CN114982639A - Efficient breeding method for salvia miltiorrhiza tissue culture seedlings - Google Patents
Efficient breeding method for salvia miltiorrhiza tissue culture seedlings Download PDFInfo
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Abstract
The invention discloses a culture medium for high-efficiency breeding of salvia miltiorrhiza tissue culture seedlings and a method for high-efficiency breeding of salvia miltiorrhiza tissue culture seedlings. The invention improves the callus induction rate in the tissue culture seedling cultivation process of the salvia miltiorrhiza bunge, the optimized culture medium composition improves the regeneration frequency of adventitious buds and root systems and the salvia miltiorrhiza bunge propagation coefficient, reduces the tissue culture seedling cultivation time, and greatly meets the requirement of the efficient propagation of the salvia miltiorrhiza bunge. The method has great popularization potential.
Description
Technical Field
The invention relates to the field of plant tissue culture, in particular to a method for efficiently breeding tissue culture seedlings of salvia miltiorrhiza.
Background
The Salvia miltiorrhiza is dry roots and rhizomes of Salvia miltiorrhiza bge of Salvia of labiate, is a medicine for promoting blood circulation and removing blood stasis, is mainly used for treating various symptoms such as pain, sadness, abdominal mass, sore, ulcer, carbuncle, swelling, palpitation and insomnia and premature ejaculation treatment cost caused by blood stasis, and has a long history of nearly 2000 years by utilizing the Salvia miltiorrhiza in China. According to statistics, more than one hundred Chinese patent medicines using salvia miltiorrhiza as raw materials exist. Pharmacological and clinical researches show that the salvia miltiorrhiza has very obvious effects on blood sweat duct systems, blood systems, digestive systems and the like.
At present, in the domestic salvia tissue culture and breeding, explants such as naturally growing leaves and the like are mainly used for directly inducing callus and then differentiating and sprouting to establish an indirect regeneration system, and the explants have browning and high mortality rate, and callus induction rate and proliferation coefficient are low; meanwhile, the tissue culture breeding mostly adopts a sterile solid culture medium to induce the root system of the regeneration seedling, the root system induction time is long, the seedling hardening and transplanting are still needed after the root system induction, the production cost is high, the transplanting survival rate is low, and the large-scale propagation and popularization of the tissue culture seedling are influenced.
Therefore, the method solves the problems of explant browning and death in the tissue culture process of the salvia miltiorrhiza bunge, improves the callus, adventitious bud and root induction rate of the salvia miltiorrhiza bunge and the transplanting survival rate, is a key for optimizing the efficient propagation of the salvia miltiorrhiza bunge, and has important significance for the industrial production and breeding of the salvia miltiorrhiza bunge.
Patent CN107094630A discloses a salvia miltiorrhiza tissue culture method, which obtains 100% of bud differentiation rate of salvia miltiorrhiza callus by a single hormone, and makes regeneration vigorous from bud growth, but realizes high-efficiency breeding of salvia miltiorrhiza tissue culture seedlings, so that the salvia miltiorrhiza tissue culture seedlings can be produced industrially, besides callus bud differentiation rate, single callus induction coefficient, rooting rate and transplanting survival rate are also key factors, thus it is not suitable for practical production and application; CN110178728A discloses a tissue culture and rapid propagation method of Salvia miltiorrhiza Bunge, which uses Salvia miltiorrhiza Bunge root for tissue culture, and although the rooting rate is 100%, the Salvia miltiorrhiza Bunge needs to be disinfected by carbendazim, ethanol and mercuric chloride for many times, and then undergoes bud induction and strong seedling culture for about 55 days, the formed tissue culture seedling is required to be subjected to complicated seedling hardening in the following, and then can be transplanted, the production cost is high, and the method is not suitable for industrialized production and propagation of Salvia miltiorrhiza Bunge.
Disclosure of Invention
In order to solve the problems, the invention provides a culture medium for the efficient breeding of salvia miltiorrhiza tissue culture seedlings, which comprises a callus induction culture medium, a bud regeneration induction culture medium and a root system induction water culture nutrient solution;
the callus induction culture medium comprises the following components: MS culture medium, which is added with plant growth regulator with final concentration of 2-4 mg/L, 8-10 g/L agar and 20-40 g/L sucrose;
the components of the bud regeneration induction culture medium comprise: adding a plant growth regulator with the final concentration of 0-2 mg/L, 8-12 g/L agar and 10-30 g/L sucrose into an MS culture medium;
the root system induction water culture solution comprises the following components: 1/2MS culture medium is added with plant growth regulator with final concentration of 0.1-3 mg/L and bactericide of 2-4 g/L.
