CN114287346A - Tissue culture and rapid propagation method for red peony roots - Google Patents
Tissue culture and rapid propagation method for red peony roots Download PDFInfo
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Abstract
The invention relates to a method for tissue culture and rapid propagation of red paeony root, which solves the problems of difficult rooting, pollution, browning and the like by sterilizing the surface of a peony explant, culturing callus and optimally adjusting the composition of culture mediums at different stages, and avoids development deformity caused by stimulation of high-concentration gibberellin by using lower-concentration gibberellin GA3 at a bud induction stage in the process of tissue culture and rapid propagation of the red paeony root; the tissue culture efficiency can be improved by replacing auxin and cytokinin in the proliferation culture stage, the growth of red peony tissue culture seedlings is promoted, the phenomena of browning and pollution are avoided, the browning problem existing in the red peony tissue culture process can be greatly improved by adding PVP, a good foundation is laid for the later differentiation of red peony callus and domestication of plants, the operation is simple, the cost is low, the market prospect is good, and good help can be provided for the breeding and growth of the peony.
Description
Technical Field
The invention belongs to the field of plant tissue culture, and particularly relates to a method for tissue culture and rapid propagation of red paeony root.
Background
The peony is a perennial herb of Paeonia of Ranunculaceae, is a famous ornamental and medicinal plant in China, is used as a medicine, and is divided into red peony and white peony root, wherein the red peony and the white peony root are used as medicines, and the red peony and the white peony root are good at clearing heat and cooling blood, eliminating stasis and relieving pain and clearing liver fire; the latter is good at nourishing blood, regulating menstruation, nourishing liver and relieving pain. The traditional breeding method of the Chinese herbaceous peony is a plant division method and a sowing method, but the traditional method has the defects of long period, low efficiency and low breeding coefficient, can not well keep the excellent properties of a female parent and seriously influences the large-scale production of the Chinese herbaceous peony.
Compared with the traditional propagation method, the tissue culture method can not only keep the original variety and excellent characters, enlarge the propagation coefficient and shorten the breeding period, but also can be operated annually without being influenced by seasons, greatly improves the propagation efficiency and is convenient for large-scale production. However, the problems of difficult rooting of tissue culture seedlings, easy bacterial or fungal pollution, serious browning and the like still exist in the tissue culture process of the peony at present, and the development of the peony tissue culture is severely restricted.
Therefore, the research on a new method for tissue culture and rapid propagation of red paeony root solves the technical problems of difficult rooting, pollution, browning and the like in the prior art, and is a technical problem which needs to be solved urgently at present.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a method for tissue culture and rapid propagation of red paeony root, which solves the problems of difficult rooting, pollution, browning and the like by adjusting the tissue culture and rapid propagation process of the red paeony root, lays a good foundation for the later differentiation of callus and domestication of plants of the red paeony root, has simple operation, low cost and good market prospect, and can provide good help for the breeding and growth of the red paeony root.
