CN112450075A - Tissue culture and rapid propagation method for hypocotyl of Qinghai rhubarb - Google Patents
Tissue culture and rapid propagation method for hypocotyl of Qinghai rhubarb Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
- A01G24/10—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material
- A01G24/12—Growth substrates; Culture media; Apparatus or methods therefor based on or containing inorganic material containing soil minerals
- A01G24/15—Calcined rock, e.g. perlite, vermiculite or clay aggregates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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Abstract
The invention discloses a tissue culture and rapid propagation method of hypocotyl of Qinghai rhubarb, which comprises the following steps: (1) sterilizing the explant; (2) screening a primary culture medium; (3) inducing hypocotyl callus; (4) culturing hypocotyl callus in a proliferation manner; (5) differentiation of hypocotyl callus buds; (6) rooting culture; (7) transplanting tissue culture seedlings; the invention takes high-quality Qinghai rhubarb seeds as explants, and leads the callus induction rate of hypocotyl to reach 90 percent, the callus multiplication coefficient to reach 4.3 and the tissue culture seedling transplanting survival rate to reach 95 percent through sterilization treatment, primary culture, callus induction, callus multiplication culture, callus bud differentiation, rooting culture and tissue culture seedling transplanting.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and rapid propagation method of the hypocotyl of the Qinghai rhubarb.
Background
Rhubarb (Rheum palmatum L.) is a perennial herb of Rheum genus in Polygonaceae family, is a traditional Chinese herbal medicine in China, and has extremely high medicinal value. Dried roots and rhizome of rhubarb, bitter and cold in nature and wide in drug effect, and the efficacy of the rhubarb, namely purgation and accumulation elimination, heat clearing and fire purging, blood cooling and detoxification, stasis removal and meridian dredging, dampness removing and jaundice treating, is described in the 2015 edition of Chinese pharmacopoeia. Ancient times, rhubarb is usually taken by plaster or water decoction, and the rhubarb prescription has obvious efficacy in treating clinical common diseases such as pediatrics, surgery, gynecology and the like and acute, dangerous, severe and difficult diseases at present, and mainly comprises constipation, gastrointestinal stagnation, damp-heat diarrhea and dysentery, conjunctival congestion, carbuncle swelling and furuncle, water and fire scald, blood circulation promotion and stasis removal, damp-heat jaundice, acute pancreatitis and the like.
The Qinghai rhubarb is a high-quality variety of Tanggute rhubarb, and has the limitations of long period, low propagation coefficient and the like due to the fact that the Qinghai rhubarb grows in Qinghai and Tibet areas with high altitude and the limitation of regions is large. In recent years, with the increasing demand of the Qinghai rhubarb, the traditional seeding and seedling raising means can not meet the market demand. Therefore, there is an urgent need to develop a tissue culture and rapid propagation method for the hypocotyl of the green sea rhubarb to solve the above technical problems.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a method for tissue culture and rapid propagation of hypocotyl of Qinghai rhubarb, which takes high-quality seed of Qinghai rhubarb as an explant, and adopts different hormone culture medium proportions to ensure that the callus induction rate of the hypocotyl of Qinghai rhubarb can reach 90 percent, the callus propagation coefficient can reach 4.3 and the tissue culture seedling transplantation survival rate can reach 95 percent by sterilizing, primary culture, callus induction, callus propagation culture, callus bud differentiation, rooting culture and tissue culture seedling transplantation.
