CN111631136B - Tissue culture and rapid propagation method of dormant buds of eyedrops - Google Patents
Tissue culture and rapid propagation method of dormant buds of eyedrops Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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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
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Abstract
The invention discloses a tissue culture and rapid propagation method of dormant buds of eyebright, which comprises the following steps: (1) explant collection and pretreatment; (2) sterilizing; (3) adventitious bud induction; (4) propagation culture; and (5) rooting culture and (6) hardening off and transplanting. The method can be free from the influence of seasons, temperature and regions, can produce a large amount of eyebright aseptic seedlings, and provides theoretical basis and technical support for directly cultivating the eyebright application part in a subsequent laboratory through establishing an eyebright dormant bud aseptic propagation system; in addition, the method has important significance for carrying out systematic research on the eyedrops, protecting and restoring the wetland and restoring the ecological environment.
Description
Technical Field
The invention relates to a tissue culture and rapid propagation method of dormant buds of eyebright, belonging to the technical field of plant tissue culture.
Background
Herba Zosterae Marinae (herba Zosterae Marinae)Potamogeton distinctusA. Bennett J. Bot.) is a perennial aquatic herb of the genus Eugenia (Potamoectacenaee) of the family Eziaceae (Potamoectonaceae), which has developed, white, 1.5-2 mm in diameter, multiple branches, often forming spindle-shaped dormant buds at the tip and somewhat dense fibrous roots at the nodes. The stem is cylindrical, 1.5-2 mm in diameter, and is generally unbranched. The floating water She Gezhi is in a shape of needle, wide needle to egg, 2-10 cm long, 1-4 cm wide, sharp or blunt tip, blunt base or sometimes wedge, and has a handle 5-20 cm long; the veins are multiple and the top ends are connected; the shape of the leaves of the Chinese mosla herb is from needle to needle, the grass is herbaceous and has a handle, and the leaves of the Chinese mosla herb always fall early; the leaf supporting membrane is 2-7 cm long, has a sharp top end and is in a sheath shape like a corm. The spike-shaped inflorescence grows at the top, has a plurality of flowers, extends out of the water surface when flowering, and sinks in the water after flowering; the inflorescence stalk is slightly enlarged and thicker than the stem, and the flower stands uprightBending from the base after the flowers are bloomed, and the length is 3-10 cm; small flower, quilt sheet 4, green; 2 pistils (or 1 or 3 diluted pistils). The fruit is wide and inverted egg-shaped, and about 3.5 mm long, 3 ridges are obvious on the back, the ridge is sharp, the upper part of the fruit is obviously raised, the side ridge is slightly blunt, the base and the upper part are respectively provided with 2 bulges, and the beak is slightly sunken and obliquely extended.
The eyedrops are widely distributed in most provinces in the south and north of China and mainly grow in static water such as ponds, paddy fields, ditches and the like. The herba Eziae is commonly called as water floating, and is also called as a water chopping board. Particularly, the seedlings are distributed in each rice growing area and mainly depend on chick paw buds (dormant buds) for vegetative propagation, so that the seedlings cause harm all the year round. Ming Dynasty blue Mao recorded in Dian nan materia Medica, tooth grass, also named tooth picking grass. In the raw field, it is bitter and astringent in taste and cold in nature. Stop red and white dysentery, large intestine bleeding, woman red metrorrhagia, metrostaxis and aversion to blood. It is recorded in Chinese medicine dictionary that Ezicai has the functions of clearing away heat, promoting diuresis, arresting bleeding, eliminating swelling and expelling ascaris, and has the pharmacological actions of promoting urination, arresting bleeding, killing parasite, etc. and is used in treating piles, ascaris, edema and other diseases. Earlier researches show that the gynura divaricata contains components such as flavonoids, volatile oils, amino acids, saccharides, tannins, phenols and the like, and has a certain medicinal value.
