CN114009342B - Tissue culture and rapid propagation medium and tissue culture and rapid propagation method for viola tinctoria bamboo - Google Patents
Tissue culture and rapid propagation medium and tissue culture and rapid propagation method for viola tinctoria bamboo Download PDFInfo
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- CN114009342B CN114009342B CN202111553293.2A CN202111553293A CN114009342B CN 114009342 B CN114009342 B CN 114009342B CN 202111553293 A CN202111553293 A CN 202111553293A CN 114009342 B CN114009342 B CN 114009342B
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- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a violin silk bamboo tissue culture rapid propagation culture medium, which comprises: the culture medium comprises an induction culture medium, a proliferation culture medium and a rooting culture medium, wherein the induction culture medium comprises an MS minimal medium; the following components were included in the induction medium at the following concentrations: 25-35 g/L of sugar and 3-4 g/L of agar; the proliferation culture medium comprises an MS minimal medium and a plant growth regulator; the proliferation medium included the following components in concentrations: 0.03-3 mg/L, TDZ 0.001.001-0.5 mg/L of 6-BA, 25-35 g/L of sugar and 3-4 g/L of agar; the rooting culture medium comprises an MS minimal medium and a plant growth regulator; the root medium included the following components at the following concentrations: 0.01-0.1 mg/L of NAA, 0.1-3.0 mg/L of 6-BA, 25-35 g/L of sugar and 3-4 g/L of agar. The invention obtains the important technical effects of rapid propagation and stable rooting of the bamboos, and achieves the purposes of high propagation coefficient, stable technology and realization of industrial production.
Description
Technical Field
The invention relates to the technical field of tissue culture, in particular to a tissue culture and rapid propagation medium and a tissue culture and rapid propagation method for violin bamboo.
Background
The tissue culture research of bamboo is started in the sixties of the last century, and dozens of kinds of bamboo can obtain complete test-tube plantlet plants in a short time through tissue culture so far. However, the tissue culture technology of bamboo is difficult to tissue culture compared with other gramineous plants because the related adventitious bud proliferation and rooting links are particularly easy to brown, wither and die, and the formulas of the culture medium used in each culture link have larger difference due to different bamboo species, so that the tissue culture technology of bamboo is not widely applied to actual seedling breeding production.
Bamboo filament of Xiaoqin (Bambusa multiplex f. alphonso-karri) Is a bamboo of family of Gramineae (B)ambusa Retz. corr. Schreber) The plants and underground stems are clustered, the stems of the new bamboos are light red when sprouting, are yellow after ripening, are accompanied by green stripes with different widths and lengths, have high ornamental value, are usually planted in courtyards, green roads, parks, lakesides and the like for ornamental, belong to rare ornamental bamboos, and have wide market application prospect.
The traditional viola bamboo breeding mainly adopts a parent bamboo division or cuttage mode, needs large-area land resources, has low survival rate and is limited by seasons, and has the problems of low speed, high cost, low land utilization rate, incapability of quickly and inexpensively meeting the industrialization requirement of bamboo seedlings. Leading to the failure to meet the requirements of the bamboo seedling market on the bamboo seedling of the violin silk. The tissue culture rapid propagation technology is utilized to carry out mass and rapid propagation on the viola bamboo, but the tissue culture rapid propagation technology of mature viola bamboo is not available at present.
Disclosure of Invention
The invention aims to solve the technical problems that the traditional viola tinctoria bamboo breeding mainly adopts a parent bamboo division or cuttage mode, the survival rate is not high, the breeding is limited by seasons, and the problems of low speed, high cost, incapability of quickly solving the industrialization requirement of bamboo seedlings and low cost exist. Aims to provide a tissue culture and rapid propagation method of violin bamboo to solve the problems.
The invention is realized by the following technical scheme:
a violin bamboo tissue culture rapid propagation culture medium comprises: an induction medium, a proliferation medium and a rooting medium, wherein,
the induction medium comprises an MS minimal medium; the following concentrations of components were included in the induction medium: 25-35 g/L of sugar and 3-4 g/L of agar;
the proliferation culture medium comprises an MS minimal medium and a plant growth regulator; the proliferation medium included the following components in concentrations: 0.03-3 mg/L, TDZ 0.001.001-0.5 mg/L of 6-BA, 25-35 g/L of sugar and 3-4 g/L of agar;
the rooting culture medium comprises an MS minimal medium and a plant growth regulator; the rooting medium comprises the following components in concentration: 0.01-0.1 mg/L of NAA, 0.1-3.0 mg/L of 6-BA, 25-35 g/L of sugar and 3-4 g/L of agar.
