CN108064690B - Tissue culture method of herba Amaranthi Tricoloris - Google Patents
Tissue culture method of herba Amaranthi Tricoloris Download PDFInfo
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- 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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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
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Abstract
The invention discloses a tissue culture method of amaranthus sanguinea, which comprises the following steps: 1) taking the current-year stem section of the amaranthus mangostanus, sterilizing with alcohol, cleaning, and sterilizing with mercuric chloride to obtain a sterilized explant; 2) inoculating the sterilized explant obtained in the step 1) into a primary culture medium for primary culture to obtain axillary buds; inoculating the axillary buds to an induction culture medium for induction culture to obtain rooted seedlings; cutting stem segments with buds by taking the rooted seedlings as female parents, inoculating the cut stem segments to a subculture multiplication culture medium, and carrying out subculture multiplication culture to obtain tissue culture multiplication seedlings; the primary culture medium, the induction culture medium and the secondary multiplication culture medium are composed of: 1/2MS minimal medium, 0.05-0.2 mg/LNAA, 6-8 g/L agar is supplemented; 3) and 3) hardening and transplanting the tissue culture seedlings obtained in the step 2) to obtain the acclimatized seedling of the amaranthus sanguinea. The tissue culture method improves the survival rate of the hardening seedlings of the amaranthus palmeri, has short culture period, high propagation coefficient, simple and convenient culture process, time and labor conservation and no limitation of seasons.
Description
Technical Field
The invention relates to the technical field of vegetative propagation of plants, in particular to a tissue culture method of amaranthus sanguinea.
Background
Sang amaranth Iresineherbistii hook.f. is a perennial herb of the genus sang amaranth of the family Amaranthaceae, 1-2 meters high; the stem is thick, red, branched, with soft hair at the beginning, and almost no hair except the nodes, with longitudinal edges and furrows. The leaves are wide, oval to nearly circular, all around, and have hair on both sides, purple red, and yellow veins if green or light green; green white or yellow white, and the outer base is white and soft; the sterile stamens are tiny; the ovary is spherical, the side is flat, and the style is extremely short. Male flowers and fruits are not seen.
The whole plant of the bloody amaranth is purple red, the leaves are green and bright, the veins are yellow or red, the leaves are gorgeous, and the bloody amaranth is very durable to see. The method is suitable for pot culture, suspended pot culture and flower bed and flower diameter configuration. Becoming a new greening plant with colorful leaves in recent years. At present, the cutting propagation is mainly used for the haemophilus asiaticus, but the propagation is limited by seasons and has low propagation efficiency, so that the domestic demand on seedlings can not be met.
Disclosure of Invention
In view of this, the application provides a tissue culture method of the amaranthus sanguinea, the tissue culture improves the survival rate of acclimatized seedling of the amaranthus sanguinea, the culture period is short, the propagation coefficient is high, the tissue culture process is simple and convenient, time and labor are saved, and the method is not limited by seasons.
In order to solve the technical problems, the technical scheme provided by the invention is a tissue culture method of the amaranthus tricolor, which comprises the following steps:
1) taking the current-year stem segment of the amaranthus sanguinea as an explant, and carrying out alcohol disinfection, cleaning and mercury bichloride sterilization to obtain the disinfected and sterilized explant;
2) inoculating the sterilized explant obtained in the step 1) into a primary culture medium for primary culture to obtain axillary buds; inoculating the axillary buds to an induction culture medium for induction culture to obtain rooted seedlings; cutting stem segments with buds by taking the rooted seedlings of the rooted seedlings as female parents, inoculating the cut stem segments to a subculture multiplication medium, and carrying out subculture multiplication culture to obtain tissue culture multiplication seedlings; the primary culture medium, the induction culture medium and the secondary multiplication culture medium are composed of: 1/2MS minimal medium, 0.05-0.2 mg/LNAA, 6-8 g/L agar is supplemented;
3) and (3) hardening and transplanting the tissue culture proliferated seedlings obtained in the step 2) to obtain acclimated seedlings of the amaranthus tricolor.