The eyedrops are also prepared from the whole herbs, and the stems are easy to break during collection, so that a large amount of medicinal materials are difficult to collect. The traditional Chinese medicine survey in the Anshun area finds that: the dormant buds (locally commonly called as 'Harpagophytum seven') of people in the peaceful region (Tunburg people) have good effect of treating hemorrhoids and hepatitis, while the dormant buds of the eyedrops are mainly formed by overwintering dormancy and are difficult to collect in ponds, paddy fields and ditches.
Disclosure of Invention
The invention aims at solving the technical problems in the background technology, adopts dormant buds of the eyedrops as explants, can directly bud and form seedlings without callus culture stage by tissue culture technology under the aseptic condition, thereby establishing a plant aseptic seedling system, greatly shortening the culture time, obtaining a large amount of aseptic seedlings in a short time, and having the advantages of easy method, convenient operation and high yield, in particular to a tissue culture and rapid propagation method of the dormant buds of the eyedrops.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows: a tissue culture and rapid propagation method of dormant buds of eyebright comprises the following steps:
(1) Explant collection and pretreatment: selecting strong dormant buds of the eyedrops as explants, directly washing the explants for 1 hour by using tap water, and filtering the explants by using sterile gauze; then placing the buds on a sterile operation table, wiping the buds for 1 time by using sterile absorbent cotton soaked with 75% ethanol, removing attachments such as redundant bud scales, adventitious roots and the like, cutting off the clustered dormant buds from the base part by using a sterile scalpel, and repeatedly washing the dormant buds cut into the single buds for 4-5 times by using sterile water for later use;
(2) And (3) disinfection treatment: placing the dormant buds of the gynura divaricata obtained in the step (1) on a sterile operation table, fully soaking the dormant buds of the gynura divaricata in 75% of alcohol for 30-40 s, fully soaking the dormant buds of the gynura divaricata in 0.1% of mercuric chloride for 10 min, repeatedly washing the buds with sterile water for 4-5 times after being taken out, and soaking the buds in a glass filled with sterile water for later use;
(3) Adventitious bud induction: the sterile dormant buds of the eyedrops obtained in the step (2) are dried by sterile filter paper, inoculated into a sterile induction culture medium and irradiated at the temperature of 25 +/-1 ℃ for 12h/d with the illumination intensity of 60-80 mu mol/(m) 2 S) culturing in a light incubator to induce adventitious buds;
(4) And (3) proliferation culture: cutting the adventitious buds induced in the step (3) into individual plants by using an aseptic scalpel, and reserving 2-3 leaves for each plant; inoculating the strain in a proliferation culture medium in a sterile environment, wherein the temperature is 25 +/-1 ℃, the illumination time is 12h/d, and the illumination intensity is 60-80 mu mol/(m) 2 S) culturing in a light incubator to obtain an eyeweed plant with dark green leaf color and good growth vigor;
(5) Rooting culture: cutting the eyedrop plants induced in the step (4) into individual plants by using a sterile scalpel, and reserving 3-4 leaves for each plant; selecting a good-growth eyedrops, inoculating the eyedrops into a rooting culture medium in a sterile environment, and culturing at the temperature of 25 +/-1 ℃ for 12h/d with the illumination intensity of 60-80 mu mol/(m) 2 S) culturing in a light incubator for one week to obtain robust rooted seedlings;
(6) Hardening and transplanting seedlings: removing the rooted seedlings obtained in the step (5) and the culture container from the illumination culture box, opening a bottle cover of the rooted tissue culture seedlings of the gynura divaricata, placing the rooted tissue culture seedlings in a room with normal room temperature, and standing and hardening the seedlings for 2-3 days at the room temperature; then taking out the plants from the culture medium, removing the culture medium at the root of the seedlings, and transplanting the plants into a water tank with soil.
Further, in the step (3), the induction culture medium selects an MS culture medium added with 0.05-0.1 mg/L KT (kinetin) and 0.05-0.1 mg/L NAA (naphthylacetic acid), wherein the MS culture medium contains 3% of sucrose mass concentration and 6g/L of agar mass concentration, and the pH of the MS culture medium is adjusted to be 5.8-6.0.