Further, the plant growth regulator is any one or more of 6-benzylpurine, alpha-naphthylacetic acid and thidiazuron; the bactericide is carbendazim.
Still further, the callus induction medium comprises the following components: adding thidiazuron with the final concentration of 2-3 mg/L, alpha-naphthylacetic acid with the final concentration of 0.1-0.15 mg/L, agar with the final concentration of 9.5g/L and cane sugar with the final concentration of 30g/L into an MS culture medium;
the components of the bud regeneration induction culture medium comprise: adding 6-benzylpurine (6-BA) with the final concentration of 0-0.6 mg/L, alpha-naphthylacetic acid (NAA) with the final concentration of 0-0.5 mg/L, agar with the final concentration of 10g/L and cane sugar with the final concentration of 20g/L into an MS culture medium;
the root system induced water culture nutrient solution comprises the following components: 1/2MS culture medium, added with alpha-naphthylacetic acid (NAA) with final concentration of 0.25-2 mg/L, 3g/L carbendazim.
Further, the radix salviae miltiorrhizae is salvia miltiorrhiza bunge.
The invention also provides a method for efficiently breeding the salvia miltiorrhiza tissue culture seedlings, which utilizes the culture medium for cultivation and comprises the following specific steps:
a. cutting Saviae Miltiorrhizae radix seedling, inoculating into callus induction culture medium, and culturing to obtain Saviae Miltiorrhizae radix callus;
b. b, taking the salvia miltiorrhiza callus obtained in the step a, shearing, inoculating into a bud regeneration induction culture medium, and culturing to obtain salvia miltiorrhiza regeneration cluster buds;
c. and c, dividing the red sage root regenerated cluster buds obtained in the step b into single plants, fixing the single plants in a fixed matrix, and placing the single plants in a root system induction culture medium for culture to obtain red sage root regenerated seedlings.
The salvia miltiorrhiza seedling in the step a is a petiole or a leaf of a healthy sterile salvia miltiorrhiza seedling plant; the petioles are cut into sections with the length of 1-2 cm, and the blades are cut into crushed leaves with the length of 1 multiplied by 1 cm; the culture conditions are as follows: the temperature is 20-22 ℃, and the illumination period is as follows: 24h dark, time 21 d.
Further, the salvia miltiorrhiza callus tissues in the step b are sheared into blocks with the diameter of 1-2 cm; the culture conditions are as follows: the temperature is 20-22 ℃, the illumination period is 16h (light)/8 h (dark), and the time is as follows: culturing to 3-4 cm high.
Further, in the step c, the fixing matrix is prepared from the following components in a mass ratio of 1: 1-5 parts of perlite and vermiculite.
Further, the fixing matrix is prepared from the following components in a mass ratio of 1: 1 perlite and vermiculite.
Further, the culture conditions are: the temperature is 22 +/-1 ℃, the humidity is 70-90%, the illumination period is 18h (light)/6 h (dark), the illumination intensity is 2500-3000 Lux, the root system induction culture medium is replaced once every 3 days, the water culture nutrient solution is kept in contact with a fixed matrix, indoor clean ventilation is kept, and the culture is carried out until the root length is 6-8 cm and the seedling height is 7-8 cm.
The 1/2MS culture medium is a culture medium with half of macroelements in the MS culture medium formula and unchanged content of other components.
The MS culture medium is one of the most common culture media in plant tissues, and the formula of the MS culture medium comprises the following components:
compared with the prior art, the invention has the following remarkable effects:
1) sterile petioles or leaves are adopted for callus induction, compared with the method of directly selecting plant tissues in the natural environment, the method can cancel the explant disinfection process in the conventional tissue culture process, simplify the operation procedure, avoid the explant damage caused by disinfection treatment and improve the survival rate of the explant;
2) the callus induction culture medium composition and culture conditions can avoid material browning and death, and the induction rate of the salvia miltiorrhiza bunge callus is improved to more than 85%, compared with the prior art, the induction rate of the callus is improved by more than 30%;
3) the regeneration induction culture medium can obviously improve the regeneration frequency of the salvia miltiorrhiza bunge buds, the number of the adventitious buds of a single callus can reach 8.5-11.2, and compared with the prior art, the propagation rate is improved by more than 2 times;
4) compared with the prior art, the invention provides the root system induction mode, the rooting rate can reach more than 85 percent, and the transplanting survival rate can reach more than 88 percent;
5) the invention omits the complicated aseptic operation of the traditional tissue culture root system induction stage, simplifies the steps of root system induction and aseptic seedling domestication, and the improved water culture environment also provides good environmental conditions for the adaptation and transition of the tissue culture seedling from the aseptic environment to the natural environment.