Preferably, the primary culture medium, the induction culture medium and the secondary propagation culture medium are composed of: 1/2MS minimal medium, 0.08-0.15 mg/LNAA, 6-8 g/L agar.
Preferably, the primary culture medium, the induction culture medium and the secondary propagation culture medium are composed of: 1/2MS minimal medium, supplemented with 0.1mg/L NAA, 7g/L agar.
Preferably, the pH values of the primary culture medium, the induction culture medium and the secondary propagation culture medium are 6.0-6.5.
Preferably, the primary culture, the rooting culture and the propagation culture are carried out at a culture temperature of 22-24 ℃, a culture humidity of 65-75% and a light-dark period of 12h/12 h.
Preferably, in the hardening-seedling transplanting process, the culture temperature is 24-26 ℃, and the culture humidity is 60-80%.
Preferably, the rooted seedlings have 3-4 segments. Preferably, the height of the tissue culture propagation seedling is 4-6 cm.
Preferably, the step 1) is specifically: taking the current-year stem of 2-3cm of the amaranthus mangostanus as an explant, carrying out alcohol disinfection for 25-45 s, cleaning for 1-3 times, and carrying out mercuric chloride sterilization for 8-12 min to obtain the disinfected and sterilized explant.
Preferably, the step 3) is specifically: placing the tissue culture proliferated seedlings obtained in the step 2) in an outdoor environment, hardening off the seedlings through a closed bottle, hardening off the seedlings through an open bottle, transplanting the seedlings into a hardening-off substrate, and culturing to obtain the acclimated seedlings of the amaranthus.
Preferably, the seedling exercising substrate comprises: the volume ratio of the grass carbon to the perlite is 7: 3.
Preferably, the closed bottle seedling exercising time is 2 d.
Preferably, the time for opening the bottle and hardening seedlings is 3 d.
Preferably, the seedling exercising substrate is placed in a 50-hole plug tray.
Compared with the prior art, the invention has the beneficial effects that:
the tissue culture method disclosed by the invention is used for propagating the amaranthus sanguinea, the propagation time is short, the propagation coefficient is 3-4, the propagation coefficient is high, and the propagation efficiency of the amaranthus sanguinea is improved; can quickly obtain the tissue culture proliferated seedlings of the amaranthus tricolor with consistent genetic characters, and lays a foundation for further developing variety improvement. In the primary culture process, the germination rate is high, and the average axillary bud length is long; the rooting seedlings obtained in the induction culture process have more rooting numbers and high rooting rate; the tissue culture proliferated seedling obtained in the process of subculture proliferation grows strongly.
The primary culture medium, the induction culture medium and the secondary multiplication culture medium used by the tissue culture method are the same culture medium, namely only one culture medium is needed in the invention, multiplication and rooting culture can be simultaneously carried out on the same culture medium, the process is simple and convenient, the operation is simple, time and labor are saved, the cost is low, the segmental propagation is adopted, the seedling is directly hardened and transplanted after the secondary multiplication culture, the transfer to the rooting culture medium is not needed, and the tissue culture multiplication seedlings of the amaranthus can be easily obtained; in addition, the culture medium is simple in composition, sugar-free culture is adopted, namely, sucrose does not need to be added into the culture medium, the cost is saved, the pollution rate is reduced, and good economic benefits are achieved.
According to the tissue culture method, the tissue culture proliferated seedlings of the amaranthus sanguinea tissue culture grow strongly, and after acclimated seedlings obtained after acclimatization and transplantation are transplanted, the survival rate is high and can reach over 90%.
The tissue culture method is not affected by seasonal climate change and natural disasters, and can be produced all the year round. The method can well keep the characteristic characteristics of excellent strains, can be directly applied to actual production, and has better economic, social and ecological benefits.