In order to achieve the aim, the tissue culture and rapid propagation method of the hypocotyl of the Qinghai rhubarb provided by the invention comprises the following steps:
(1) and (3) explant sterilization treatment: selecting plumpSoaking seed of radix et rhizoma Rhei of Qinghai as explant in distilled water for 3 hr, peeling off wings in operation table, draining with sterile filter paper, placing in 50ml centrifugal tube, soaking with 75% ethanol for 20s, and adding ddH2O(double distilled H2O) 3 times, soaking in 2% NaClO (sodium hypochlorite) disinfectant for 20min, and adding ddH2Repeatedly washing for 4-5 times, and then placing the seeds in a sterile culture dish paved with sterile filter paper to filter out the water on the surfaces of the seeds for later use;
(2) primary culture medium screening: inoculating the to-be-used sterile explant in the step (1) into a primary culture medium, with the embryonic axis of the seed facing downwards, immersing 1/3 seeds in the culture medium, wherein the temperature of an illumination incubator is 20 +/-1 ℃, the illumination culture is carried out for 16h/d, the dark culture is carried out for 8h/d, the light intensity threshold value is 4800-6000 lx, and the explant is germinated and grown into seedlings after being cultured for 30 d;
(3) hypocotyl callus induction: placing the aseptic seedlings obtained in the step (2) into an aseptic culture dish, sucking surface moisture by using aseptic filter paper, cutting by using an aseptic scalpel, taking off hypocotyls, inoculating the hypocotyls into an induction culture medium, culturing in dark for 7 days and in light for 8 days at the temperature of 20 +/-1 ℃ in an illumination incubator, and inducing callus after culturing for 15 days, wherein the light intensity threshold value is 4800-60001 x;
(4) and (3) propagation culture of hypocotyl callus: transplanting the hypocotyl callus induced in the step (3) into a proliferation culture medium for culturing for 20 d;
(5) hypocotyl callus bud differentiation: transplanting the hypocotyl callus of the step (4) into a bud differentiation culture medium, and inducing germination after culturing for 15 d;
(6) rooting culture: cutting the aseptic buds obtained in the step (5) into single plants, wherein each plant has 2-3 leaves, transplanting the single plants into a rooting culture medium, culturing in an illumination incubator at the temperature of 20 +/-1 ℃ for 16h/d in illumination and 8h/d in dark for 8h/d, and culturing for 14d to obtain tissue culture seedlings of the Qinghai rhubarb with good growth vigor;
(7) transplanting tissue culture seedlings: and (4) removing the root culture medium of the rooted seedlings obtained in the step (6), transplanting the rooted seedlings into a flowerpot filled with nutrient matrix soil, spraying more than 1 flower once every week at the room temperature of 25 ℃, illuminating for 12h/d and the humidity of 55-65%, and diluting by 2000 times.
Preferably, in the step (2), the primary medium is: MS4.43g/L +6-BA (6-benzylaminopurine) 1.0mg/L + NAA (naphthalene acetic acid) 0.1mg/L + agar 3g/L + sucrose 30g/L, and the pH value is 5.8.
Preferably, in the step (3), the induction medium is: MS4.43g/L +6-BA1.5mg/L + NAA1.5mg/L + agar 3g/L + sucrose 30g/L, pH 5.8.
Preferably, in the step (4), the proliferation medium is: MS4.43g/L + KT (6-furfurylaminopurine) 2.0mg/L +6-BA1.0mg/L + NAA1.5g/L + agar 3g/L + sucrose 30g/L, and the pH value is 5.8.
Preferably, in the step (5), the bud differentiation medium is: MS4.43g/L +6-BA1.0mg/L + NAA1.0mg/L + agar 3g/L + cane sugar 30g/L, and the pH value is 5.8.
Preferably, in the step (6), the rooting medium is: MS2.215g/L + agar 5g/L + sucrose 15g/L, pH 5.8.
Preferably, in the step (7), the ratio of the matrix soil to the vermiculite to the perlite is 3: 1, and the pH value is 6.5-7.0.
Preferably, in the steps (2) to (6), a solvent ddH is added to each medium2O, adjusting the pH value to 5.8 by NaOH and HCl, and sterilizing at 121 ℃ for 20 min.
The tissue culture and rapid propagation method of the hypocotyl of the Qinghai rhubarb provided by the invention has the following beneficial effects.
The invention establishes a sterile propagation system for in vitro culture of the Qinghai rhubarb, takes high-quality Qinghai rhubarb seeds as explants, and adopts different hormone culture medium proportions to ensure that the induction rate of the hypocotyl callus of the Qinghai rhubarb can reach 90 percent, the multiplication coefficient of the callus can reach 4.3 and the survival rate of the transplanted tissue culture seedling can reach 95 percent by sterilization treatment, primary culture, callus induction, callus multiplication culture, callus bud differentiation, rooting culture and tissue culture seedling transplantation.
