The W6 adventitious bud does not elongate, and the W2 adventitious bud grows weak and gradually turns yellow. Therefore, in the adventitious bud elongation culture stage of the fraxinus mandshurica, the W1 and the W5 light are combined, the fraxinus mandshurica is cultured under the W5 light at the initial stage of elongation, and the fraxinus mandshurica is transferred to the W1 light for continuous growth after about 15 days.
Sampling and freezing at 14d (when adventitious buds are transferred from induction culture to elongation culture), 18d (elongation culture 4 d), 22d (elongation culture 8 d) and 26d (elongation culture 12 d), respectively, extracting water yeast Liu Cailiao RNA by using a CTAB method, synthesizing cDNA by using a Baozhen reverse transcription kit, diluting 10 times, and then using the cDNA for fluorescence quantitative PCR (qRT-PCR), and designing specific quantitative primers for the adventitious bud elongation related genes FmPHV, fmWOX4 and FmCUC1 of the ash buds (Table 9), wherein FmTUB is the water yeast Liu Nacan gene. As a result, as shown in FIG. 4, the expression levels of FmPHV, fmWOX4 and FmCUC1 genes were increased during the bud elongation of 14-26d by using the initial expression levels of each gene of W1 as a control. At 26d, the FmPHV gene expression amount of W5 is 7.54 times that of the control, and the FmCUC1 gene expression amount is 13.52 times that of the control; WOX4 gene expression was 8.54 times that of the control. The result shows that red light can inhibit the elongation of the adventitious bud, blue light can promote the elongation of the adventitious bud, and the expression level of the bud elongation related gene is highest when the ratio of blue light to red light is 3:2.
Table 9 real-time fluorescent quantitative primers for fraxinus mandshurica bud regeneration genes
Example 4: influence of different lights on rooting of fraxinus mandshurica
Roots of tissue culture seedlings of fraxinus mandshurica with a seedling age of 15d are excised and transferred to a rooting medium of WPM+1mg/L IAA+1mg/L IBA+20g/L sucrose+7g/L agar (pH=5.8-6.0), and the rooting medium is respectively prepared in white light (W1), blue light (W2), red light (W3) and blue light: red = 2:3 (W4), blue: red = 3:2 (W5), blue: red = 1:4 (W6) and blue: red = 4:1 (W7) seven different light quality combinations. 15 seedlings were inoculated for each light source combination at a temperature of 23.+ -. 2 ℃ and photoperiod of 16h light/8 h dark, and the average root number and root length were counted after 20d (FIGS. 5, 6).
The result shows that each group starts to root on the fifth day of rooting culture, the W2 roots faster and the rooting number is more, and then the W5; the rooting of other light quality treatment groups is faster than that of the W1 light quality, which shows that red light and blue light can promote the rooting of the fraxinus mandshurica. At 20d, W2 has an average root number of at most 8.57, followed by W6 and W4 of 8.26 and 7.43, respectively; but the length of the W2 root is 0.99cm, which is far lower than 3.88cm of W4 and 3.45cm of W6; w1 has the longest average root length of 4.89cm, but has a smaller average root number of only 5.28. From the growth state of the root, the W1 root is stronger, and the color is light green; w3 is slender, and the diameters of W4 and W6 are slightly smaller than W1. The result shows that the red-blue light combination can promote the rooting of the fraxinus mandshurica, but single red light or blue light cannot meet the growth requirement of roots, and the rooting culture promoting effect of the fraxinus mandshurica is better when the ratio of the blue light to the red light is 2:3 and 1:4.
Example 5: influence of different lights on quality of tissue culture seedlings of fraxinus mandshurica
The aquatic yeast Liu Gezi embryos were grown in WPM medium with white light until distinct roots appeared (about 0.5 cm), and seedlings were transferred to Erlenmeyer flasks in white light (W1), blue light (W2), red light (W3), blue light, respectively: red = 2:3 (W4), blue: red = 3:2 (W5), blue: red = 1:4 (W6) and blue: red = 4:1 (W7) seven different light quality combinations. 10 seedlings are inoculated in each light quality combination, the culture temperature is 23+/-2 ℃, the photoperiod is 16h illumination/8 h darkness, and the plant height, leaf area and chlorophyll content of the tissue culture seedlings are counted after the culture.
The 25d state of the tissue culture Miao Shengchang of the fraxinus mandshurica under different light quality is shown in fig. 7, and obvious growth difference exists among the groups: the hypocotyls of W3 and W6 are obviously longer and stronger, and the root development of W4 and W6 is better. Measurement of leaf area found that W4, W5, W6 were larger, where W4 was 1.97 times W1, W5 was 1.61 times W1, and W7 was 1.72 times W1, i.e. blue-red light combination (blue light: red light=3:2, 2:3, 1:4) promoted an increase in leaf area of the fraxinus mandshurica tissue culture seedlings (fig. 8 a). Leaf photosynthetic pigments are measured, and the result shows that the total chlorophyll content of W4 and W7 is 1.22 times of that of W1; the total chlorophyll content of W2 and W3 is 0.92 times and 0.95 times of W1; the chlorophyll a/b values of W5 and W7 are significantly higher than those of other groups; namely blue light: red = 2:3, 4:1 combination can effectively promote chlorophyll synthesis, blue: the red = 3:2, 4:1 combination can significantly increase leaf chlorophyll a/b values (fig. 8b, c). The carotenoid content of W7 is 1.1 times of W1, and the carotenoid contents of W2 and W3 are 0.49 times and 0.39 times of W1, namely blue light: the red = 4:1 combination is effective in promoting carotenoid synthesis (figure 8 d).