Drawings
FIG. 1 shows the case of tissue-cultured seedlings in step 2) of example 1 of the present invention;
FIG. 2 shows the condition of the root system of the tissue-cultured seedling in step 2) of example 1 of the present invention;
FIG. 3 shows the acclimatized seedling of the amaranthus tricolor in step 3) of example 1 of the present invention.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the present invention is provided with reference to specific embodiments.
Example 1
The tissue culture method of the amaranthus sanguinea comprises the following steps:
1) taking the current-year stem segment of 2-3cm of the acalypha australis as an explant, carrying out 75 vol% alcohol disinfection for 25-45 s, washing with sterile water for 1-3 times, and carrying out 0.1 vol% mercuric chloride disinfection for 8-12 min to obtain the disinfected and sterilized explant;
2) inoculating the sterilized explant obtained in the step 1) into a primary culture medium for primary culture to obtain axillary buds; inoculating the axillary buds to an induction culture medium for induction culture to obtain rooted seedlings; cutting stem segments with buds by taking the rooted seedlings as female parents, inoculating the cut stem segments to a subculture multiplication culture medium, and carrying out subculture multiplication culture to obtain tissue culture multiplication seedlings; the primary culture medium, the induction culture medium and the secondary multiplication culture medium are composed of: 1/2MS minimal medium supplemented with 0.1mg/LNAA, 7g/L agar; the primary culture, the rooting culture and the proliferation culture are carried out at the culture temperature of 22-24 ℃, the culture humidity of 65-75% and the light-dark period of 12h/12 h; the tissue culture proliferated seedling is shown in figure 1, and the root system of the tissue culture proliferated seedling is shown in figure 2;
3) placing the tissue culture proliferated seedlings obtained in the step 2) in an outdoor environment, hardening the seedlings for 2d through closing a bottle under the conditions that the culture temperature is 24-26 ℃ and the culture humidity is 60-80%, and transplanting the seedlings into a hardening substrate for culture after the seedlings are hardened for 3d through opening the bottle to obtain the acclimated seedlings of the amaranthus tricolor; wherein, the hardening seedling substrate is arranged in a 50-hole plug tray and comprises: the volume ratio of the grass peat to the vermiculite is 7: 3; the acclimatized seedling of the amaranthus tricolor is shown in figure 3.
In this example, the time for the first axillary bud to grow was 13 days during the primary culture; after the primary culture, the axillary bud is extended by 2-3 cm. In the process of induction culture, inoculating axillary buds to start expanding germination after 15-20 days; the time for obtaining 3-4 segments of rooted seedlings is 25-30 days; the average number of roots of each rooted seedling is about 14, and the root length is 10 cm. And in the process of subculture proliferation, the height of the obtained tissue culture proliferated seedling is 4-6 cm, the time is 25-30 days, and the proliferation coefficient is 4.
Example 2
The present invention differs from example 1 only in that the composition of the primary culture medium, the induction medium and the secondary propagation medium is: 1/2MS minimal medium supplemented with 0.05mg/LNAA, 7g/L agar.
In this example, the time for the first axillary bud to grow was 18 days during the primary culture; after the primary culture, the axillary bud is extended by 2-3 cm. In the process of induction culture, inoculating axillary buds to start expanding germination after 20-30 days; the time for obtaining 3-4 segments of rooted seedlings is 30-35 days; the average number of roots of each rooted seedling is about 10, and the root length is 8 cm. And in the process of subculture proliferation, the height of the obtained tissue culture proliferated seedling is 4-6 cm, the time is 30-35 days, and the proliferation coefficient is 3.
Example 3
The present invention differs from example 1 only in that the composition of the primary culture medium, the induction medium and the secondary propagation medium is: 1/2MS minimal medium supplemented with 0.08mg/LNAA, 7g/L agar.
In this example, the time for the first axillary bud to grow was 22 days during the primary culture; after the primary culture, the axillary bud is extended by 2-3 cm. In the process of induction culture, inoculating axillary buds to start expanding germination after 20-30 days; the time for obtaining 3-4 segments of rooted seedlings is 30-35 days; the average number of roots of each rooted seedling is about 10, and the root length is 10 cm. And in the process of subculture proliferation, the height of the obtained tissue culture proliferated seedling is 4-6 cm, the time is 30-35 days, and the proliferation coefficient is 3.