Detailed Description
The present invention will be further described with reference to specific examples to assist understanding of the invention.
Example 1
The invention provides a tissue culture and rapid propagation method of hypocotyl of Qinghai rhubarb, which comprises the following steps:
(1) and (3) explant sterilization treatment: selecting plump Qinghai rhubarb seeds as explants with average length of 7.1mm and width of 5.5mm, soaking in distilled water for 3h, peeling off the seeds on an operation table, draining with sterile filter paper, placing in a 50ml centrifugal tube, soaking with 75% ethanol for 20s, and adding ddH2Rinsing with O for 3 times, soaking in 2% NaClO disinfectant for 20min, and adding ddH2Repeatedly washing for 4-5 times, and then placing the seeds in a sterile culture dish paved with sterile filter paper to filter out the water on the surfaces of the seeds for later use;
(2) primary culture medium screening: inoculating the to-be-used sterile explant in the step (1) into a primary culture medium, wherein the hypocotyl of the seed is downward, 1/3 seeds are immersed in the culture medium, the temperature of an illumination incubator is 20 +/-1 ℃, the illumination culture is performed for 16h/d, the dark culture is performed for 8h/d, the light intensity threshold value is 4800-60001 x, the seed germinates and grows into seedlings after being cultured for 30d, the hypocotyl slightly expands, and the primary culture medium is as follows: MS4.43g/L +6-BA1.0mg/L + NAA0.1mg/L + agar 3g/L + sucrose 30g/L, and the pH value is 5.8;
(3) hypocotyl callus induction: placing the aseptic seedlings obtained in the step (2) into an aseptic culture dish, sucking surface moisture by using aseptic filter paper, cutting by using an aseptic scalpel, taking hypocotyls, inoculating the hypocotyls into an induction culture medium, wherein the temperature of an illumination incubator is 20 +/-1 ℃, dark culture is carried out for 7d, illumination culture is carried out for 8d, the light intensity threshold value is 4800-60001 x, the hypocotyls are emerald green callus after 15d culture, the callus induction rate is 90%, and the induction culture medium is as follows: MS4.43g/L +6-BA1.5mg/L + NAA1.5mg/L + agar 3g/L + sucrose 30g/L, pH 5.8;
(4) and (3) propagation culture of hypocotyl callus: transplanting the hypocotyl callus induced in the step (3) into a multiplication culture medium, wherein the hypocotyl callus is dark green callus after being cultured for 20 days, the multiplication coefficient of the callus is 4.3, and the multiplication culture medium is as follows: MS4.43g/L + KT2.0mg/L +6-BA1.0mg/L + NAA1.5g/L + agar 3g/L + sucrose 30g/L, and the PH value is 5.8;
(5) hypocotyl callus bud differentiation: transplanting the hypocotyl callus of the step (4) into a bud differentiation culture medium, and inducing a plurality of green buds after culturing for 15 days, wherein the bud differentiation culture medium is MS4.43g/L +6-BA1.0mg/L + NAA1.0mg/L + agar 3g/L + sucrose 30g/L, and the pH value is 5.8;
(6) rooting culture: cutting the aseptic buds in the step (5) into single plants, wherein each plant has 2-3 leaves, transplanting the single plants into a rooting culture medium, culturing in an illumination incubator at the temperature of 20 +/-1 ℃ for 16h/d and 8h/d in a dark environment, wherein the light intensity threshold is 4800-60001 x, and after culturing for 14d, rooting to obtain tissue culture seedlings of the Qinghai rhubarb with emerald green leaves and good growth vigor, wherein the rooting culture medium is as follows: MS2.215g/L + agar 5g/L + sucrose 15g/L, pH 5.8;
(7) transplanting tissue culture seedlings: removing the root culture medium of the rooted seedlings obtained in the step (6), transplanting the rooted seedlings into a flowerpot filled with nutrient matrix soil, carrying out illumination for 12h/d at room temperature of 25 ℃, carrying out dilution by 2000 times, spraying more than 1 flower once every week, and transplanting the rooted seedlings for 30d, wherein the survival rate of the transplanted seedlings is 95%, the ratio of the matrix soil to vermiculite to perlite is 3: 1, and the pH value is 6.5-7.0.