Further, the induction medium comprises MS minimal medium; the following concentrations of components were included in the induction medium: sugar 30g/L and agar 3.5 g/L; the pH was 5.8.
Further, the multiplication medium comprises an MS minimal medium and a plant growth regulator; the proliferation medium included the following components in concentrations: 6-BA3mg/L, TDZ 0.05.05 mg/L, sugar 30g/L and agar 3.5 g/L; the pH was 5.8.
Further, the rooting medium comprises an MS minimal medium and a plant growth regulator; the rooting medium comprises the following components in concentration: NAA0.1mg/L, 6-BA0.3mg/L, sugar 30g/L and agar 3.5 g/L; the pH was 5.8.
A tissue culture and rapid propagation method of violin bamboo comprises the following steps of:
(1) selecting and processing explants: selecting the internode bud part of the current-year branch as a test material, and cleaning and disinfecting the test material;
(2) and (3) induction culture: inoculating the cleaned and disinfected internode bud parts of the current-year branches into an induction culture medium for induction culture for 7-15 days;
(3) and (3) proliferation culture: inoculating the well-grown buds obtained by culture into a multiplication culture medium for adventitious bud multiplication culture for 14-21 days;
(4) rooting culture: dividing cluster buds obtained by induced proliferation culture into 2-3 clusters, and inoculating the clusters into a rooting culture medium for induced rooting for 14-21 days;
(5) transplanting and domesticating: after the test-tube plantlet is subjected to rooting culture, when the root grows to 1.5-3cm, the test-tube plantlet is placed in a domestication room for hardening for 7d, and then is transplanted into a culture medium for culturing for 15d, so that a regeneration plant is obtained.
Further, the cleaning and disinfecting method in the step (1) is as follows: washing the cut explants with detergent for 1 time, slightly brushing internode stains with a fine hair brush after washing, putting the washed explants in a beaker, washing the washed explants with clean water for 2 hours, disinfecting the washed explants with 75% alcohol for 30 seconds on a superclean workbench, then disinfecting the washed explants with 10 times of sodium hypochlorite solution for 4 to 8 minutes, taking out the explants after slight yellowing, and washing the explants with sterile water for 5 times.
Further, the conditions of the induction culture in the step (2) are as follows: temperature: dark culture for the first 24 hours at 24 +/-2 ℃, and illumination intensity of subsequent culture: 1400-1600lx, the light period is 16 h/d.
Further, the culture conditions of the proliferation culture in the step (3) are as follows: temperature: 24. + -.2 ℃ and light intensity: 2400-2600lx, the photoperiod is 16 h/d.
Further, the culture conditions of the rooting culture in the step (4) are as follows: temperature: 24. + -.2 ℃ and light intensity: 2400-2600lx, the photoperiod is 16 h/d.
Further, the matrix in the step (5) is prepared by mixing peat soil and perlite according to the volume ratio of 3: 7.
Further, the plant regeneration in step (5) is carried out under the following culture conditions: temperature: 26 ± 2 ℃, light intensity: 3900-.
The method takes internode bud parts of branches growing in the current year as test materials, screens various culture mediums through the influence of different growth regulating substances on the steps of induction, proliferation, rooting and the like of adventitious buds to obtain the optimal culture medium and the optimal culture condition, and realizes the factory rapid breeding of seedlings through the tissue culture of the rapid propagation Hemikania violacea by the method.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1. the invention provides a tissue culture and rapid propagation medium and a tissue culture and rapid propagation method for violin bamboo, which take an MS culture medium as a base, and are added with plant growth regulators: 6-benzyl adenosine (6-BA), Thidiazuron (TDZ) and naphthylacetic acid (NAA) are used for tissue culture of two key stages of induction proliferation and rooting of the adventitious bud of the Piano silk bamboo. The invention realizes the industrial seedling raising of the violin bamboo, solves the important technical links of the rapid propagation and the stable rooting of the violin bamboo, and achieves the purposes of high propagation coefficient, stable technology and realization of industrial production.