In order to realize the purpose, the invention adopts the following technical method, namely a method for tissue culture and rapid propagation of red paeony root, which comprises the following steps:
(1) obtaining explants
Selecting a stem section with buds of the peony, washing the stem section with buds clean with clear water, soaking the stem section with buds in 75 wt% of ethanol for 10-20s, taking out the stem section, soaking the stem section with buds in 0.8 wt% of mercuric chloride solution for 5-10min, and cutting the stem section with buds into small sections with the length of 2-3cm after taking out;
(2) preparation of callus
Placing the small section obtained in the step (1) in a germination culture medium for inducing germination of callus, wherein the germination culture medium takes 1/2MS culture medium as basal culture medium, and CaCl2 with the final concentration of 500-600mg/L, 6-BA with the final concentration of 0.5-1.5mg/L, NAA with the final concentration of 0.3-0.8mg/L, GA3 with the final concentration of 0.2-0.5mg/L, PVP with the final concentration of 0.5-1.5g/L, agar with the final concentration of 5-10g/L and glucose with the final concentration of 30-40g/L are additionally added; firstly, dark culture is carried out for 7-10 days at the temperature of 24-26 ℃, and then illumination culture is carried out for 23-28 days under the illumination culture conditions that the illumination time is 10-14h/d and the illumination intensity is 1300-;
(3) inducing adventitious bud
Cutting the callus obtained by induction in the step (2) from the explant, and placing the cut callus in a differentiation medium for adventitious bud differentiation, wherein the differentiation medium takes 1/2MS medium as a basic medium, and is added with CaCl2 with the final concentration of 500-600mg/L, 6-BA with the final concentration of 3-5mg/L, NAA with the final concentration of 0.3-0.8mg/L, GA3 with the final concentration of 0.1-0.2mg/L, agar with the final concentration of 5-10g/L and sucrose with the final concentration of 30-40 g/L; performing illumination culture for 25-30d, wherein the illumination culture conditions comprise illumination time of 10-14h/d and illumination intensity of 1300-1500 lux;
(4) proliferation culture
Cutting the adventitious bud obtained by induction in the step (3), and then placing the cut adventitious bud into a multiplication culture medium for multiplication subculture for 25-30d to obtain a rootless seedling, wherein the multiplication culture medium takes 1/2MS culture medium as a basic culture medium, and is added with CaCl2 with the final concentration of 500-600mg/L, TDZ with the final concentration of 3-5mg/L, IBA with the final concentration of 0.3-0.8mg/L, GA3 with the final concentration of 0.1-0.2mg/L, PVP with the final concentration of 0.5-1.5g/L, agar with the final concentration of 5-10g/L and sucrose with the final concentration of 30-40 g/L;
(5) root and seedling strengthening
Placing the rootless seedlings obtained in the step (4) into a rooting culture medium for culturing, wherein the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and is additionally added with 800mg/L CaCl2, 3-5mg/L TDZ, 0.3-0.8mg/L IBA, 0.1-0.2mg/L GA3, 0.5-1.5g/L PVP, 5-10g/L agar and 30-40g/L sucrose in final concentration;
(6) hardening off seedlings
Opening the bottle mouth virtual cover to harden the seedling for 3-5d when the adventitious root grows to be full of the bottle bottom and the whole plant grows steadily, and opening the bottle mouth for 3-5d after the tissue culture seedling is adapted, so that the tissue culture seedling is adapted to the external environment completely; pulling out the tissue culture seedling, and soaking the root in the rooting preparation for 20-30 min;
(7) transplanting
And (3) transplanting the tissue culture seedlings obtained in the step (6) into a plug tray, filling a small amount of water into the plug tray, and placing the plug tray in a greenhouse for growth, wherein the volume ratio of imported peat, perlite, vermiculite, biochar and fine sand is 6:1:1:3:2, the temperature in the greenhouse is 20 +/-2 ℃, and the humidity is 85 +/-5%.
Preferably, in the step (1), after the ethanol soaking or the mercuric chloride soaking is finished, the washing is carried out for 4 to 5 times by using sterile water;
preferably, in the step (2), the illumination is red light;
preferably, in the step (3), the illumination is performed by red light;
preferably, activated carbon with the weight of 0.5-1% of that of the rooting medium is added into the rooting medium in the step (5);
preferably, the rooting agent in the step (6) is a microorganism rooting promoter.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention solves the problems of difficult rooting, pollution, browning and the like by carrying out surface disinfection on the peony explant, callus culture and optimization and adjustment on the composition of culture mediums at different stages, lays a good foundation for the differentiation of the peony root callus and the domestication of plants, and has the advantages of simple operation, low cost and good market prospect. Can provide good help for the breeding and growth of the Chinese herbaceous peony.
(2) In the process of tissue culture and rapid propagation of red paeony root, the invention avoids developmental deformity caused by stimulation of high-concentration gibberellin by using lower-concentration gibberellin GA3 at the bud induction stage; meanwhile, the tissue culture efficiency can be improved by replacing auxin and cytokinin in the propagation culture stage, the growth of the radix paeoniae rubra tissue culture seedling is promoted, the phenomena of browning and pollution are avoided, and the browning problem in the radix paeoniae rubra tissue culture process can be greatly improved by adding PVP.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the present invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the present invention and is not intended to limit the scope of the claims which follow. All of the starting materials of the present invention, without particular limitation as to their source, may be purchased commercially or prepared according to conventional methods well known to those skilled in the art.