In the above-mentioned steps (2) to (6), a solvent ddH is added to each medium2O, adjusting the pH value to 5.8 by NaOH and HCl, and sterilizing at 121 ℃ for 20 min.
Example 2
The invention provides a tissue culture and rapid propagation method of hypocotyl of Qinghai rhubarb, which comprises the following steps:
(1) and (3) explant sterilization treatment: selecting plump Qinghai rhubarb seeds as explants with average length of 7.1mm and width of 5.5mm, soaking in distilled water for 3h, peeling off the seeds on an operation table, draining with sterile filter paper, placing in a 50ml centrifugal tube, soaking with 75% ethanol for 20s, and adding ddH2Rinsing with O for 3 times, soaking in 2% NaClO disinfectant for 20min, and adding ddH2Repeatedly washing for 4-5 times, and then placing the seeds in a sterile culture dish paved with sterile filter paper to filter out the water on the surfaces of the seeds for later use;
(2) primary culture medium screening: inoculating the to-be-used sterile explant in the step (1) into a primary culture medium, wherein the embryonic axis of the seed is downward, 1/3 seeds are immersed in the culture medium, the temperature of an illumination incubator is 20 +/-1 ℃, the illumination culture is 16h/d, the dark culture is 8h/d, the light intensity threshold value is 6000-72001 x, the seed germinates and grows into seedlings after being cultured for 25d, the hypocotyl slightly expands, the color is yellow green, the primary culture medium is MS4.43g/L +6-BA1.0mg/L + NAA0.1mg/L + agar 3g/L + sucrose 30g/L, and the pH value is 5.8;
(3) hypocotyl callus induction: placing the aseptic seedling obtained in the step (2) into an aseptic culture dish, sucking surface moisture by using aseptic filter paper, cutting by using an aseptic scalpel, taking a hypocotyl, inoculating into an induction culture medium, wherein the temperature of an illumination incubator is 20 +/-1 ℃, dark culture is carried out for 7d, illumination culture is carried out for 8d, the light intensity threshold value is 6000-72001 x, the hypocotyl is yellow green callus after 15d culture, the callus induction rate is 87%, and the induction culture medium is: MS4.43g/L +6-BA1.5mg/L + NAA1.0mg/L + agar 3g/L + sucrose 30g/L, and the PH value is 5.8;
(4) and (3) propagation culture of hypocotyl callus: transplanting the hypocotyl callus induced in the step (3) into a proliferation culture medium, culturing for 20d to ensure that the hypocotyl is dark green callus, wherein the proliferation coefficient of the callus is 4.0, and the proliferation culture medium is as follows: MS4.43g/L + KT0.5mg/L +6-BA0.5mg/L + NAA1.5g/L + agar 3g/L + sucrose 30g/L, and the PH value is 5.8;
(5) hypocotyl callus bud differentiation: transplanting the hypocotyl callus of the step (4) into a bud differentiation culture medium, and inducing a plurality of yellow-green buds after culturing for 15 days, wherein the bud differentiation culture medium is as follows: MS4.43g/L +6-BA1.0mg/L + NAA0.5mg/L + agar 3g/L + sucrose 30g/L, and the PH value is 5.8;
(6) rooting culture: cutting the aseptic buds in the step (5) into single plants, wherein each plant has 2-3 leaves, transplanting the single plants into a rooting culture medium, culturing in an illumination incubator at the temperature of 20 +/-1 ℃ for 16h/d and 8h/d in a dark environment, wherein the light intensity threshold value is 6000-72001 x, and after culturing for 14d, rooting to obtain tissue culture seedlings of the Qinghai rhubarb with emerald green leaves and good growth vigor, wherein the rooting culture medium is as follows: MS4.43g/L + agar 5g/L + sucrose 15g/L, pH 5.8;
(7) transplanting tissue culture seedlings: and (3) removing the root culture medium from the rooted seedlings obtained in the step (6), transplanting the rooted seedlings into a flowerpot filled with nutrient matrix soil, spraying more than 1 flower once every week at the room temperature of 25 ℃, illuminating for 12h/d and the humidity of 55-65%, diluting by 2000 times, and transplanting the rooted seedlings for 30d until the survival rate of the transplanted seedlings is 90%. The nutrient matrix soil comprises matrix soil, vermiculite and perlite in a ratio of 2: 1, and has a pH value of 6.5-7.0.