Furthermore, in the step (4), the multiplication culture medium selects an MS culture medium added with 0.5-1 mg/L6-BA (6-benzylaminopurine) and 0.05-0.1 mg/L NAA (naphthalene acetic acid), wherein the MS culture medium contains 3% of sucrose mass concentration and 6g/L of agar mass concentration, and the pH value of the MS culture medium is adjusted to be 5.8-6.0.
Furthermore, the invention relates to a tissue culture and rapid propagation method of dormant buds of eyedrops, wherein sterile water and/or sterile nutrient solution are added into the propagation medium.
Furthermore, in the step (5), the rooting medium selects a 1/2MS medium added with 0.5-1 mg/L IBA (indolebutyric acid), wherein the MS medium contains 1.5% of sucrose by mass and 6g/L of agar by mass, and the pH value of the MS medium is adjusted to be 5.8-6.0.
Furthermore, the invention relates to a tissue culture and rapid propagation method of dormant buds of eyedrops, wherein sterile water and/or sterile nutrient solution are added into the rooting medium.
Further, the invention relates to a tissue culture and rapid propagation method of dormant buds of eyedrops, wherein the sterile water is prepared by sterilizing distilled water at the temperature of 121 ℃ for 25 minutes, and the sterile nutrient solution is prepared by sterilizing the nutrient solution without adding auxin and agar at the temperature of 121 ℃ for 25 minutes.
Further, in the tissue culture and rapid propagation method of dormant buds of eyebright, the water depth in the water tank is 2cm, and the soil depth is 10cm in the transplanting process in the step (6).
Further, the invention relates to a tissue culture and rapid propagation method of dormant buds of eyebright, wherein the induction culture medium, the proliferation culture medium and the rooting culture medium are sterilized at the temperature of 121 ℃ for 25 minutes, and the pH value is adjusted to be 5.6-5.8 before sterilization.
The MS culture medium in the culture medium is a common basic culture medium formula in the plant tissue culture technology, has higher inorganic salt concentration, can ensure mineral nutrition required by tissue growth, can accelerate the growth of callus, is a more stable ion balance solution, has high nitrate content and proper nutrient quantity and proportion, can meet the nutritional and physiological needs of plant cells, has wider application range, and can be used as the basic culture medium for the rapid propagation of most plant tissue cultures.
KT is named as kinetin/6-furfuryl amino purine in Chinese, and is one kind of plant cytokinin, which is non-natural cytokinin, has a chemical name of 6-glycosyl amino purine (or N6-furan methyl adenine) and a molecular formula of C10H9N5O. Is insoluble in water and soluble in strong acid, alkali and glacial acetic acid; besides the function of promoting cell division, it also has the functions of delaying the senility of in vitro leaf and cut flower, inducing bud differentiation and development and increasing stomatal aperture.
NAA is named as naphthylacetic acid and one of auxin, can promote cell division and expansion, induce to form adventitious roots, increase fruit bearing, prevent fruit drop, change the ratio of male flowers to female flowers, enter plants through the tender epidermis and seeds of leaves and branches, and is guided to the whole plant along with nutrient flow.
The Chinese name of 6-BA benzyl purine/6-benzyl amino purine, one kind of plant cytokinin, has the functions of inhibiting the decomposition of chlorophyll, nucleic acid and protein in plant leaf, protecting green and resisting senility, regulating amino acid, auxin, inorganic salt, etc. to the treated part, etc. and is used widely in various stages of harvesting agricultural, tree and garden crops.
The Chinese name of IBA, indolebutyric acid, one of the auxins, is mainly used for cutting and rooting, can induce the formation of root protomer, promote cell differentiation and division, is beneficial to the generation of new roots and the differentiation of vascular bundle systems, promotes the formation of adventitious roots of the cutting, and is widely applied to the cutting and rooting of trees and flowers.