Example 1
A method for tissue culture and rapid propagation of radix paeoniae rubra comprises the following steps:
(1) obtaining explants
Selecting a stem section with buds of the Chinese herbaceous peony, washing the stem section with buds by clear water, soaking the stem section with the ethanol with 75 wt% for 20s, taking out the stem section, washing the stem section with sterile water for 4 times, soaking the stem section with mercuric chloride solution with 0.8 wt% for 5min, taking out the stem section with buds, and then, washing the stem section with the sterile water for 5 times, and cutting the stem section with buds into small sections with the length of 3 cm;
(2) preparation of callus
Placing the small section obtained in the step (1) in a germination culture medium for inducing germination of callus, wherein the germination culture medium takes 1/2MS culture medium as a basal culture medium, and CaCl2 with the final concentration of 600mg/L, 1.5 mg/L6-BA, 0.8mg/L NAA, 0.2mg/L GA3, 1.5g/L PVP, 10g/L agar and 40g/L glucose are additionally added; firstly, dark culture is carried out for 10 days at the temperature of 26 ℃, and then illumination culture is carried out for 28 days under the illumination culture condition that red light is used for illumination, the illumination time is 14h/d, and the illumination intensity is 1500 lux;
(3) inducing adventitious bud
Cutting the callus obtained by induction in the step (2) from an explant, and placing the cut callus in a differentiation medium for adventitious bud differentiation, wherein the differentiation medium takes 1/2MS medium as a basic medium, and CaCl2 with the final concentration of 550mg/L, 6-BA with the final concentration of 5mg/L, NAA with the final concentration of 0.3mg/L, GA3 with the final concentration of 0.1mg/L, agar with the final concentration of 10g/L and sucrose with the final concentration of 30g/L are added; carrying out illumination culture for 25-30d, wherein the illumination culture condition is that the illumination time is 14h/d and the illumination intensity is 1500 lux;
(4) proliferation culture
Cutting the adventitious bud obtained by induction in the step (3), and then placing the cut adventitious bud into a multiplication culture medium for multiplication subculture for 30d to obtain a rootless seedling, wherein the multiplication culture medium takes 1/2MS culture medium as a basic culture medium, and CaCl2 with the final concentration of 600mg/L, TDZ with the final concentration of 5mg/L, IBA with the final concentration of 0.5mg/L, GA3 with the final concentration of 0.1mg/L, PVP with the final concentration of 0.5g/L, agar with the final concentration of 8g/L and sucrose with the final concentration of 35g/L are added;
(5) root and seedling strengthening
Putting the rootless seedlings obtained in the step (4) into a rooting culture medium for culture, wherein the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and CaCl2 with the final concentration of 800mg/L, TDZ with the final concentration of 5mg/L, IBA with the final concentration of 0.3mg/L, GA3 with the final concentration of 0.1mg/L, PVP with the final concentration of 1.2g/L, agar with the final concentration of 9g/L and sucrose with the final concentration of 40g/L are additionally added; adding activated carbon accounting for 1 percent of the weight of the rooting culture medium into the rooting culture medium;
(6) hardening off seedlings
Opening the bottle mouth virtual cover to harden the seedling for 5d when the adventitious root grows to be full of the bottle bottom and the whole plant grows steadily, and opening the bottle mouth for 5d after the tissue culture seedling is adapted, so that the tissue culture seedling is adapted to the external environment completely; pulling out the tissue culture seedling, and soaking the root in the rooting preparation for 30 min;
(7) transplanting
And (3) transplanting the tissue culture seedlings obtained in the step (6) into a plug tray, filling a small amount of water into the plug tray, and placing the plug tray in a greenhouse for growth, wherein the volume ratio of imported peat, perlite, vermiculite, biochar and fine sand is 6:1:1:3:2, the temperature in the greenhouse is 20 +/-2 ℃, and the humidity is 85 +/-5%.