Example 4
The present invention differs from example 1 only in that the composition of the primary culture medium, the induction medium and the secondary propagation medium is: 1/2MS minimal medium supplemented with 0.15mg/LNAA, 7g/L agar.
In this example, the time for the first axillary bud to grow was 22 days during the primary culture; after the primary culture, the axillary bud is extended by 2-3 cm. In the process of induction culture, inoculating axillary buds to start expanding germination after 20-30 days; the time for obtaining 3-4 segments of rooted seedlings is 30-35 days; the average number of roots of each rooted seedling is about 12, and the root length is 10 cm. And in the process of subculture proliferation, the height of the obtained tissue culture proliferated seedling is 4-6 cm, the time is 30-35 days, and the proliferation coefficient is 3.
Example 5
The present invention differs from example 1 only in that the composition of the primary culture medium, the induction medium and the secondary propagation medium is: 1/2MS minimal medium supplemented with 0.2mg/LNAA, 7g/L agar.
In this example, the time for the first axillary bud to grow was 23 days during the primary culture; after the primary culture, the axillary bud is extended by 2-3 cm. In the process of induction culture, inoculating axillary buds to start expanding germination after 20-30 days; the time for obtaining 3-4 segments of rooted seedlings is 30-35 days; the average number of roots of each rooted seedling is about 12, and the root length is 10 cm. And in the process of subculture proliferation, the height of the obtained tissue culture proliferated seedling is 4-6 cm, the time is 30-35 days, and the proliferation coefficient is 3.
Example 6
The present invention differs from example 1 only in that the composition of the primary culture medium, the induction medium and the secondary propagation medium is: 1/2MS minimal medium supplemented with 0.1mg/LNAA, 6g/L agar.
In this example, the time for the first axillary bud to grow was 16 days during the primary culture; after the primary culture, the axillary bud is extended by 2-3 cm. In the process of induction culture, the axillary buds are inoculated for 20-25 days to start expanding germination; the time for obtaining 3-4 segments of rooted seedlings is 25-30 days; the average number of roots of each rooted seedling is about 13, and the root length is 10 cm. And in the process of subculture proliferation, the height of the obtained tissue culture proliferated seedling is 4-6 cm, the time is 25-30 days, and the proliferation coefficient is 4.
Example 7
The present invention differs from example 1 only in that the composition of the primary culture medium, the induction medium and the secondary propagation medium is: 1/2MS minimal medium supplemented with 0.2mg/LNAA, 8g/L agar.
In this example, the time for the first axillary bud to grow was 16 days during the primary culture; after the primary culture, the axillary bud is extended by 2-3 cm. In the process of induction culture, the axillary buds are inoculated for 20-25 days to start expanding germination; the time for obtaining 3-4 segments of rooted seedlings is 25-30 days; the average number of roots of each rooted seedling is about 13, and the root length is 10 cm. And in the process of subculture proliferation, the height of the obtained tissue culture proliferated seedling is 4-6 cm, the time is 25-30 days, and the proliferation coefficient is 4.
Example 8
Effect of growth hormone concentration in Primary Medium on axillary bud growth
Taking the current-year stem segment of 2-3cm of the acalypha australis as an explant, carrying out 75 vol% alcohol disinfection for 25-45 s, washing with sterile water for 1-3 times, and carrying out 0.1 vol% mercuric chloride disinfection for 8-12 min to obtain the disinfected and sterilized explant; inoculating the sterilized explant to a primary culture medium for primary culture, wherein the culture temperature is 22-24 ℃, the culture humidity is 65-75%, and the light-dark period is 12h/12 h; the primary culture medium adopts 1/2MS minimal medium, NAA is supplemented, grouping is carried out according to the concentration of NAA, the culture condition of the explant in the primary culture medium is observed and recorded, and the specific grouping and culture results are shown in table 1.