2. The tissue culture and rapid propagation medium and the tissue culture and rapid propagation method for the violin bamboo provided by the invention can be carried out under the condition of manual control, are not limited by factors such as seasons, lands and the like, and strictly control the quality of seedlings; the individual difference of the tissue culture seedlings is small, the production period is short, the equipment is simple, the occupied area is small, the labor, the material resources and the like can be saved, and the industrialized production is facilitated; the tissue culture of the Piano silkworms can quickly obtain new plants, thereby reducing the damage to natural resources.
Drawings
In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the drawings that are required to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and that for those skilled in the art, other related drawings can be obtained from these drawings without inventive effort. In the drawings:
FIG. 1 is a bamboo picture of a Piano silk cultivated by the present invention.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.
The tissue culture and rapid propagation method of the violin bamboo comprises the following steps:
(1) selecting and processing explants: and (3) selecting an internode bud-bearing part of the current-year branch, wherein the upper part of the internode is reserved for 0.5cm, and the lower part of the internode is reserved for 1.0 cm. Washing the cut explants with detergent for 1 time, slightly brushing internode stains with a fine hair brush after washing, putting the washed explants in a beaker, washing the washed explants with clean water for 2 hours, disinfecting the washed explants with 75% alcohol on a superclean workbench for 30 seconds, then disinfecting the washed explants with 10 times of sodium hypochlorite solution for 4 minutes, 6 minutes and 8 minutes respectively, taking out the explants after slight yellowing, and washing the explants with sterile water for 5-6 times.
Tests show that when the disinfection time is 8 minutes, the contamination rate and the withering rate of the explant are the lowest, and the survival ratio of the sterile explant reaches more than 60%.
(2) And (3) induction culture: and (3) sucking the surface moisture of the internode bud part of the disinfected current-year branch by using sterile absorbent paper, inoculating the sterilized internode bud part to an induction culture medium, carrying out dark culture for 24 hours, and then carrying out culture for 7-15 days in an environment with illumination intensity of 1500lx, temperature of 24 ℃ and illumination time of 16 h/d.
The induction medium was based on MS medium with pH of 5.8, and a combination of sucrose (25, 30, 35 g/L) and agar (3, 3.5, 4 g/L) was added.
Through tests: when the concentration of sucrose is 30g/L and the concentration of agar is 3.5g/L, the induction rate is 98% at the highest, and the growth is the best.
Among these, MS medium is common knowledge known to those skilled in the art.
(3) And (3) proliferation culture: and (3) inoculating the explant which is obtained in the step (2) and has good growth to a proliferation culture medium for proliferation culture, wherein the illumination intensity is 2500lx, the illumination time is 16h/d, and the explant is cultured at 25 ℃ for 14-21 days, so that the bud proliferation and germination are promoted.
The proliferation culture medium is based on MS culture medium with pH of 5.8, and plant growth regulator 6-BA, TDZ, sugar and agar are added. Setting the concentration of 6-BA as 0.03mg/L, 0.1mg/L, 0.3mg/L, 1mg/L and 3 mg/L; the concentration of the plant growth regulator TDZ is 0.001mg/L, 0.01mg/L, 0.05mg/L, 0.1mg/L and 0.5 mg/L; plant growth regulator 6-BA (1, 2, 3 mg/L) and TDZ (0.01, 0.05 mg/L); the sugar concentration is 30 g/L; the agar concentration was 3.5 g/L.
Tests show that in the concentration range, the average number of the proliferated buds is between 2.38 and 4.80, and the average bud length is between 1.80 and 2.96 cm. When 6-BA3mg/L + TDZ0.05mg/L, the cluster buds are the most, the average multiplication bud number reaches 4.8, the average bud length reaches 1.95cm, the buds are strong, the leaves are green, and the leaves are spread.
(4) Rooting culture: dividing the cluster buds obtained in the step (3) into 2-3 clusters, inoculating the clusters into a rooting culture medium to induce rooting, and culturing at 24 ℃ for 14-21 days under the illumination intensity of 2500lx and the illumination time of 16h/d to induce the cluster buds to root.