Example 2
A method for tissue culture and rapid propagation of radix paeoniae rubra comprises the following steps:
(1) obtaining explants
Selecting a stem section with buds of the Chinese herbaceous peony, washing the stem section with buds by clear water, soaking the stem section with 75 wt% of ethanol for 10s, taking out the stem section, washing the stem section with sterile water for 4 times, soaking the stem section with 0.8 wt% of mercuric chloride solution for 10min, taking out the stem section with buds, and cutting the stem section with buds into small sections with the length of 2cm by using the sterile water for 5 times;
(2) preparation of callus
Placing the small section obtained in the step (1) in a germination culture medium for inducing germination of callus, wherein the germination culture medium takes 1/2MS culture medium as a basal culture medium, and CaCl2 with the final concentration of 500mg/L, 0.8 mg/L6-BA, 0.5mg/L NAA, 0.2-0.5mg/L GA3, 1.5g/L PVP, 10g/L agar and 30g/L glucose are additionally added; firstly, dark culture is carried out for 7 days at the temperature of 26 ℃, and then illumination culture is carried out for 23 days under the illumination culture conditions that red light is selected for illumination, the illumination time is 10h/d, and the illumination intensity is 1500 lux;
(3) inducing adventitious bud
Cutting the callus obtained by induction in the step (2) from an explant, and placing the cut callus in a differentiation medium for adventitious bud differentiation, wherein the differentiation medium takes 1/2MS medium as a basic medium, and CaCl2 with the final concentration of 500mg/L, 6-BA with the final concentration of 3mg/L, NAA with the final concentration of 0.8mg/L, GA3 with the final concentration of 0.2mg/L, agar with the final concentration of 10g/L and sucrose with the final concentration of 40g/L are added; carrying out illumination culture for 25-30d, wherein the illumination culture condition is that red light is selected for illumination, the illumination time is 14h/d, and the illumination intensity is 1300 lux;
(4) proliferation culture
Cutting the adventitious bud obtained by induction in the step (3), and then placing the cut adventitious bud into a multiplication culture medium for multiplication subculture for 25d to obtain a rootless seedling, wherein the multiplication culture medium takes 1/2MS culture medium as a basic culture medium, and CaCl2 with the final concentration of 600mg/L, TDZ with the final concentration of 5mg/L, IBA with the final concentration of 0.8mg/L, GA3 with the final concentration of 0.1mg/L, PVP with the final concentration of 0.5g/L, agar with the final concentration of 10g/L and sucrose with the final concentration of 35g/L are added;
(5) root and seedling strengthening
Putting the rootless seedlings obtained in the step (4) into a rooting culture medium for culturing, wherein the rooting culture medium takes 1/2MS culture medium as a basal culture medium, and CaCl2 with the final concentration of 700mg/L, TDZ with the final concentration of 5mg/L, IBA with the final concentration of 0.8mg/L, GA3 with the final concentration of 0.2mg/L, PVP with the final concentration of 1.5g/L, agar with the final concentration of 5g/L and sucrose with the final concentration of 40g/L are additionally added; adding active carbon which accounts for 0.5 percent of the weight of the rooting culture medium into the rooting culture medium;
(6) hardening off seedlings
Opening the bottle mouth virtual cover to harden the seedling for 3d when the adventitious root grows to be full of the bottle bottom and the whole plant grows steadily, and opening the bottle mouth for 5d after the tissue culture seedling is adapted, so that the tissue culture seedling is adapted to the external environment completely; pulling out the tissue culture seedling, and soaking the root in the rooting preparation for 30 min; the rooting agent is a microorganism rooting promoting agent;
(7) transplanting
And (3) transplanting the tissue culture seedlings obtained in the step (6) into a plug tray, filling a small amount of water into the plug tray, and placing the plug tray in a greenhouse for growth, wherein the volume ratio of imported peat, perlite, vermiculite, biochar and fine sand is 6:1:1:3:2, the temperature in the greenhouse is 20 +/-2 ℃, and the humidity is 85 +/-5%.