TABLE 1
As can be seen from the above table, when the NAA concentration is 0.1mg/L, the number of the germinated axillary buds is the largest, the germination rate is high, and the average axillary bud length is also the longest, which is the most suitable growth hormone composition condition of the primary culture medium.
Example 9
Effect of growth hormone concentration in Induction Medium on axillary bud growth
Taking the current-year stem segment of 2-3cm of the acalypha australis as an explant, carrying out 75 vol% alcohol disinfection for 25-45 s, washing with sterile water for 1-3 times, and carrying out 0.1 vol% mercuric chloride disinfection for 8-12 min to obtain the disinfected and sterilized explant; inoculating the obtained sterilized explant to a primary culture medium for primary culture to obtain axillary buds; taking a plurality of axillary buds with consistent growth conditions, inoculating the axillary buds to an induction culture medium for induction culture to obtain rooted seedlings; wherein in the primary culture and induction culture processes, the culture temperature is 22-24 ℃, the culture humidity is 65-75%, and the light-dark period is 12h/12 h; the composition of the primary medium was: 1/2MS minimal medium supplemented with 0.1 mg/LNAA; the induction culture medium adopts 1/2MS minimal medium, NAA and 7g/L agar are supplemented, grouping is carried out according to the concentration of the NAA, the culture condition of axillary buds in the induction culture medium is observed and recorded, and the specific grouping and culture results are shown in table 2.
TABLE 2
As can be seen from the above table, the concentration of NAA is 0.1mg/L, the root number is the most, the rooting rate is the highest, and the NAA is the most suitable growth hormone composition condition of the induction culture medium.
Example 10
Effect of growth hormone concentration in multiplication Medium on axillary bud growth
Taking the current-year stem segment of 2-3cm of the acalypha australis as an explant, carrying out 75 vol% alcohol disinfection for 25-45 s, washing with sterile water for 1-3 times, and carrying out 0.1 vol% mercuric chloride disinfection for 8-12 min to obtain the disinfected and sterilized explant; inoculating the obtained sterilized explant to a primary culture medium for primary culture to obtain axillary buds; inoculating axillary buds to an induction culture medium for induction culture to obtain rooted seedlings; cutting stem sections with buds by taking the rooted seedlings as female parents, taking a plurality of stem sections with consistent growth conditions, inoculating the stem sections to a subculture multiplication culture medium for subculture multiplication culture to obtain tissue culture multiplication seedlings; wherein the primary culture, the induction culture and the secondary multiplication culture are carried out at the culture temperature of 22-24 ℃, the culture humidity of 65-75% and the light-dark period of 12h/12 h; the primary culture medium and the induction culture medium comprise the following components: 1/2MS minimal medium supplemented with 0.1mg/LNAA, 7g/L agar; the subculture multiplication medium adopts 1/2MS minimal medium, NAA and 7g/L agar are supplemented, grouping is carried out according to the NAA concentration, the culture condition of the rooted seedlings in the subculture multiplication medium is observed and recorded, and the specific grouping and culture results are shown in table 2.
TABLE 3
As can be seen from the above table, when the NAA concentration is 0.1mg/L, the multiplication coefficient is the largest, the time for obtaining the tissue culture multiplication seedlings by the subculture multiplication is the shortest, and the plant height of the tissue culture multiplication seedlings is the highest, so that the tissue culture multiplication seedlings are the most suitable growth hormone composition conditions of the subculture multiplication medium.
Example 11
Placing the tissue culture proliferated seedlings obtained in the embodiments 1-5 in an outdoor environment, hardening the seedlings for 2d through closing a bottle under the conditions that the culture temperature is 24-26 ℃ and the culture humidity is 60-80%, opening the bottle, hardening the seedlings for 3d, transplanting the seedlings into a hardening seedling substrate, and culturing to obtain the acclimated seedlings of the amaranthus hypochondriacus. Survival rate was measured 45 days after transplantation and the results are shown in table 4.