Compared with the prior art, the tissue culture and rapid propagation method of the dormant buds of the eyedrops has the beneficial effects that: the eyedrops are aquatic plants, the whole grass and dormant buds of the eyedrops can be used for medicines, but the whole grass is difficult to collect in a large quantity, the dormant buds are difficult to pick up, the aquatic environment of the eyedrops is simulated in a culture medium to form gradient culture and propagation, a large number of eyedrops can be propagated in a short period, and the eyedrops have important significance in further research on medicinal components of the eyedrops, exploration on pharmacological action and the like. The method can be free from the influence of seasons, temperature and regions, can produce a large amount of aseptic seedlings of the eyedrops, and provides theoretical basis and technical support for directly cultivating the medicinal parts of the eyedrops in a subsequent laboratory through establishing an aseptic propagation system of dormant buds of the eyedrops; in addition, the method has important significance for carrying out systematic research on the eyedrops, protecting and restoring the wetland and restoring the ecological environment.
Drawings
FIG. 1 is a diagram showing the cultivation of EZICAI in the induction culture stage in example 1 of the present invention;
FIG. 2 is a diagram showing the cultivation of Ezikia rossica in the multiplication cultivation stage in example 1 of the present invention;
FIG. 3 is a diagram showing the cultivation of Ezikia indica during the rooting culture stage in example 1 of the present invention.
Detailed Description
The present invention is further illustrated by the following specific examples, and it should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the principle of the present invention, and these should be construed as falling within the scope of the present invention.
Reagents used in the embodiments of the present invention may be commercially available to carry out the present invention, if not specifically described, and technical solutions related thereto may be generally used in the art, if not specifically described.
The formulations of the MS media used in the examples are as follows:
wherein the culture medium, the propagation medium and the rooting medium are formed by adding 6g/L of agar and 1.5-3% of sucrose into the culture formula.
Example 1
A tissue culture and rapid propagation method of dormant buds of eyebright comprises the following steps:
(1) Explant collection and pretreatment: selecting strong dormant buds of potamogeton mukurossi as explants, directly washing the explants for 1 hour by using tap water, and draining the explants by using sterile gauze; then placing the buds on a sterile operation table, wiping the buds for 1 time by sterile absorbent cotton soaked in 75% ethanol, removing attachments such as redundant bud scales and adventitious roots, cutting off single tufted dormant buds from a base part by a sterile scalpel, repeatedly washing the dormant buds cut into single dormant buds for 4-5 times by sterile water, and then entering the next step;
(2) And (3) disinfection treatment: placing the dormant buds of the gynura divaricata obtained in the step (1) on a sterile operation table, fully soaking the dormant buds of the gynura divaricata in 75% of alcohol for 30-40 s, fully soaking the dormant buds of the gynura divaricata in 0.1% of mercuric chloride for 10 min, repeatedly washing the buds with sterile water for 4-5 times after being taken out, soaking the buds in a glass filled with the sterile water, and entering the next step;
(3) Adventitious bud induction: absorbing surface water of the sterile dormant buds of the eyedrops obtained in the step (2) by using sterile filter paper, inoculating the sterile dormant buds into a sterile induction culture medium, wherein the induction culture medium is MS + KT 0.05mg/L + NAA 0.1mg/L, the agar mass concentration is 6g/L, the sucrose mass concentration is 3%, the pH value is 5.8-6.0, and the temperature is (25 +/-1)The illumination time is 12h/d, and the illumination intensity is 60-80 mu mol/(m) 2 S) in an illumination incubator, after 16 days adventitious buds of 4-5cm height can grow, proceeding to the next step, the situation is shown in FIG. 1;