In the above-mentioned steps (2) to (6), a solvent ddH is added to each medium2O, adjusting the pH value to 5.8 by NaOH and HCl, and sterilizing at 121 ℃ for 20 min.
Example 3
The invention provides a tissue culture and rapid propagation method of hypocotyl of Qinghai rhubarb, which comprises the following steps:
(1) and (3) explant sterilization treatment: selecting plump Qinghai rhubarb seeds as explants with average length of 7.1mm and width of 5.5mm, soaking in distilled water for 3h, peeling off the seeds on an operation table to remove the wings, draining the seeds with sterile filter paper, placing the seeds in a 50ml centrifuge tube, soaking with 75% ethanol for 20s, and using ddH2Rinsing with O for 3 times, soaking in 2% NaClO disinfectant for 20min, and adding ddH2Repeatedly washing for 4-5 times, and then placing the seeds in a sterile culture dish paved with sterile filter paper to filter out the water on the surfaces of the seeds for later use;
(2) primary culture medium screening: inoculating the to-be-used sterile explant in the step (1) into a primary culture medium, enabling the hypocotyl of the seed to face downwards, immersing 1/3 seeds in the culture medium, enabling the temperature of an illumination incubator to be 20 +/-1 ℃, carrying out illumination culture for 16h/d, carrying out dark culture for 8h/d, enabling the light intensity threshold value to be 3600-48001 x, enabling the seeds to germinate and grow into seedlings after being cultured for 40d, and enabling the hypocotyl to slightly expand. The primary culture medium is as follows: MS4.43g/L +6-BA1.0mg/L + NAA0.1mg/L + agar 3g/L + sucrose 30g/L, and the pH value is 5.8;
(3) hypocotyl callus induction: and (3) placing the sterile seedlings obtained in the step (2) into a sterile culture dish, sucking surface moisture by using sterile filter paper, cutting by using a sterile scalpel, taking hypocotyls, inoculating the hypocotyls into an induction culture medium, culturing at the temperature of 20 +/-1 ℃ in a dark environment for 7 days, culturing for 8 days in a light environment with the light intensity threshold of 3600-48001 x, culturing for 15 days, and then enabling the hypocotyls to be yellow green callus, wherein the callus induction rate is 80%. The induction culture medium is as follows: MS4.43g/L +6-BA1.5mg/L + NAA0.5mg/L + agar 3g/L + sucrose 30g/L, pH 5.8;
(4) and (3) propagation culture of hypocotyl callus: transplanting the hypocotyl callus induced in the step (3) into a proliferation culture medium, and culturing for 20 days to obtain dark green hypocotyl callus with the callus proliferation coefficient of 3.5. The proliferation culture medium is as follows: MS4.43g/L +6-BA0.5mg/L + NAA1.5g/L + agar 3g/L + sucrose 30g/L, and the PH value is 5.8;
(5) hypocotyl callus bud differentiation: transplanting the hypocotyl callus of the step (4) into a bud differentiation culture medium, and inducing a plurality of green buds after culturing for 15 days, wherein the bud differentiation culture medium is as follows: MS4.43g/L +6-BA1.5mg/L + NAA0.5mg/L + agar 3g/L + sucrose 30g/L, pH 5.8;
(6) rooting culture: and (3) cutting the sterile buds obtained in the step (5) into single plants, transplanting the single plants with 2-3 leaves into a rooting culture medium, culturing in an illumination incubator at the temperature of 20 +/-1 ℃ for 16h/d in illumination, culturing in dark for 8h/d, and rooting after 14d to obtain the tissue culture seedlings of the Qinghai rhubarb with emerald green leaves and good growth vigor. The rooting culture medium comprises: MS3.32g/L + agar 5g/L + sucrose 15g/L, and pH value is 5.8;
(7) transplanting tissue culture seedlings: and (3) removing the root culture medium from the rooted seedlings obtained in the step (6), transplanting the rooted seedlings into a flowerpot filled with nutrient matrix soil, spraying more than 1 flower once every week at the room temperature of 25 ℃, illuminating for 12h/d and the humidity of 55-65%, diluting by 2000 times, and transplanting the transplanted seedlings for 30d until the survival rate of the transplanted seedlings is 85%. The nutrient matrix soil comprises matrix soil, vermiculite and perlite in a ratio of 1: 1, and has a pH value of 6.5-7.0.