The efficient breeding method of the tissue culture seedlings of the salvia miltiorrhiza bunge improves the callus induction rate in the tissue culture seedling culture process of the salvia miltiorrhiza bunge, improves the regeneration frequency of adventitious buds and root systems and the propagation coefficient of the salvia miltiorrhiza bunge by the optimized culture medium composition, reduces the tissue culture seedling culture time, and greatly meets the demand of efficient breeding of the salvia miltiorrhiza bunge. The method has great popularization potential.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Drawings
FIG. 1 schematic representation of callus growth
FIG. 2 is a schematic diagram of adventitious bud induction (red arrow indicates adventitious bud obtained by induction)
FIG. 3 schematic diagram of hydroponics
FIG. 4 is a schematic diagram of the effect of hydroponic growth and root induction
FIG. 5 is a schematic view of a seedling raising box for hydroponics
Detailed Description
The raw materials, reagents and equipment used in the embodiment of the invention are all known products; may be obtained by commercial purchase.
Example 1 efficient breeding of Salvia miltiorrhiza
1) Material selection and treatment: selecting stems of robust aseptic seedlings of salvia miltiorrhiza bunge, and shearing the stems into 1cm long stem sections in an aseptic environment for later use;
2) callus induction: and (3) aseptically inoculating the petiole segments processed in the step (1) in a super clean workbench until the components are as follows: MS culture medium, 2mg/L Thidiazuron (TDZ), 0.1mg/L alpha-naphthylacetic acid (NAA), 9.5g/L agar and 30g/L sucrose culture medium to induce callus, then placing the inoculated culture medium in a constant temperature clean culture room for 24h dark culture, keeping the temperature of the culture room at 22 +/-1 ℃, and culturing for 21d to obtain yellow-green leaf stalk callus of the Sichuan salvia miltiorrhiza (figure 1);
3) regeneration and induction of buds: and (3) cutting the salvia miltiorrhiza bunge callus obtained in the step (2) into blocks with the diameter of 1-2 cm under the aseptic condition, and then inoculating the cut blocks to the medium-density salvia miltiorrhiza bunge callus containing the following components: carrying out bud induction culture on an MS culture medium, a culture medium of 0.25 mg/L6-benzylpurine (6-BA), 0.1mg/L alpha-naphthylacetic acid (NAA), 10g/L agar and 20g/L sucrose, keeping the temperature of a culture room at 22 +/-1 ℃, illuminating at 3000Lux and illuminating for 16h (light)/8 h (dark) in an illuminating period, thus obtaining a large amount of regenerated cluster buds of the leaf stalk calluses of the salvia miltiorrhiza bunge through culture (figure 2), and dividing the regenerated cluster buds into single plants in a sterile operating platform for later use when the cluster buds grow to be 3-4 cm high;
4) inducing a hydroponic root system: adding a water culture nutrient solution containing 1/2MS culture medium, 0.25mg/L alpha-naphthylacetic acid (NAA) and 3g/L carbendazim into a seedling raising box, placing a seedling raising hole tray containing a fixed matrix into the water culture nutrient solution, inoculating the regenerated single bud obtained in the step 3) into the fixed matrix for fixed culture (figure 3), keeping the temperature of a culture room at 22 +/-1 ℃, the illumination intensity at 3000Lux, the illumination period at 16h (light)/8 h (dark), and the humidity at 80%; after rooting for 30 days, obtaining the tissue culture seedling which has the root length of 6-8 cm and the seedling height of 7-8 cm and can be used for transplanting (as shown in figure 4);
wherein the seedling raising box (figure 5) is a plastic box body with the length of 36.5cm, the width of 23.0cm and the height of 11.0cm, the plastic box body is provided with a vent cover, a seedling raising hole tray is arranged in the container, the seedling raising hole tray is a plastic plate with the width of 20-30cm, the length of 30-40cm and the thickness of 0.5cm, the size of the plastic plate just covers the bottom of the cuttage container, and small seedling raising holes with the length, the width and the height of 5 multiplied by 10cm are arranged on the seedling raising hole tray and are used for seedling raising and fixing.