Example 3
A method for tissue culture and rapid propagation of radix paeoniae rubra comprises the following steps:
(1) obtaining explants
Selecting a stem section with buds of the Chinese herbaceous peony, washing the stem section with buds by clear water, soaking the stem section with 75 wt% of ethanol for 15s, taking out the stem section, washing the stem section with sterile water for 5 times, soaking the stem section with 0.8 wt% of mercuric chloride solution for 5min, taking out the stem section, washing the stem section with the sterile water for 5 times, and cutting the stem section with buds into small sections with the length of 3 cm;
(2) preparation of callus
Placing the small section obtained in the step (1) in a germination culture medium for inducing germination of callus, wherein the germination culture medium takes 1/2MS culture medium as a basal culture medium, and CaCl2 with the final concentration of 500mg/L, 1.2 mg/L6-BA, 0.6mg/L NAA, 0.4mg/L GA3, 0.5g/L PVP, 10g/L agar and 40g/L glucose are additionally added; firstly, dark culture is carried out for 8 days at the temperature of 24 ℃, and then illumination culture is carried out for 26 days under the illumination culture conditions that red light illumination is selected, the illumination time is 13h/d, and the illumination intensity is 1400 lux;
(3) inducing adventitious bud
Cutting the callus obtained by induction in the step (2) from an explant, and placing the cut callus in a differentiation medium for adventitious bud differentiation, wherein the differentiation medium takes 1/2MS medium as a basic medium, and CaCl2 with the final concentration of 580mg/L, 6-BA with the final concentration of 5mg/L, NAA with the final concentration of 0.8mg/L, GA3 with the final concentration of 0.2mg/L, agar with the final concentration of 9g/L and sucrose with the final concentration of 40g/L are added; carrying out illumination culture for 25-30d, wherein the illumination culture condition is that red light is selected for illumination, the illumination time is 13h/d, and the illumination intensity is 1400 lux;
(4) proliferation culture
Cutting the adventitious bud obtained by induction in the step (3), and then placing the cut adventitious bud into a multiplication culture medium for multiplication subculture for 28d to obtain a rootless seedling, wherein the multiplication culture medium takes 1/2MS culture medium as a basic culture medium, and CaCl2 with the final concentration of 600mg/L, TDZ with the final concentration of 5mg/L, IBA with the final concentration of 0.8mg/L, GA3 with the final concentration of 0.1mg/L, PVP with the final concentration of 0.8g/L, agar with the final concentration of 7g/L and sucrose with the final concentration of 40g/L are added;
(5) root and seedling strengthening
Putting the rootless seedlings obtained in the step (4) into a rooting culture medium for culturing, wherein the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and CaCl2 with the final concentration of 800mg/L, TDZ with the final concentration of 5mg/L, IBA with the final concentration of 0.3mg/L, GA3 with the final concentration of 0.2mg/L, PVP with the final concentration of 0.5g/L, agar with the final concentration of 10g/L and cane sugar with the final concentration of 40g/L are additionally added; adding active carbon which accounts for 0.8 percent of the weight of the rooting culture medium into the rooting culture medium;
(6) hardening off seedlings
Opening the bottle mouth virtual cover to harden the seedling for 4d when the adventitious root grows to be full of the bottle bottom and the whole plant grows steadily, and opening the bottle mouth for 3d after the tissue culture seedling is adapted, so that the tissue culture seedling is adapted to the external environment completely; pulling out the tissue culture seedling, and soaking the root in the rooting preparation for 20 min;
(7) transplanting
And (3) transplanting the tissue culture seedlings obtained in the step (6) into a plug tray, filling a small amount of water into the plug tray, and placing the plug tray in a greenhouse for growth, wherein the volume ratio of imported peat, perlite, vermiculite, biochar and fine sand is 6:1:1:3:2, the temperature in the greenhouse is 20 +/-2 ℃, and the humidity is 85 +/-5%.