In the above-mentioned steps (2) to (6), a solvent ddH is added to each medium2O, adjusting the pH value to 5.8 by NaOH and HCl, and sterilizing at 121 ℃ for 20 min.
The growth conditions of the blue-sea rhubarb of the three groups of examples are shown in table 1, and the growth conditions of the blue-sea rhubarb are comprehensively judged according to three aspects of callus induction rate, callus proliferation coefficient and tissue culture seedling transplanting survival rate.
TABLE 1 growth of Rheum palmatum in examples 1-3
| Examples | Example 1 | Example 2 | Example 3 |
| Callus induction rate | 90％ | 87％ | 80％ |
| Callus proliferation coefficient | 4.3 | 4.0 | 3.5 |
| Survival rate of tissue culture seedling transplantation | 95％ | 90％ | 85％ |
By comparison, the method for tissue culture and rapid propagation of the hypocotyl of the Qinghai rhubarb provided by the invention has the advantages that the induction rate of the hypocotyl callus of the Qinghai rhubarb can reach 90%, the multiplication coefficient of the callus can reach 4.3, the transplanting survival rate of tissue culture seedlings can reach 95%, and the growth condition of the tissue culture seedlings is far better than that of the Qinghai rhubarb adopting other tissue culture and rapid propagation methods. Therefore, the method can obviously shorten the breeding cycle of the Qinghai rhubarb and greatly improve the breeding efficiency of the Qinghai rhubarb.
The invention establishes a Qinghai rhubarb in-vitro culture sterile propagation system, which takes high-quality Qinghai rhubarb seeds as explants, and adopts different hormone culture medium proportions to ensure that the induction rate of hypocotyl callus of the Qinghai rhubarb can reach 90 percent, the multiplication coefficient of the callus can reach 4.3 and the survival rate of tissue culture seedling transplantation can reach 95 percent by sterilization treatment, primary culture, callus induction, callus multiplication culture, callus bud differentiation, rooting culture and tissue culture seedling transplantation.
The inventive concept is explained in detail herein using specific examples, which are given only to aid in understanding the core concepts of the invention. It should be understood that any obvious modifications, equivalents and other improvements made by those skilled in the art without departing from the spirit of the present invention are included in the scope of the present invention.