TABLE 4
| Seedling book | Survival rate (%) |
| Example 1 | 95 |
| Example 2 | 75 |
| Example 3 | 80 |
| Example 4 | 80 |
| Example 5 | 89 |
As can be seen from the table above, the survival rate of the acclimatized amaranth seedling obtained by the tissue culture method is high and is more than 90%.
Example 12
1) Taking the current-year stem segment of 2-3cm of the acalypha australis as an explant, carrying out 75 vol% alcohol disinfection for 25-45 s, washing with sterile water for 1-3 times, and carrying out 0.1 vol% mercuric chloride disinfection for 8-12 min to obtain the disinfected and sterilized explant;
2) inoculating the sterilized explant obtained in the step 1) into a primary culture medium for primary culture to obtain axillary buds; inoculating the axillary buds to an induction culture medium for induction culture to obtain rooted seedlings; cutting stem segments with buds by taking the rooted seedlings as female parents, inoculating the cut stem segments to a subculture multiplication culture medium, and carrying out subculture multiplication culture to obtain tissue culture multiplication seedlings; the primary culture, the rooting culture and the proliferation culture are carried out at the culture temperature of 22-24 ℃, the culture humidity of 65-75% and the light-dark period of 12h/12 h;
3) placing the tissue culture proliferated seedlings obtained in the step 2) in an outdoor environment, hardening the seedlings for 2d through closing a bottle under the conditions that the culture temperature is 24-26 ℃ and the culture humidity is 60-80%, and transplanting the seedlings into a hardening substrate for culture after the seedlings are hardened for 3d through opening the bottle to obtain the acclimated seedlings of the amaranthus tricolor; wherein, the hardening seedling substrate is arranged in a 50-hole plug tray and comprises: the volume ratio of the grass peat to the vermiculite is 7: 3;
in a specific embodiment of this example, the media were grouped according to sucrose content:
experimental groups: the primary culture medium, the induction culture medium and the secondary multiplication culture medium are composed of: 1/2MS minimal medium supplemented with 0.1mg/LNAA, 7g/L agar;
control group 1: the primary culture medium, the induction culture medium and the secondary multiplication culture medium are composed of: 1/2MS minimal medium, supplemented with 0.1mg/LNAA, 10mg/L sucrose, 7g/L agar;
control group 2: the primary culture medium, the induction culture medium and the secondary multiplication culture medium are composed of: 1/2MS minimal medium supplemented with 0.1mg/LNAA, 20mg/L sucrose, 7g/L agar;
control group 3: the primary culture medium, the induction culture medium and the secondary multiplication culture medium are composed of: 1/2MS minimal medium supplemented with 0.1mg/LNAA, 30mg/L sucrose, 7g/L agar;
control group 4
The cutting propagation method is characterized in that the cutting propagation method is adopted for the seedlings of the red amaranth, firstly, the nursery garden is loosened, fertilizer is added, then the red amaranth is processed into a female parent capable of being cut, and the female parent is cut on the nursery garden. And in the later stage, the seedlings are subjected to the working procedures of illumination, fertilization and the like to obtain the seedlings.
Observing and recording the culture conditions of explants in primary culture media of the experimental group and the control group 1-3, the culture conditions of axillary buds in an induction culture medium and the culture conditions of rooting seedlings in a subculture multiplication culture medium, transplanting the seedlings obtained from the experimental group and the control group 1-3 and the seedlings obtained from the control group 4, and managing in a conventional water and fertilizer mode until the seedlings are out of nursery. The survival rate was measured 45 days after transplantation and the results are shown in table 5;
TABLE 5
From the above table, it can be seen that the addition and concentration of sucrose have no influence on the culture conditions of the explant in the primary culture medium, the axillary buds in the induction culture medium and the rooting seedlings in the secondary multiplication culture medium. Compared with the hardening seedling of the cutting propagation of the amaranthus sanguinea, the tissue culture method of the amaranthus sanguinea provided by the invention improves the survival rate of the hardening seedling.