Culturing until 60d, wherein the heights of the tissue culture seedlings are obviously different, and the heights of the tissue culture seedlings of W1, W2, W3, W4, W5, W6 and W7 are respectively 4.47cm, 1.69cm, 5.40cm, 4.46cm, 2.92cm, 4.84cm and 1.92cm; the W3 strain is highest and the W2 strain is lowest (fig. 8 e); and the W3 root system grows well, and the lateral root number is obviously increased (figure 7); that is, the single-color red light can promote the growth of the strain type of the tissue culture seedling of the fraxinus mandshurica, the single-color blue light can obviously inhibit the growth, and the red light with a certain proportion can partially inhibit the effect.
In conclusion, the sterile hypocotyl is obtained by culturing the water yeast Liu Gezi embryo 5d under the dark condition, and the generation of the adventitious buds of the hypocotyl is induced in a germination medium of WPM+1.0mg/L TDZ+3.0mg/L6-BA+30 g/L sucrose+7g/L agar (pH=5.8-6.0) under monochromatic red light; in blue: red = 3:2 light source, wpm+0.025mg/L tdz+1.0mg/L GA 3 Inducing adventitious bud elongation in +30g/L sucrose +7g/L agar (pH=5.8-6.0) elongation culture medium, white light continues to induce elongation after 15 d; in blue: red = 2:3 or blue: rooting is induced in a rooting medium with a light source of red light=1:4, WPM+1mg/L IAA+1mg/L IBA+20g/L sucrose+7 g/L agar (pH=5.8-6.0). The treatment of different light qualities and combinations of the LED light sources advances the appearance time of the hypocotyl bud point of the fraxinus mandshurica to 3 days, shortens the cultivation time of the bud stage to about 14 days, the cultivation time of the elongation stage to about 15 days and advances the rooting time to about 5 days.
Finally, it should be noted that: while the foregoing describes specific embodiments of the present invention, it will be appreciated by those skilled in the art that the specific embodiments described are illustrative only and not limiting of the scope of the invention, as modifications and variations may be made by those skilled in the art without departing from the principles of the invention, and such modifications and variations are to be regarded as being within the scope of the invention as claimed.
Claims (7)
1. A method for efficiently regenerating fraxinus mandshurica based on light quality regulation comprises the following steps:
(1) Obtaining the hypocotyl of fraxinus mandshurica;
(2) Budding culture;
(3) Elongation culture;
(4) Rooting culture;
the method is characterized in that the step (2) uses monochromatic red light as a light source, and the step (3) uses blue light: red light=3:2 and white light as light sources, step (4) uses blue light: red = 2:3 or blue: red = 1:4 is a light source.
2. The efficient fraxinus mandshurica regeneration method based on light quality control as in claim 1, wherein the LED lamp is used as an artificial light source, and the illumination intensity is 12 mu mol/m 2 S, the power is 40W, the culture temperature is 23+/-2 ℃, and the photoperiod is 16h illumination/8 h darkness; the wavelength of the white light is 410-690nm; the wavelength of the red light is 600-900nm, and the peak is 612nm; the blue light wavelength is 410-540nm, and the peak is 435nm.
3. The efficient fraxinus mandshurica regeneration method based on light quality control as in claim 1, wherein the step (1) specifically comprises: soaking the fraxinus mandshurica seeds in tap water for 48 hours, removing seed coats, and washing for 24 hours; sterilizing with 75% (v/v) alcohol in an ultra clean bench for 2min, and cleaning with sterile water once; sterilizing with 10% sodium hypochlorite for 15min, repeating twice, and cleaning with sterile water for three to five times; taking out the zygotic embryo, inoculating in a culture medium of WPM+20g/L sucrose+7g/L agar (pH=5.8-6.0), and culturing in darkness for 5d; the embryo and radicle are excised by a scalpel, the hypocotyl is obtained, and the ultrasonic treatment is carried out for 90s.
4. The efficient fraxinus mandshurica regeneration method based on light quality control as in claim 1, wherein the step (2) uses wpm+1.0mg/L tdz+3.0 mg/L6-ba+30 g/L sucrose+7 g/L agar (ph=5.8-6.0) as a germination medium, and cultures for 14d.
5. The efficient photo-quality control-based fraxinus mandshurica regeneration method according to claim 1, wherein the step (3) uses WPM+0.025mg/L TDZ+1.0mg/L GA 3 +30g/L sucrose+7 g/L agar (ph=5.8-6.0) as elongation medium, blue light: culturing under red light=3:2 for 15d, and then culturing under white light.
6. The efficient photo-quality control-based fraxinus mandshurica regeneration method according to claim 1, wherein the step (4) uses wpm+1mg/L iaa+1mg/L iba+20g/L sucrose+7 g/L agar (ph=5.8-6.0) as a culture medium.
7. The application of the photo-quality control-based fraxinus mandshurica efficient regeneration method in fraxinus mandshurica tissue culture rapid propagation and genetic transformation.
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