(4) And (3) proliferation culture: cutting the adventitious buds induced in the step (3) into individual plants by using an aseptic scalpel, and reserving 2-3 leaves for each plant; inoculating the strain into a multiplication culture medium under a sterile environment, wherein the multiplication culture medium is MS +6-BA 1mg/L + NAA 0.05mg/L, the agar mass concentration is 6g/L, the sucrose mass concentration is 3%, and the pH value is 5.8-6.0, and sterile nutrient solution is added into the multiplication culture medium; wherein the sterile nutrient solution is a nutrient solution without auxin and agar, and is prepared by sterilizing at the temperature of 121 ℃ for 25 minutes; the temperature is (25 +/-1) DEG C, the illumination time is 12h/d, and the illumination intensity is 60-80 mu mol/(m) 2 S), the growth vigor is good after 8 days, more leaves and rootstocks can grow out, more branches can grow out of the nodes of the rootstocks, and the next step is carried out, wherein the situation is shown in figure 2;
(5) Rooting culture: cutting the eyedrop plants induced in the step (4) into single plants by using a sterile scalpel, and reserving 3-4 leaves for each plant; selecting a good-growth-vigor Eyemenu seedling, inoculating the good-growth-vigor Eyemenu seedling to a rooting culture medium in a sterile environment, wherein the rooting culture medium is 1/2MS + IBA 0.5mg/L, the agar mass concentration is 6g/L, the sucrose mass concentration is 1.5%, the pH value is 5.8-6.0, and sterile water is added into a propagation culture medium; the sterile water is prepared by sterilizing distilled water at the temperature of 121 ℃ for 25 minutes; the illumination time is 12h/d and the illumination intensity is 60 to 80 mu mol/(m) at the temperature of (25 +/-1) ° C 2 S) in a light incubator, culturing for 6-7 days to obtain robust rooted seedlings, the conditions of which are shown in figure 3;
(6) Hardening and transplanting seedlings: removing the rooted seedlings obtained in the step (5) and the culture container from the illumination culture box, opening a bottle cover of the rooted tissue culture seedlings of the gynura divaricata, placing the rooted tissue culture seedlings in a room with normal room temperature, and standing and hardening the seedlings for 2-3 days at the room temperature; and then taking out the plants from the culture medium, removing the culture medium at the roots of the seedlings, and transplanting the plants into a water tank with soil, wherein in the transplanting process, the water depth in the water tank is 2cm, the soil depth is 10cm, and the final transplanting survival rate reaches 97%.
Example 2
A tissue culture and rapid propagation method of dormant buds of eyebright comprises the following steps:
(1) Explant collection and pretreatment: selecting strong dormant buds of the eyedrops as explants, directly washing the explants for 1 hour by using tap water, and filtering the explants by using sterile gauze; then placing the buds on a sterile operation table, wiping the buds for 1 time by sterile absorbent cotton soaked in 75% ethanol, removing attachments such as redundant bud scales and adventitious roots, cutting off single tufted dormant buds from a base part by a sterile scalpel, repeatedly washing the dormant buds cut into single dormant buds for 4-5 times by sterile water, and then entering the next step;
(2) And (3) disinfection treatment: placing the dormant buds of the gynura divaricata obtained in the step (1) on a sterile operation table, fully soaking the dormant buds of the gynura divaricata in 75% of alcohol for 30-40 s, fully soaking the dormant buds of the gynura divaricata in 0.1% of mercuric chloride for 10 min, repeatedly washing the buds with sterile water for 4-5 times after being taken out, soaking the buds in a glass filled with the sterile water, and entering the next step;