Example 4
A method for tissue culture and rapid propagation of radix paeoniae rubra comprises the following steps:
(1) obtaining explants
Selecting a stem section with buds of the Chinese herbaceous peony, washing the stem section with buds by clear water, soaking the stem section with the ethanol with 75 wt% for 20s, taking out the stem section, washing the stem section with sterile water for 4 times, soaking the stem section with mercuric chloride solution with 0.8 wt% for 8min, taking out the stem section with buds, and then soaking the stem section with the sterile water for 5 times, and cutting the stem section with buds into small sections with the length of 2 cm;
(2) preparation of callus
Placing the small section obtained in the step (1) in a germination culture medium for inducing germination of callus, wherein the germination culture medium takes 1/2MS culture medium as a basal culture medium, and CaCl2 with the final concentration of 580mg/L, 0.9 mg/L6-BA, 0.6mg/L NAA, 0.4mg/L GA3, 0.9g/L PVP, 8g/L agar and 33g/L glucose are additionally added; firstly, dark culture is carried out for 9d at the temperature of 25 ℃, and then illumination culture is carried out for 27d, wherein the illumination culture conditions are that red light is adopted for illumination, the illumination time is 14h/d, and the illumination intensity is 1500 lux;
(3) inducing adventitious bud
Cutting the callus obtained by induction in the step (2) from an explant, and placing the cut callus in a differentiation medium for adventitious bud differentiation, wherein the differentiation medium takes 1/2MS medium as a basic medium, and CaCl2 with the final concentration of 500mg/L, 6-BA with the final concentration of 5mg/L, NAA with the final concentration of 0.3mg/L, GA3 with the final concentration of 0.2mg/L, agar with the final concentration of 10g/L and sucrose with the final concentration of 30g/L are added; performing illumination culture for 30 days under the conditions of red light irradiation, wherein the illumination time is 10h/d and the illumination intensity is 1300 lux;
(4) proliferation culture
Cutting the adventitious bud obtained by induction in the step (3), and then placing the cut adventitious bud into a multiplication culture medium for multiplication subculture for 30d to obtain a rootless seedling, wherein the multiplication culture medium takes 1/2MS culture medium as a basic culture medium, and CaCl2 with the final concentration of 600mg/L, TDZ with the final concentration of 5mg/L, IBA with the final concentration of 0.3mg/L, GA3 with the final concentration of 0.2mg/L, PVP with the final concentration of 1.5g/L, agar with the final concentration of 10g/L and sucrose with the final concentration of 30g/L are added;
(5) root and seedling strengthening
Putting the rootless seedlings obtained in the step (4) into a rooting culture medium for culturing, wherein the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and adding CaCl2 with the final concentration of 600mg/L, TDZ with the final concentration of 5mg/L, IBA with the final concentration of 0.8mg/L, GA3 with the final concentration of 0.2mg/L, PVP with the final concentration of 1.5g/L, agar with the final concentration of 10g/L and sucrose with the final concentration of 30 g/L; adding activated carbon which accounts for 0.9 percent of the weight of the rooting culture medium into the rooting culture medium;
(6) hardening off seedlings
Opening the bottle mouth virtual cover to harden the seedling for 5d when the adventitious root grows to be full of the bottle bottom and the whole plant grows steadily, and opening the bottle mouth for 5d after the tissue culture seedling is adapted, so that the tissue culture seedling is adapted to the external environment completely; pulling out the tissue culture seedling, and soaking the root in the rooting preparation for 20-30 min;
(7) transplanting
And (3) transplanting the tissue culture seedlings obtained in the step (6) into a plug tray, filling a small amount of water into the plug tray, and placing the plug tray in a greenhouse for growth, wherein the volume ratio of imported peat, perlite, vermiculite, biochar and fine sand is 6:1:1:3:2, the temperature in the greenhouse is 20 +/-2 ℃, and the humidity is 85 +/-5%.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (6)
1. A method for tissue culture and rapid propagation of red paeony root is characterized in that: the method comprises the following steps:
(1) obtaining explants
Selecting a stem section with buds of the peony, washing the stem section with buds clean with clear water, soaking the stem section with buds in 75 wt% of ethanol for 10-20s, taking out the stem section, soaking the stem section with buds in 0.8 wt% of mercuric chloride solution for 5-10min, and cutting the stem section with buds into small sections with the length of 2-3cm after taking out;
