CN115250919A - Eucalyptus robusta tissue culture method - Google Patents
Eucalyptus robusta tissue culture method Download PDFInfo
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- A—HUMAN NECESSITIES
- 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 eucalyptus grandis tissue culture method, which comprises the following steps: the eucalyptus robusta seeds are taken as explants and are subjected to refrigeration dark treatment, heat treatment and disinfection in sequence; inoculating the treated eucalyptus robusta seeds into an MS minimal medium, and culturing under a dark condition until the seeds are planted and germinated; after the seeds germinate to 4-5cm high tissue culture seedlings, taking stems with 1-2 axillary buds, and placing the stems in a cluster bud induction culture medium for cluster bud induction; when the cluster buds grow to be 2-3 cm high, taking out the cluster buds, and inoculating the cluster buds into a culture medium containing 6-BA and NAA for strong seedling culture; when the tissue culture seedling grows to 4-5cm, transferring the tissue culture seedling into a rooting culture medium containing rooting phytohormone and riboflavin, and carrying out rooting culture. The invention not only realizes the rapid propagation of the eucalyptus robusta in a short time, but also makes the engineered propagation of the eucalyptus robusta possible.
Description
Technical Field
The invention belongs to plant tissue culture, and particularly relates to a eucalyptus grandis tissue culture method.
Background
Eucalyptus robusta (Eucalyptus robusta Smith) Eucalyptus plants of Myrtaceae are native to Australian offshore fertile and slightly salty places, and are respectively called mosquito young trees. The eucalyptus robusta can be as high as 20 m, and the bark is slightly soft and has irregular oblique furrows; the leaves are oval and thick leathery, and the two surfaces of the leaves are provided with glandular spots and handles; the florescence is 9-10 months, and the inflorescence is umbrella-shaped; the peel of the eucalyptus robusta is dark in color, speckles and plaques when the eucalyptus robusta fruit is ripe. The eucalyptus robusta grows rapidly, has strong updating capability, poor cold resistance and drought resistance and short growth period, and the growth wheel of the eucalyptus robusta is easily influenced by the growth environment in the growth process, but has uniform and hard structure and corrosion resistance of the core material. In the traditional Chinese pharmacology, the eucalyptus robusta is pungent and bitter in taste and cold in nature, has the effects of dispelling wind and relieving exterior syndrome, clearing heat and removing toxicity, killing parasites and relieving itching and the like, and is often applied to the aspects of eliminating bacteria, relieving inflammation, resisting insects and the like. Due to the influence of factors such as low branch cutting rooting capacity, weak seed reproductive capacity and the like of the eucalyptus robusta, the demand of the eucalyptus robusta for realizing engineered propagation is increased. At present, in the domestic and foreign researches on the eucalyptus robusta, no research on the tissue culture of the eucalyptus robusta plant is found, so that the research provided by the invention not only can provide theoretical reference for researching the tissue culture technology of the eucalyptus robusta plant, but also can promote the research progress of the rapid propagation engineering of the eucalyptus robusta. The research on the eucalyptus robusta can maximize the medicinal value and the economic value of the eucalyptus robusta and benefit the mankind.
Disclosure of Invention
The invention aims to provide a tissue culture method of eucalyptus robusta, which not only realizes the rapid propagation of the eucalyptus robusta in a short time, but also makes the engineered propagation of the eucalyptus robusta possible. Not only provides theoretical reference for researching the eucalyptus robusta plant tissue culture technology, but also can promote the research progress of the eucalyptus robusta for realizing rapid propagation engineering.
In order to realize the purpose, the invention adopts the following technical scheme:
the tissue culture method of the eucalyptus robusta comprises the following steps:
(1) Treatment of explants: taking the eucalyptus robusta seeds as explants, and sequentially carrying out cold storage dark treatment, heat treatment and disinfection;
(2) Seed germination: inoculating the treated eucalyptus robusta seeds into an MS minimal medium, and culturing under a dark condition until the seeds are planted and germinated;
(3) Inducing cluster buds: when the seeds germinate to 4-5cm high tissue culture seedlings, taking stems with 1-2 axillary buds, and placing the stems in a cluster bud induction culture medium for cluster bud induction;
(4) Strong seedling culture: when the cluster buds grow to be 2-3 cm high, taking out the cluster buds, and inoculating the cluster buds into a strong seedling culture medium containing 6-BA and NAA for strong seedling culture;
(5) Rooting culture: when the tissue culture seedling grows to 4-5cm, transferring the tissue culture seedling into a rooting culture medium containing rooting phytohormone and riboflavin for rooting culture.