(3) Adventitious bud induction: the sterile dormant buds of the potamogeton indicum obtained in the step (2) are dried by using sterile filter paper, and inoculated into a sterile induction culture medium, wherein the induction culture medium comprises MS + KT 0.05mg/L + NAA 0.05mg/L, the mass concentration of agar is 6g/L, the mass concentration of sucrose is 3%, the pH value is 5.8-6.0, the illumination time is 12h/d at the temperature of (25 +/-1) DEG C, and the illumination intensity is 60-80 mu mol/(m mol/d 2 S) culturing in a light incubator under the condition that adventitious buds with the height of 4-5cm can grow after 16 days, and carrying out the next step;
(4) And (3) proliferation culture: cutting the adventitious bud induced in the step (3) into individual plants by using a sterile scalpel, and reserving 2-3 leaves for each plant; inoculating a proliferation culture medium in a sterile environment, wherein the proliferation culture medium is MS +6-BA 1mg/L + NAA 0.05mg/L, the agar mass concentration is 6g/L, the sucrose mass concentration is 3 percent, the pH value is 5.8-6.0, and sterile water is added into the proliferation culture medium; the sterile water is prepared by sterilizing distilled water at the temperature of 121 ℃ for 25 minutes; the temperature is (25 +/-1) DEG C, the illumination time length is 12h/d, and the illumination intensity is 60-80 mu mol/(m) 2 S) conditionCulturing in a light incubator for 8 days, wherein the growth vigor is good, more leaves and rhizomes can grow, more branches can grow on the node part of the rhizomes, and the next step is carried out;
(5) Rooting culture: cutting the eyedrop plants induced in the step (4) into single plants by using a sterile scalpel, and reserving 3-4 leaves for each plant; selecting a good-growth-vigor Menispermum plant seedling, inoculating the good-growth-vigor Menispermum plant seedling into a rooting culture medium under a sterile environment, wherein the rooting culture medium is 1/2MS + IBA 0.5mg/L, the agar mass concentration is 6g/L, the sucrose mass concentration is 1.5%, the pH value is 5.8-6.0, and sterile nutrient solution is added into a propagation culture medium; wherein the sterile nutrient solution is a nutrient solution without auxin and agar, and is prepared by sterilizing at the temperature of 121 ℃ for 25 minutes; at the temperature of (25 +/-1) DEG C, the illumination time length of 12h/d and the illumination intensity of 60-80 mu mol/(m) 2 S) culturing in an illumination incubator for 6-7 days to obtain robust rooted seedlings;
(6) Hardening and transplanting seedlings: moving the rooted seedlings obtained in the step (5) together with the culture container out of the illumination culture box, opening a bottle cap of the rooted tissue culture seedlings of the gynura divaricata, placing the seedlings in a room with normal room temperature, and placing the seedlings for hardening for 2-3 days at the room temperature; and then taking out the plants from the culture medium, removing the culture medium at the roots of the seedlings, and transplanting the plants into a water tank with soil, wherein in the transplanting process, the water depth in the water tank is 2cm, the soil depth is 10cm, and the final transplanting survival rate reaches 97%.
Example 3
A tissue culture and rapid propagation method of dormant buds of eyebright comprises the following steps:
(1) Explant collection and pretreatment: selecting strong dormant buds of the eyedrops as explants, directly washing the explants for 1 hour by using tap water, and filtering the explants by using sterile gauze; then placing the buds on a sterile operation table, wiping the buds for 1 time by using sterile absorbent cotton soaked with 75% ethanol, removing attachments such as redundant bud scales, adventitious roots and the like, cutting off the clustered dormant buds from the base part by using a sterile scalpel, repeatedly washing the dormant buds cut into the single buds for 4-5 times by using sterile water, and then entering the next step;