(2) preparation of callus
Placing the small section obtained in the step (1) in a germination culture medium for inducing germination of callus, wherein the germination culture medium takes 1/2MS culture medium as basal culture medium, and CaCl2 with the final concentration of 500-600mg/L, 6-BA with the final concentration of 0.5-1.5mg/L, NAA with the final concentration of 0.3-0.8mg/L, GA3 with the final concentration of 0.2-0.5mg/L, PVP with the final concentration of 0.5-1.5g/L, agar with the final concentration of 5-10g/L and glucose with the final concentration of 30-40g/L are additionally added; firstly, dark culture is carried out for 7-10 days at the temperature of 24-26 ℃, and then illumination culture is carried out for 23-28 days under the illumination culture conditions that the illumination time is 10-14h/d and the illumination intensity is 1300-;
(3) inducing adventitious bud
Cutting the callus obtained by induction in the step (2) from the explant, and placing the cut callus in a differentiation medium for adventitious bud differentiation, wherein the differentiation medium takes 1/2MS medium as a basic medium, and is added with CaCl2 with the final concentration of 500-600mg/L, 6-BA with the final concentration of 3-5mg/L, NAA with the final concentration of 0.3-0.8mg/L, GA3 with the final concentration of 0.1-0.2mg/L, agar with the final concentration of 5-10g/L and sucrose with the final concentration of 30-40 g/L; performing illumination culture for 25-30d, wherein the illumination culture conditions comprise illumination time of 10-14h/d and illumination intensity of 1300-1500 lux;
(4) proliferation culture
Cutting the adventitious bud obtained by induction in the step (3), and then placing the cut adventitious bud into a multiplication culture medium for multiplication subculture for 25-30d to obtain a rootless seedling, wherein the multiplication culture medium takes 1/2MS culture medium as a basic culture medium, and is added with CaCl2 with the final concentration of 500-600mg/L, TDZ with the final concentration of 3-5mg/L, IBA with the final concentration of 0.3-0.8mg/L, GA3 with the final concentration of 0.1-0.2mg/L, PVP with the final concentration of 0.5-1.5g/L, agar with the final concentration of 5-10g/L and sucrose with the final concentration of 30-40 g/L;
(5) root and seedling strengthening
Placing the rootless seedlings obtained in the step (4) into a rooting culture medium for culturing, wherein the rooting culture medium takes 1/2MS culture medium as a basic culture medium, and is additionally added with 800mg/L CaCl2, 3-5mg/L TDZ, 0.3-0.8mg/L IBA, 0.1-0.2mg/L GA3, 0.5-1.5g/L PVP, 5-10g/L agar and 30-40g/L sucrose in final concentration;
(6) hardening off seedlings
Opening the bottle mouth virtual cover to harden the seedling for 3-5d when the adventitious root grows to be full of the bottle bottom and the whole plant grows steadily, and opening the bottle mouth for 3-5d after the tissue culture seedling is adapted, so that the tissue culture seedling is adapted to the external environment completely; pulling out the tissue culture seedling, and soaking the root in the rooting preparation for 20-30 min;
(7) transplanting
And (3) transplanting the tissue culture seedlings obtained in the step (6) into a plug tray, filling a small amount of water into the plug tray, and placing the plug tray in a greenhouse for growth, wherein the volume ratio of imported peat, perlite, vermiculite, biochar and fine sand is 6:1:1:3:2, the temperature in the greenhouse is 20 +/-2 ℃, and the humidity is 85 +/-5%.
2. The method for tissue culture and rapid propagation of red peony root according to claim 1, characterized in that: in the step (1), after ethanol soaking or mercury bichloride soaking is finished, the solution is washed for 4-5 times by using sterile water.
3. The method for tissue culture and rapid propagation of red peony root according to claim 1, characterized in that: in the step (2), red light is selected for illumination.
4. The method for tissue culture and rapid propagation of red peony root according to claim 1, characterized in that: in the step (3), red light is selected for illumination.
5. The method for tissue culture and rapid propagation of red peony root according to claim 1, characterized in that: and (3) adding activated carbon accounting for 0.5-1% of the weight of the rooting culture medium into the rooting culture medium in the step (5).
6. The method for tissue culture and rapid propagation of red peony root according to claim 1, characterized in that: the rooting preparation in the step (6) is a microorganism rooting promoter.
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