Example 2 efficient breeding of Salvia miltiorrhiza
1) Material selection and treatment: selecting stems of robust aseptic seedlings of salvia miltiorrhiza bunge, and shearing the stems into 1.5cm long stem sections in an aseptic environment for later use;
2) callus induction: and (3) aseptically inoculating the petiole segments processed in the step (1) in a super clean workbench until the components are as follows: performing callus induction on an MS culture medium, namely 3mg/L Thidiazuron (TDZ), 0.15mg/L alpha-naphthylacetic acid (NAA), 9.5g/L agar and 30g/L sucrose, placing the inoculated culture medium in a constant-temperature clean culture room, performing dark culture for 24 hours, keeping the temperature of the culture room at 22 +/-1 ℃, and performing culture for 21 days to obtain yellow-green callus of the salvia miltiorrhiza bunge;
3) and (3) bud regeneration induction: cutting the callus of the salvia miltiorrhiza bunge obtained in the step 2) into blocks with the diameter of 1-2 cm under the aseptic condition, and then inoculating the cut blocks to the mixture with the following components: carrying out bud induction culture on an MS culture medium, a culture medium of 0.5 mg/L6-benzylpurine (6-BA), 0.25mg/L alpha-naphthylacetic acid (NAA), 10g/L agar and 20g/L sucrose, keeping the temperature of a culture room at 22 +/-1 ℃, the illumination intensity at 3000Lux and the illumination period at 16h (light)/8 h (dark), thus obtaining a large amount of regenerated cluster buds of the salvia miltiorrhiza bunge through culture, and dividing the regenerated cluster buds into single plants in a sterile operating platform for later use when the cluster buds grow to be 3-4 cm high;
4) inducing a hydroponic root system: adding a water culture nutrient solution containing 1/2MS culture medium with the final concentration of 1mg/L alpha-naphthylacetic acid (NAA) and 3g/L carbendazim into a seedling raising box, placing a seedling raising hole tray containing a fixed matrix into the water culture nutrient solution, inoculating the regenerated single bud obtained in the step 3) into the fixed matrix for fixed culture, keeping the temperature of a culture room at 22 +/-1 ℃, the illumination intensity at 3000Lux, the illumination period at 16h (light)/8 h (dark), and the humidity at 80%; after rooting for 30 days, obtaining the tissue culture seedling with the root length of 6-8 cm and the seedling height of 7-8 cm and capable of being used for transplanting,
the nursery box was the same as in example 1.
Example 3 efficient breeding of Salvia miltiorrhiza
1) Material selection and treatment: selecting leaves of healthy and sterile seedlings of salvia miltiorrhiza bunge, and shearing the leaves into square crushed leaves of 1cm multiplied by 1cm in a sterile environment for later use;
2) callus induction: aseptically inoculating the crushed leaves processed in the step 1 in an ultra-clean workbench until the components are as follows: MS culture medium, 2mg/L Thidiazuron (TDZ), 0.1mg/L alpha-naphthylacetic acid (NAA), 9.5g/L agar and 30g/L sucrose culture medium are used for callus induction, then the inoculated culture medium is placed in a constant-temperature clean culture room for 24h dark culture, the temperature of the culture room is kept at 22 +/-1 ℃, and a large amount of yellow-green leaf callus is generated on the edge of the leaf after 21d (figure 1);
3) regeneration and induction of buds: and (3) cutting the salvia miltiorrhiza bunge callus obtained in the step (2) into blocks with the diameter of 1-2 cm under the aseptic condition, and then inoculating the cut blocks to the medium-density salvia miltiorrhiza bunge callus containing the following components: carrying out bud induction culture on an MS culture medium, a culture medium of 0.25 mg/L6-benzylpurine (6-BA), 0.25mg/L alpha-naphthylacetic acid (NAA), 10g/L agar and 20g/L sucrose, keeping the temperature of a culture room at 22 +/-1 ℃, the illumination intensity at 3000Lux and the illumination period at 16h (light)/8 h (dark), thus obtaining a large amount of regenerated cluster buds of salvia miltiorrhiza leaves callus (figure 2), and dividing the regenerated cluster buds into single plants in a sterile operation platform for later use when the cluster buds grow to be 3-4 cm high;
4) inducing a hydroponic root system: adding 1/2MS culture medium with the liquid level being 5cm high, alpha-naphthylacetic acid (NAA) with the final concentration being 1.5mg/L and water culture nutrient solution of carbendazim with the final concentration being 3g/L into a seedling raising box, placing a seedling raising hole tray containing a fixed matrix into the water culture nutrient solution, then inoculating the regenerated single bud obtained in the step 3 into the fixed matrix for fixed culture, keeping the temperature of a culture room at 22 +/-1 ℃, the illumination intensity of 3000Lux, the illumination period of 16h (light)/8 h (dark), and the humidity of 80%; after rooting for 30 days, obtaining the tissue culture seedlings which have the root length of 6-8 cm and the seedling height of 7-8 cm and can be used for transplanting;
the seedling raising box was the same as in example 1.