(2) And (3) disinfection treatment: placing the dormant buds of the gynura divaricata obtained in the step (1) on a sterile operation table, fully soaking the dormant buds of the gynura divaricata in 75% of alcohol for 30-40 s, fully soaking the dormant buds of the gynura divaricata in 0.1% of mercuric chloride for 10 min, repeatedly washing the buds with sterile water for 4-5 times after being taken out, soaking the buds in a glass filled with the sterile water, and entering the next step;
(3) Adventitious bud induction: absorbing surface moisture of the sterile dormant buds of the eyedrops obtained in the step (2) by using sterile filter paper, and inoculating the sterile dormant buds into a sterile induction culture medium, wherein the induction culture medium is MS + KT 0.05mg/L + NAA 0.1mg/L, the agar mass concentration is 6g/L, the sucrose mass concentration is 3%, the pH is 5.8-6.0, the illumination time is 12h/d at the temperature of (25 +/-1) DEG C, and the illumination intensity is 60-80 mu mol/(m +/-1) 2 S) culturing in a light incubator under the condition that adventitious buds with the height of 4-5cm can grow after 16 days, and carrying out the next step;
(4) And (3) proliferation culture: cutting the adventitious bud induced in the step (3) into individual plants by using a sterile scalpel, and reserving 2-3 leaves for each plant; inoculating the strain into a proliferation culture medium under a sterile environment, wherein the proliferation culture medium is MS +6-BA 1mg/L + NAA 0.05mg/L, the agar mass concentration is 6g/L, the sucrose mass concentration is 3%, and the pH value is 5.8-6.0; the temperature is (25 +/-1) DEG C, the illumination time is 12h/d, and the illumination intensity is 60-80 mu mol/(m) 2 S), the cultivation is carried out in a light incubator under the condition of 8 days, the growth vigor is good, more leaves and rhizomes can grow, more branches can grow on the nodes of the rhizomes, and the next step is carried out;
(5) Rooting culture: cutting the eyedrop plants induced in the step (4) into single plants by using a sterile scalpel, and reserving 3-4 leaves for each plant; selecting a good-growth-vigor Evosimus plant seedling, and inoculating the good-growth-vigor Evosimus plant seedling to a rooting culture medium under a sterile environment, wherein the rooting culture medium is 1/2MS + IBA 0.5mg/L, the agar mass concentration is 6g/L, the sucrose mass concentration is 1.5%, and the pH value is 5.8-6.0; the illumination time is 12h/d and the illumination intensity is 60 to 80 mu mol/(m) at the temperature of (25 +/-1) ° C 2 S) culturing in an illumination incubator for 6-7 days to obtain robust rooted seedlings;
(6) Hardening and transplanting seedlings: moving the rooted seedlings obtained in the step (5) together with the culture container out of the illumination culture box, opening a bottle cap of the rooted tissue culture seedlings of the gynura divaricata, placing the seedlings in a room with normal room temperature, and placing the seedlings for hardening for 2-3 days at the room temperature; and then taking out the plant from the culture medium, removing the culture medium at the root of the seedling, transplanting the plant into a water tank with soil, wherein in the transplanting process, the water depth in the water tank is 2cm, the soil depth is 10cm, and the final transplanting survival rate reaches 97%.
The tissue culture and rapid propagation method of the present invention is adopted, wherein the induction medium, the proliferation medium and the rooting medium of examples 1 to 3 are sterilized at 121 ℃ for 25 minutes, and the pH value is adjusted to 5.6 to 5.8 before sterilization.
In order to illustrate the method for tissue culture and rapid propagation, the dormant bud of the eyeweed is used as an explant, and the growth condition of the eyeweed is judged from two aspects of multiplication multiple and rhizome multiple in three embodiments by using a tissue culture technology under aseptic conditions, wherein the specific conditions are shown in table 1.
Table 1 shows the growth of Ezikia rossica in three examples of the present invention.