In the present invention, medium composition is explained:
1/2MS minimal medium is a medium formed by halving macroelements and keeping the rest unchanged in MS minimal medium. The MS minimal medium 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 nutrition and physiological requirements of plant cells, has wider application range, and can be used as a minimal medium for rapid propagation of most plant tissues.
NAA: NAA is naphthylacetic acid, is a plant growth hormone, is used when plants are propagated by a cutting method, can also be used for plant tissue culture, can promote cell division and expansion, induce to form adventitious roots to increase fruit setting, prevent fruit drop, change the ratio of female flowers and male flowers and the like, can enter the plants through the tender epidermis of leaves and branches, and seeds enter the plants to be guided to the whole plants along with nutrient flow.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Claims (10)
1. A tissue culture method of the amaranthus tricolor is characterized by comprising the following steps:
1) taking the current-year stem segment of the amaranthus sanguinea as an explant, and carrying out alcohol disinfection, cleaning and mercury bichloride sterilization to obtain the disinfected and sterilized explant;
2) inoculating the sterilized explant obtained in the step 1) into a primary culture medium for primary culture to obtain axillary buds; inoculating the axillary buds to an induction culture medium for induction culture to obtain rooted seedlings; cutting stem segments with buds by taking the rooted seedlings as female parents, inoculating the cut stem segments to a subculture multiplication culture medium, and carrying out subculture multiplication culture to obtain tissue culture multiplication seedlings; the primary culture medium, the induction culture medium and the secondary multiplication culture medium are composed of: 1/2MS minimal medium, 0.05-0.2 mg/LNAA, 6-8 g/L agar is supplemented;
3) and (3) hardening and transplanting the tissue culture proliferated seedlings obtained in the step 2) to obtain acclimated seedlings of the amaranthus tricolor.
2. The tissue culture method according to claim 1, wherein the composition of the primary medium, the induction medium and the secondary propagation medium is: 1/2MS minimal medium, 0.08-0.15 mg/LNAA, 6-8 g/L agar.
3. The tissue culture method according to claim 2, wherein the composition of the primary medium, the induction medium and the secondary propagation medium is: 1/2MS minimal medium, supplemented with 0.1mg/L NAA, 7g/L agar.
4. The tissue culture method according to claim 1, wherein the primary culture medium, the induction culture medium and the secondary propagation culture medium have a pH value of 6.0 to 6.5.
5. The tissue culture method according to claim 1, wherein the primary culture, the induction culture and the propagation culture are carried out at a culture temperature of 22 to 24 ℃, a culture humidity of 65 to 75% and a light-dark period of 12h/12 h.
6. The tissue culture method according to claim 1, wherein in the hardening seedling transplanting process, the culture temperature is 24-26 ℃ and the culture humidity is 60-80%.
7. The tissue culture method according to claim 1, wherein the rooted shoots have 3 to 4 segments.
8. The tissue culture method according to claim 1, wherein the step 1) is specifically: taking the current-year stem of 2-3cm of the amaranthus mangostanus as an explant, carrying out alcohol disinfection for 25-45 s, cleaning for 1-3 times, and carrying out mercuric chloride sterilization for 8-12 min to obtain the disinfected and sterilized explant.
9. The tissue culture method according to claim 1, wherein the step 3) is specifically: placing the tissue culture proliferated seedlings obtained in the step 2) in an outdoor environment, hardening off the seedlings through a closed bottle, hardening off the seedlings through an open bottle, transplanting the seedlings into a hardening-off substrate, and culturing to obtain the acclimated seedlings of the amaranthus.
10. The tissue culture method of claim 9, wherein the seeding matrix comprises: the volume ratio of the grass carbon to the perlite is 7: 3.
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| CN105638464B (en) * | 2015-12-30 | 2018-01-09 | 四川七彩林业开发有限公司 | A kind of tissue culture and rapid propagation method of silk ribbon grass |
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