Example 4 efficient Breeding of Salvia miltiorrhiza
1) Material selection and treatment: selecting leaves of healthy and sterile seedlings of salvia miltiorrhiza bunge, and shearing the leaves into 1 in a sterile environment for later use, namely 1cm × 1cm square crushed leaves;
2) callus induction: aseptically inoculating the crushed leaves processed in the step 1 in an ultra-clean workbench until the components are as follows: MS culture medium, 3mg/L Thidiazuron (TDZ), 0.15mg/L alpha-naphthylacetic acid (NAA), 9.5g/L agar and 30g/L sucrose culture medium are used for callus induction, then the inoculated culture medium is placed in a constant-temperature clean culture room for 24h dark culture, the temperature of the culture room is kept at 22 +/-1 ℃, and a large amount of yellow-green callus is generated on the edge of the leaf after 21 d;
3) and (3) regeneration and induction of buds: cutting the salvia miltiorrhiza bunge callus obtained in the step 2 into blocks with the diameter of 1-2 cm under an aseptic condition, and then inoculating the cut blocks to the culture medium with the following components: carrying out bud induction culture on an MS culture medium, a culture medium of 0 mg/L6-benzylpurine (6-BA), 0.5mg/L alpha-naphthylacetic acid (NAA), 10g/L agar and 20g/L sucrose, keeping the temperature of a culture room at 22 +/-1 ℃, the illumination intensity at 3000Lux and the illumination period at 16h (light)/8 h (dark), and then culturing to obtain a large amount of regenerated cluster buds of the salvia miltiorrhiza bunge, and dividing the regenerated cluster buds into single plants for later use in a sterile operating platform when the cluster buds grow to be 3-4 cm high;
4) inducing a hydroponic root system: adding 1/2MS culture medium with the liquid level being 5cm high, alpha-naphthylacetic acid (NAA) with the final concentration being 2mg/L and water culture nutrient solution of carbendazim with the final concentration being 3g/L into a seedling raising box, placing a seedling raising hole tray containing a fixed matrix into the water culture nutrient solution, then inoculating the regenerated single bud obtained in the step 3 into the fixed matrix for fixed culture, keeping the temperature of a culture room at 22 +/-1 ℃, the illumination intensity of 3000Lux, the illumination period of 16h (light)/8 h (dark), and the humidity of 80%; and rooting for 30 days to obtain the tissue culture seedling which has the root length of 6-8 cm and the seedling height of 7-8 cm and can be used for transplanting.
The nursery box was the same as in example 1.
The following test examples illustrate the advantageous effects of the present invention:
experimental example 1 tissue culture, propagation and culture results of the present invention
(first) culture results of example 1
60 sterile stems of the Sichuan salvia miltiorrhiza which are 1-2 cm long are selected to be subjected to callus induction according to the embodiment 1, 53 yellow green healthy Sichuan salvia miltiorrhiza callus tissues are obtained by culturing in an induction culture medium for 21 days, the callus induction rate is 88.33 percent, and the callus induction rate is improved by 30 percent compared with that of the traditional material taking. The salvia miltiorrhiza callus is subjected to bud regeneration induction culture in a bud induction culture medium for 45 days to obtain salvia miltiorrhiza cluster buds, and the multiplication coefficient of the single callus culture can reach 8.5 on average.