| Group of embodiments | Example 1 | Example 2 | Example 3 |
| Fold of proliferation | 5.3 | 5.0 | 4.1 |
| Multiple of rhizome | 8.3 | 7.0 | 7.0 |
Claims (4)
1. A tissue culture and rapid propagation method of dormant buds of eyebright is characterized by comprising the following steps:
(1) Explant collection and pretreatment: selecting strong dormant buds of potamogeton mukurossi as explants, directly washing the explants for 1 hour by using tap water, and draining the explants by using sterile gauze; then placing the buds on a sterile operation table, wiping the buds for 1 time by using sterile absorbent cotton soaked with 75% ethanol, removing attachments such as redundant bud scales, adventitious roots and the like, cutting off the clustered dormant buds from the base part by using a sterile scalpel, and repeatedly washing the dormant buds cut into the single buds for 4-5 times by using sterile water for later use;
(2) And (3) disinfection treatment: placing the dormant buds of the gynura bicolor obtained in the step (1) on a sterile operation table, fully soaking the dormant buds for 30-40 s by using 75% alcohol, fully soaking the dormant buds for 10 min by using 0.1% mercury bicolor, repeatedly washing the dormant buds for 4-5 times by using sterile water after taking out the dormant buds, and soaking the dormant buds in a glass filled with the sterile water for later use;
(3) Adventitious bud induction: the sterile dormant buds of the eyedrops obtained in the step (2) are dried by sterile filter paper, inoculated into a sterile induction culture medium and irradiated at the temperature of 25 +/-1 ℃ for 12h/d with the illumination intensity of 60-80 mu mol/(m) 2 S) culturing in a light incubator to induce adventitious buds; the induction culture medium selects an MS culture medium added with 0.05-0.1 mg/L KT (kinetin) and 0.05-0.1 mg/L NAA (naphthylacetic acid), wherein the MS culture medium comprises cane sugar with the mass concentration of 3% and agar with the mass concentration of 6g/L, and the pH value of the MS culture medium is adjusted to be 5.8-6.0;
(4) And (3) proliferation culture: cutting the adventitious bud induced in the step (3) into individual plants by using a sterile scalpel, and reserving 2-3 leaves for each plant; inoculating the strain in a proliferation culture medium in a sterile environment, wherein the temperature is 25 +/-1 ℃, the illumination time is 12h/d, and the illumination intensity is 60-80 mu mol/(m) 2 S) in a light incubator to obtain an eye with dark green leaf color and good growthVegetable plants; the enrichment culture medium selects an MS culture medium added with 0.5-1 mg/L6-BA (6-benzylaminopurine) and 0.05-0.1 mg/L NAA (naphthylacetic acid), wherein the MS culture medium contains cane sugar with the mass concentration of 3 percent and agar with the mass concentration of 6g/L, and the pH value of the MS culture medium is adjusted to be 5.8-6.0;
(5) Rooting culture: cutting the eyedrop plants induced in the step (4) into single plants by using a sterile scalpel, and reserving 3-4 leaves for each plant; selecting a good-growth eyedrops, inoculating the eyedrops into a rooting culture medium in a sterile environment, and culturing at the temperature of 25 +/-1 ℃ for 12h/d with the illumination intensity of 60-80 mu mol/(m) 2 S) culturing in a light incubator for one week to obtain robust rooted seedlings; selecting a 1/2MS culture medium added with 0.5-1 mg/L IBA (indolebutyric acid) from the rooting culture medium, wherein the 1/2MS culture medium comprises 1.5% of sucrose and 6g/L of agar, and adjusting the pH value to 5.8-6.0;
(6) Hardening and transplanting seedlings: moving the rooted seedlings obtained in the step (5) together with the culture container out of the illumination culture box, opening a bottle cap of the rooted tissue culture seedlings of the gynura divaricata, placing the seedlings in a room with normal room temperature, and placing the seedlings for hardening for 2-3 days at the room temperature; then taking out the plants from the culture medium, removing the culture medium at the root of the seedlings, and transplanting the plants into a water tank with soil.
2. The tissue culture and rapid propagation method of dormant buds of clitocybe maxima according to claim 1, which is characterized in that: sterile water and/or sterile nutrient solution are also added into the proliferation culture medium and the rooting culture medium, the sterile water is prepared by sterilizing distilled water at the temperature of 121 ℃ for 25 minutes, and the sterile nutrient solution is prepared by sterilizing the nutrient solution without adding auxin and agar at the temperature of 121 ℃ for 25 minutes.
3. The tissue culture and rapid propagation method of dormant buds of clitocybe maxima according to claim 1, which is characterized in that: in the step (6), during the transplanting process, the water depth in the water tank is 2cm, and the soil depth is 10cm.
4. The tissue culture and rapid propagation method of dormant buds of clitocybe maxima according to claim 1, which is characterized in that: wherein the induction medium, the proliferation medium and the rooting medium are all sterilized at 121 ℃ for 25 minutes, and the pH value is adjusted to 5.6-5.8 before sterilization.
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