The rooting culture medium is based on MS culture medium with pH value of 5.8, and plant growth regulators NAA and 6-BA, sugar and agar are added. Setting the combination of plant growth regulator NAA (0.01, 0.1 mg/L) and 6-BA (0.1, 0.3, 1, 3 mg/L); the sugar concentration is 30 g/L; the agar concentration was 3.5 g/L.
Tests show that the rooting rate is between 0 and 100 percent in the concentration range, and when NAA is 0.1mg/L and 6-BA is 0.3mg/L, the rooting rate is the highest and reaches 100 percent for rooting.
(5) Transplanting and domesticating: after rooting culture, when the root length reaches 1.5-3cm, placing the test-tube seedling in a domestication room for acclimatization, transplanting the test-tube seedling into a planting cup with the diameter of 12cm after 7 days, wherein the matrix mainly comprises peat soil and perlite, the volume ratio is 3:7, the temperature is controlled to be 25-28 ℃, bagging is carried out for keeping humidity, the illumination intensity is 4000lx, after 15 days, new roots are generated, the survival rate reaches more than 95%, the growth vigor of the survival bamboo seedling is better and consistent, and then the survival bamboo seedling can be transplanted to a field for planting or can be used for preparing potted plants.
Example 1
The tissue culture and rapid propagation method of the violin bamboo comprises the following steps:
(1) selecting and processing plant materials: and (3) selecting an internode bud-bearing part of the current-year branch, wherein the upper part of the internode is reserved for 0.5cm, and the lower part of the internode is reserved for 1.0 cm. Washing the cut explants with detergent for 1 time, slightly brushing internode stains with a fine hair brush after washing, putting the washed explants in a beaker, washing the washed explants with clear water for 2 hours, disinfecting the washed explants with 75% alcohol for 30 seconds on a superclean workbench, then disinfecting the washed explants with 10 times of sodium hypochlorite for 8 minutes, taking out the explants after slight yellowing, and washing the explants with sterile water for 5-6 times.
(2) And (3) induction culture: and (3) sucking the surface moisture of the internode bud part of the disinfected current-year branch by using sterile absorbent paper, inoculating the sterilized internode bud part to an induction culture medium, carrying out dark culture for 24 hours, and then carrying out culture for 7-15 days in an environment with illumination intensity of 1500lx, temperature of 24 ℃ and illumination time of 16 h/d.
The induction culture medium is based on MS culture medium with pH value of 5.8, and is added with 30g/L of sucrose and 3.5g/L of agar. The induction rate is 98% at the highest, and the growth vigor is the best.
(3) And (3) proliferation culture: and (3) inoculating the sterile well-grown buds obtained in the step (2) to a multiplication culture medium for multiplication culture, wherein the illumination intensity is 2500lx, the illumination time is 16h/d, and the buds are cultured for 14-21 days at 24 ℃ to induce the buds to multiply and germinate.
The proliferation culture medium is based on MS culture medium with pH of 5.8, and is added with plant growth regulator 6-BA3mg/L + TDZ0.05mg/L, sugar with concentration of 30g/L and agar with concentration of 3.5 g/L. The average number of the proliferated buds reaches 4.8, the average bud length reaches 1.95cm, the buds are strong, the leaves are green, and the leaves are spread.
(4) Rooting culture: dividing the cluster buds obtained in the step (3) into 2-3 clusters, inoculating the clusters into a rooting culture medium to induce rooting, and culturing at 24 ℃ for 14-21 days under the illumination intensity of 2500lx and the illumination time of 16h/d to induce the cluster buds to root.
The rooting culture medium is based on MS culture medium with pH value of 5.8, and plant growth regulator NAA0.1mg/L + 6-BA0.3mg/L, sugar with concentration of 30g/L and agar with concentration of 3.5g/L are added. The rooting rate reaches 100 percent.