Claims (8)
1. A tissue culture and rapid propagation method for hypocotyls of Qinghai rhubarb is characterized by comprising the following steps:
(1) and (3) explant sterilization treatment: selecting plump Qinghai rhubarb seeds as explants, soaking in distilled water for 3h, peeling and removing wings on an operation table, draining with sterile filter paper, placing in a 50ml centrifugal tube, soaking with 75% ethanol for 20s, and adding ddH2Rinsing with O for 3 times, soaking in 2% NaClO disinfectant for 20min, and adding ddH2Repeatedly washing for 4-5 times, and then placing the seeds in a sterile culture dish paved with sterile filter paper to filter out the water on the surfaces of the seeds for later use;
(2) primary culture medium screening: inoculating the to-be-used sterile explant in the step (1) into a primary culture medium, with the embryonic axis of the seed facing downwards, immersing 1/3 seeds in the culture medium, wherein the temperature of an illumination incubator is 20 +/-1 ℃, the illumination culture is carried out for 16h/d, the dark culture is carried out for 8h/d, the light intensity threshold value is 4800-6000 lx, and the explant is germinated and grown into seedlings after being cultured for 30 d;
(3) hypocotyl callus induction: placing the aseptic seedlings obtained in the step (2) into an aseptic culture dish, sucking surface moisture by using aseptic filter paper, cutting by using an aseptic scalpel, taking off embryonic axis, inoculating into an induction culture medium, culturing in dark at the temperature of 20 +/-1 ℃ in an illumination incubator for 7 days and in illumination for 8 days at the light intensity threshold of 4800-6000 lx, and inducing callus after culturing for 15 days;
(4) and (3) propagation culture of hypocotyl callus: transplanting the hypocotyl callus induced in the step (3) into a proliferation culture medium for culturing for 20 d;
(5) hypocotyl callus bud differentiation: transplanting the hypocotyl callus of the step (4) into a bud differentiation culture medium, and inducing germination after culturing for 15 d;
(6) rooting culture: cutting the aseptic buds obtained in the step (5) into single plants, wherein each plant is 2-3 leaves, transplanting the single plants into a rooting culture medium, culturing in an illumination incubator at the temperature of 20 +/-1 ℃ for 16h/d in illumination, culturing in dark for 8h/d, and culturing for 14d to obtain tissue culture seedlings of the Qinghai rhubarb with good growth vigor, wherein the light intensity threshold value is 4800-60001 x;
(7) transplanting tissue culture seedlings: and (4) removing the root culture medium of the rooted seedlings obtained in the step (6), transplanting the rooted seedlings into a flowerpot filled with nutrient matrix soil, spraying more than 1 flower once every week at the room temperature of 25 ℃, illuminating for 12h/d and the humidity of 55-65%, and diluting by 2000 times.
2. The tissue culture and rapid propagation method of hypocotyl of green-sea rhubarb according to claim 1, characterized in that in the step (2), the primary culture medium is: MS4.43g/L +6-BA1.0mg/L + NAA0.1mg/L + agar 3g/L + sucrose 30g/L, and the pH value is 5.8.
3. The tissue culture and rapid propagation method of hypocotyls of Qinghai rhubarb according to claim 1, characterized in that in the step (3), the induction culture medium is: MS4.43g/L +6-BA1.5mg/L + NAA1.5mg/L + agar 3g/L + sucrose 30g/L, pH 5.8.
4. The tissue culture and rapid propagation method of hypocotyls of Qinghai rhubarb according to claim 1, characterized in that in the step (4), the propagation medium is: MS4.43g/L + KT2.0mg/L +6-BA1.0mg/L + NAA1.5g/L + agar 3g/L + sucrose 30g/L, and the pH value is 5.8.
5. The tissue culture and rapid propagation method of hypocotyls of Qinghai rhubarb according to claim 1, characterized in that in the step (5), the bud differentiation medium is: MS4.43g/L +6-BA1.0mg/L + NAA1.0mg/L + agar 3g/L + cane sugar 30g/L, and the pH value is 5.8.
6. The tissue culture and rapid propagation method of the hypocotyl of the green-sea rhubarb according to claim 1, characterized in that in the step (6), the rooting medium is: MS2.215g/L + agar 5g/L + sucrose 15g/L, pH 5.8.
7. The tissue culture and rapid propagation method of the hypocotyl of Qinghai rhubarb according to claim 1, characterized in that in the step (7), the ratio of the components of the nutrient matrix soil is matrix soil, vermiculite and perlite is 3: 1, and the pH value is 6.5-7.0.
8. The tissue culture and rapid propagation method of hypocotyl of green-sea rhubarb as claimed in claim 1, wherein in steps (2) to (6), solvent ddH is added to each culture medium2O, adjusting the pH value to 5.8 by NaOH and HCl, and sterilizing at 121 ℃ for 20 min.
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