After the plants are separated, the root system of 24 Sichuan salvia seedlings is induced in a water culture solution, the root induction rate is up to 95 percent, and the root system is diverged. Finally, after 30 days of cultivation, the survival rate of the tissue culture seedlings of the salvia miltiorrhiza bunge reaches 93 percent.
(II) culture results of example 2
Selecting 60 sterile stems of the salvia miltiorrhiza bunge with the length of 1-2 cm, carrying out callus induction according to the example 2, and culturing in an induction culture medium for 21 days to obtain 56 yellow-green healthy salvia miltiorrhiza bunge callus, wherein the callus induction rate is 93.33 percent and is 32 percent higher than that of the traditional material. The salvia miltiorrhiza callus is subjected to 45-day bud regeneration induction culture in a bud induction culture medium to obtain salvia miltiorrhiza cluster buds, and the multiplication coefficient of the single callus culture can reach 9.2 on average.
After the plants are separated, the root system of 24 Sichuan salvia seedlings is induced in a water culture solution, the root induction rate is up to 96.83%, and the root system is diverged. Finally, after 30 days of cultivation, the survival rate of the tissue culture seedlings of the salvia miltiorrhiza bunge reaches 95 percent.
(III) culture results of example 3
60 pieces of Sichuan salvia root sterile leaves with the size of 1 multiplied by 1cm are selected to carry out callus induction according to the embodiment 3, 57 yellow green healthy Sichuan salvia root callus tissues are obtained by culturing in an induction culture medium for 21 days, the callus induction rate is 95.00 percent, and the callus induction rate is improved by 35 percent compared with the traditional material taking. The salvia miltiorrhiza callus is subjected to bud regeneration induction culture in a bud induction culture medium for 45 days to obtain salvia miltiorrhiza cluster buds, and the multiplication coefficient of the single callus culture can reach 11.5 on average.
After the plants are separated, the root system of the salvia miltiorrhiza bunge seedlings in the hydroponic culture solution is induced, the root induction rate is as high as 95 percent, and the root system is diverged. Finally, after 30 days of cultivation, the survival rate of the tissue culture seedlings of the salvia miltiorrhiza bunge reaches 94 percent.
(fourth) culture results of example 4
60 pieces of Sichuan salvia root sterile leaves with the size of 1 multiplied by 1cm are selected to carry out callus induction according to the embodiment 4, 58 yellow green healthy Sichuan salvia root callus tissues are obtained by culturing in an induction culture medium for 21 days, the callus induction rate is 96.66 percent, and the callus induction rate is improved by 36 percent compared with the traditional material. The salvia miltiorrhiza callus is subjected to bud regeneration induction culture in a bud induction culture medium for 45 days to obtain salvia miltiorrhiza cluster buds, and the multiplication coefficient of the single callus culture can reach 10.8 on average.
After the plants are separated, the root system of 24 Sichuan salvia seedlings is induced in a water culture solution, the root induction rate is up to 98 percent, and the root system is diverged. Finally, after 30 days of cultivation, the survival rate of the tissue culture seedlings of the salvia miltiorrhiza bunge reaches 94 percent.
In conclusion, the method for efficiently breeding the tissue culture seedlings of the salvia miltiorrhiza bunge improves the callus induction rate in the culture process of the tissue culture seedlings of the salvia miltiorrhiza bunge, improves the regeneration frequency of adventitious buds and root systems and the propagation coefficient of the salvia miltiorrhiza bunge by the optimized culture medium composition, reduces the tissue culture seedling culture time, and greatly meets the requirement of efficiently breeding the salvia miltiorrhiza bunge. The method has great popularization potential.
Claims (10)
1. A culture medium for efficient breeding of tissue culture seedlings of salvia miltiorrhiza is characterized in that: the method comprises a callus induction culture medium, a bud regeneration induction culture medium and a root induction hydroponic nutrient solution;
the callus induction culture medium comprises the following components: adding a plant growth regulator with the final concentration of 2-4 mg/L, 8-10 g/L agar and 20-40 g/L sucrose into an MS culture medium;
the components of the bud regeneration induction culture medium comprise: adding a plant growth regulator with the final concentration of 0-2 mg/L, 8-12 g/L agar and 10-30 g/L sucrose into an MS culture medium;
the root system induction water culture solution comprises the following components: 1/2MS culture medium is added with plant growth regulator with final concentration of 0.1-3 mg/L and bactericide of 2-4 g/L.