(5) Transplanting and domesticating: after rooting culture is carried out on test-tube seedlings, when the root length reaches 2cm, the test-tube seedlings are placed in a domestication room for acclimatization, the test-tube seedlings are transplanted into a planting cup with the diameter of 12cm after 7 days, a matrix mainly comprises peat soil and perlite, the volume ratio is 3:7, the temperature is controlled to be 25-28 ℃, the humidity is maintained by bagging, the illumination intensity is 4000lx, after 15 days, new roots are generated, the survival rate reaches more than 95%, the growth vigor of the survival bamboo seedlings is good and consistent, and then the survival bamboo seedlings can be transplanted to a field for planting or can be used for preparing potted plants.
Example 2
The tissue culture and rapid propagation method of the violin bamboo comprises the following steps:
(1) selecting and processing plant materials: and (3) selecting an internode bud-bearing part of the current-year branch, wherein the upper part of the internode is reserved for 0.5cm, and the lower part of the internode is reserved for 1.0 cm. Washing the cut explants with detergent for 1 time, after washing, lightly scrubbing internode stains with a fine hair brush, putting the cleaned explants in a beaker, washing the cleaned explants with clear water for 2 hours, disinfecting the cleaned explants with 75% alcohol for 30 seconds on a superclean bench, then disinfecting the cleaned explants with 10 times of sodium hypochlorite solution for 8 minutes, taking out the cleaned explants after the explants are slightly yellow, and washing the cleaned explants with sterile water for 5 times.
(2) And (3) induction culture: and (3) sucking the surface moisture of the internode bud part of the disinfected current-year branch by using sterile absorbent paper, inoculating the sterilized internode bud part to an induction culture medium, carrying out dark culture for 24 hours, and then carrying out culture for 7-15 days in an environment with illumination intensity of 1500lx, temperature of 24 ℃ and illumination time of 16 h/d.
The induction culture medium is based on MS culture medium with pH value of 5.8, and is added with sucrose 25g/L and agar 3 g/L. The induction rate is 90% at most, and the growth vigor is better.
(3) And (3) proliferation culture: and (3) inoculating the sterile well-grown buds obtained in the step (2) to a multiplication culture medium for multiplication culture, wherein the illumination intensity is 2500lx, the illumination time is 16h/d, and the buds are cultured for 14-21 days at 24 ℃ to induce the buds to multiply and germinate.
The proliferation culture medium is based on MS culture medium with pH of 5.8, and is added with plant growth regulator 6-BA1mg/L + TDZ0.05mg/L, sugar with concentration of 30g/L and agar with concentration of 3.5 g/L. The average number of the proliferated buds reaches 4.5, the average bud length reaches 1.80cm, the leaves are green, and the leaves are spread.
(4) Rooting culture: dividing the cluster buds obtained in the step (3) into 2-3 clusters, inoculating the clusters into a rooting culture medium to induce rooting, and culturing at 24 ℃ for 14-21 days under the illumination intensity of 2500lx and the illumination time of 16h/d to induce the cluster buds to root.
The rooting culture medium is based on MS culture medium with pH value of 5.8, and plant growth regulator NAA 0.01mg/L + 6-BA0.3mg/L, sugar 30g/L and agar 3.5g/L are added. The rooting rate is 80%.
(5) Transplanting and domesticating: after rooting culture, when the root length reaches 3cm, placing the test-tube seedling in a domestication room for acclimatization, transplanting the test-tube seedling into a planting cup with the diameter of 12cm after 7d, wherein the matrix mainly comprises peat soil and perlite, the volume ratio is 3:7, the temperature is controlled to be 25-28 ℃, bagging is carried out for keeping humidity, the illumination intensity is 4000lx, after 15d, new roots are generated, the survival rate reaches more than 90%, the survival rate of the survival bamboo seedling is better and consistent, and then the survival bamboo seedling can be transplanted to a field for planting or used for preparing potted plants.
The violin bamboo cultured in this example is shown in fig. 1: a is an explant; b is explant culture seedling; c is a proliferation test-tube plantlet; d is rooting test-tube plantlet; e is the transplanted test-tube plantlet (15 d, 30d, 60 d); f, pot culture and matching planting after 30d transplanting; g, transplanting for 60d, and then pot culture and matching planting.