2. The culture medium according to claim 1, wherein: the plant growth regulator is any one or more of 6-benzyl purine, alpha-naphthylacetic acid and thidiazuron; the bactericide is carbendazim.
3. The culture medium according to claim 2, wherein: the callus induction culture medium comprises the following components: adding thidiazuron with the final concentration of 2-3 mg/L, alpha-naphthylacetic acid with the final concentration of 0.1-0.15 mg/L, agar with the final concentration of 9.5g/L and cane sugar with the final concentration of 30g/L into an MS culture medium;
the components of the bud regeneration induction culture medium comprise: adding 6-benzylpurine (6-BA) with the final concentration of 0-0.6 mg/L, alpha-naphthylacetic acid (NAA) with the final concentration of 0-0.5 mg/L, agar with the final concentration of 10g/L and cane sugar with the final concentration of 20g/L into an MS culture medium;
the root system induced water culture nutrient solution comprises the following components: 1/2 the MS culture medium is added with alpha-naphthylacetic acid (NAA) with final concentration of 0.25-2 mg/L and carbendazim with final concentration of 3 g/L.
4. The culture medium according to any one of claims 1 to 3, wherein: the Saviae Miltiorrhizae radix is radix Salviae Miltiorrhizae.
5. An efficient breeding method of salvia miltiorrhiza tissue culture seedlings is characterized by comprising the following steps: the method is characterized by utilizing the culture medium of any one of claims 1-4 for cultivation, and comprises the following specific steps:
a. cutting Saviae Miltiorrhizae radix seedling, inoculating into callus induction culture medium, and culturing to obtain Saviae Miltiorrhizae radix callus;
b. b, taking the salvia miltiorrhiza callus obtained in the step a, shearing, inoculating into a bud regeneration induction culture medium, and culturing to obtain salvia miltiorrhiza regeneration cluster buds;
c. and c, dividing the red sage root regenerated cluster buds obtained in the step b into single plants, fixing the single plants in a fixed matrix, and placing the single plants in a root system induction culture medium for culture to obtain red sage root regenerated seedlings.
6. The efficient salvia miltiorrhiza breeding method according to claim 5, wherein the efficient salvia miltiorrhiza breeding method comprises the following steps: the salvia miltiorrhiza seedling in the step a is a petiole or a leaf of a robust sterile salvia miltiorrhiza seedling plant; the petioles are cut into segments with the length of 1-2 cm, and the blades are cut into broken leaves with the length of 1 multiplied by 1 cm; the culture conditions are as follows: the temperature is 20-22 ℃, and the illumination period is as follows: 24h dark, time 21 d.
7. The efficient salvia miltiorrhiza breeding method according to claim 5, wherein the efficient salvia miltiorrhiza breeding method comprises the following steps: b, shearing the salvia miltiorrhiza callus into blocks with the diameter of 1-2 cm; the culture conditions are as follows: the temperature is 20-22 ℃, the illumination period is 16h (light)/8 h (dark), and the time is as follows: culturing to 3-4 cm high.
8. The efficient salvia miltiorrhiza breeding method according to claim 5, wherein the efficient salvia miltiorrhiza method is characterized in that: step c, the volume ratio of the fixed matrix is 1: 1-5 parts of perlite and vermiculite.
9. The efficient salvia miltiorrhiza breeding method according to claim 8, wherein the efficient salvia miltiorrhiza breeding method comprises the following steps: the fixed matrix is prepared from the following components in a volume ratio of 1: 1 perlite and vermiculite.
10. The efficient salvia miltiorrhiza breeding method according to claim 5, wherein the efficient salvia miltiorrhiza breeding method comprises the following steps: the culture conditions in the step c are as follows: the temperature is 22 +/-1 ℃, the humidity is 70-90%, the illumination period is 18h (light)/6 h (dark), the illumination intensity is 2500-3000 Lux, the root system induction culture medium is replaced once every 3 days, the water culture nutrient solution is kept in contact with a fixed matrix, indoor clean ventilation is kept, and the culture is carried out until the root length is 6-8 cm and the seedling height is 7-8 cm.
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