The above-mentioned embodiments are intended to illustrate the objects, technical solutions and advantages of the present invention in further detail, and it should be understood that the above-mentioned embodiments are merely exemplary embodiments of the present invention, and are not intended to limit the scope of the present invention, and any modifications, equivalent substitutions, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. A violin bamboo tissue culture rapid propagation medium is characterized by comprising: an induction medium, a proliferation medium and a rooting medium, wherein,
the induction culture medium is an MS basic culture medium, 25-35 g/L sugar and 3-4 g/L agar;
the enrichment culture medium is an MS minimal medium, 1-3 mg/L, TDZ 0.05.05 mg/L of 6-BA, 25-35 g/L of sugar and 3-4 g/L of agar;
the rooting culture medium is an MS minimal medium, 0.01-0.1 mg/L of NAA, 0.1-0.3 mg/L of 6-BA, 25-35 g/L of sugar and 3-4 g/L of agar.
2. The violin bamboo tissue culture rapid propagation medium according to claim 1, wherein the induction medium is MS minimal medium, sugar 30g/L and agar 3.5 g/L.
3. The violin bamboo tissue culture rapid propagation medium as claimed in claim 1, wherein the propagation medium is MS minimal medium, 6-BA3mg/L, TDZ0.05mg/L, sugar 30g/L and agar 3.5 g/L.
4. The violin bamboo tissue culture rapid propagation medium according to claim 1, wherein the rooting medium is MS minimal medium, NAA0.1mg/L, 6-BA0.3mg/L, sugar 30g/L and agar 3.5 g/L.
5. A tissue culture and rapid propagation method of Piano bamboo based on any one of claims 1 to 4, characterized by comprising the following steps:
(1) selecting and processing explants: selecting an internode bud part of the current-year branch as a test material, and cleaning and disinfecting the test material;
(2) and (3) induction culture: inoculating the cleaned and disinfected internode bud parts of the current-year branches into an induction culture medium for induction culture for 7-15 days;
(3) and (3) proliferation culture: inoculating the well-grown buds obtained by culture into a multiplication culture medium for adventitious bud multiplication culture for 14-21 days;
(4) rooting culture: dividing cluster buds obtained by propagation culture into 2-3 clusters, inoculating the clusters into a rooting culture medium, and inducing and rooting for 14-21 days;
(5) transplanting and domesticating: after the test-tube plantlet is subjected to rooting culture, when the root grows to 1.5-3cm, the test-tube plantlet is placed in a domestication room for hardening for 7d, and then is transplanted into a culture medium for culturing for 15d, so that a regeneration plant is obtained.
6. The tissue culture and rapid propagation method of bamboo as claimed in claim 5, wherein the cleaning and sterilizing method in step (1) is: washing the cut explants with detergent for 1 time, slightly brushing internode stains with a fine hair brush after washing, putting the washed explants in a beaker, washing the washed explants with clean water for 2 hours, disinfecting the washed explants with 75% alcohol for 30 seconds on a superclean workbench, then disinfecting the washed explants with 10 times of sodium hypochlorite solution for 4 to 8 minutes, taking out the explants after slight yellowing, and washing the explants with sterile water for 5 times.
7. The method for tissue culture and rapid propagation of Piano silk bamboo according to claim 5, wherein the conditions of the induction culture in step (2) are as follows: temperature: dark culture for the first 24 hours at 24 +/-2 ℃, and illumination intensity of subsequent culture: 1400-1600lx, the light period is 16 h/d.
8. The method for tissue culture and rapid propagation of bamboo shoots as claimed in claim 5, wherein the culture conditions of the propagation culture in step (3) are as follows: temperature: 24. + -.2 ℃ and light intensity: 2400-2600lx, the light period is 16 h/d.
9. The viola excelsa tissue culture rapid propagation method according to claim 5, wherein the culture conditions of the rooting culture in the step (4) are as follows: temperature: 24. + -.2 ℃ and light intensity: 2400-2600lx, the photoperiod is 16 h/d.
10. The tissue culture and rapid propagation method of bamboo shoots as claimed in claim 5, wherein in step (5), the substrate is prepared by mixing peat soil and perlite according to a volume ratio of 3: 7; the culture conditions for plant regeneration in the step (5) are as follows: temperature: 26 ± 2 ℃, light intensity: 3900-.
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