In the step (1), the plump eucalyptus robusta seeds with high maturity and no impurities are selected as explants. The dark treatment condition is that the eucalyptus robusta seeds are put into the temperature of minus 20 ℃ to 3 ℃ for dark treatment for 24 to 48 hours. The heat treatment conditions are as follows: immersing the eucalyptus robusta seeds in water, and carrying out constant-temperature water bath at 45 ℃ for 6-8h. In the disinfection treatment, the eucalyptus robusta seeds are soaked in 75% alcohol for 30s, shaken and washed for 3 times in sterile water, then soaked in 0.1% mercuric chloride for disinfection for 2-8min, and then shaken and washed for 5 times in sterile water.
Preferably, step (1) explant treatment: storing the seeds at-18 ℃ for 48h, taking out, soaking in a constant-temperature water bath kettle at 45 ℃ for 8h to increase the germination quantity of the eucalyptus robusta seeds.
In step (2), the MS minimal medium is modified MS minimal medium, preferably containing modified MS +1mg/L GA 3 +30g/L sucrose +7g/L agar, pH 5.8 + -0.2, maximum seed germination quantity and best quality.
Preferably, explant sterilization: sterilizing with 0.1% mercuric chloride solution after 30S with 75% alcohol for 4min, with moderate seed pollution rate and highest seed germination amount.
In the step (3), the temperature of the culture for inducing the cluster buds is controlled to be 25 +/-2 ℃, the illumination intensity is 1500-2000lx, and the illumination time is 12h/d. The cluster bud induction culture medium comprises modified MS +3.2mg/L6-BA +0.8mg/LNAA +30g/L sucrose +7g/L agar, the pH value is 5.8 +/-0.2, and the average number of cluster buds in each bottle is 3.
In the step (4), tannin substances are generated due to good growth of the eucalyptus robusta tissue culture seedlings, so that the tissue culture seedlings die due to browning in the strong seedling process, and in order to prevent the situation, a proper amount of riboflavin needs to be added into a strong seedling culture medium. Strong seedling culture conditions: 3 clump buds are inoculated into each bottle of culture medium, the culture temperature is controlled at 25 +/-2 ℃, the illumination intensity is 1500-2000lx, and the illumination time is 12h/d.
The strong seedling culture medium in the step (4) is as follows: modified MS +4mg/L6-BA +2mg/L IBA +0.8mg/L riboflavin +30g/L sucrose +7g/L agar, modified MS +2mg/L6-BA +2mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, or modified MS +4mg/L6-BA +0.8mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, pH value is 5.8 (+ -0.2), tissue culture plantlets grow well, and the two culture mediums take root.
In the step (5), a few roots grow in the process of strengthening the seedlings, the roots are cut off by sterilized surgical scissors and then are transferred into a rooting culture medium, and the tissue culture seedlings are induced to grow into root systems. Rooting culture conditions are as follows: 2-3 seedlings are inoculated into each bottle of culture medium, the culture temperature is controlled at 25 +/-2 ℃, the illumination intensity is 1500-2000lx, and the illumination time is 12h/d.
The rooting culture medium in the step (5) comprises: the improved MS +2.4mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, the improved MS +3.2mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, the pH value is 5.8 (+/-0.2), the rooting rate is up to 100%, and the tissue culture seedling grows well.
The beneficial effects of the invention are:
1. according to the eucalyptus grandis tissue culture method provided by the invention, the seeds of the eucalyptus grandis are subjected to low-temperature dark treatment in the early stage, and then are placed in a constant-temperature water bath for seed soaking treatment. The low-temperature treatment can realize that the eucalyptus robusta seeds uniformly enter dormancy, and the constant-temperature seed soaking can recover the seeds, thereby effectively accelerating the germination time and the germination number of the seeds and improving the germination rate of the seeds.
2. The invention adopts a disinfection method of 75% alcohol 30S +0.1% mercuric chloride solution for 4min, the pollution rate of the seeds is moderate, and the germination quantity of the seeds is the most.
3. In the invention, the optimal hormone proportion of the culture medium for inducing seed germination is as follows: improved MS minimal medium +1mg/L GA 3 Under the hormone proportion, the germination quantity of the seeds is the highest. The optimal hormone proportion of the cluster bud induction culture medium for inducing the eucalyptus robusta to generate the cluster buds is as follows: the improved MS minimal medium +3.2mg/L6-BA +0.8mg/L NAA, the number of the cluster buds is 3, the stems and leaves of the cluster buds grow fast, the stems are thick and strong, and the leaves are green.
4. In the invention, the strong seedling culture medium hormone proportion of the eucalyptus robusta tissue culture seedling is as follows: the improved MS +4mg/L6-BA +2mg/L IBA +0.8mg/L riboflavin, the improved MS +2mg/L6-BA +2mg/LIBA +4mg/L riboflavin and the improved MS +4mg/L6-BA +0.8mg/L IBA +4mg/L riboflavin. The culture medium hormone proportion of the rooting of the eucalyptus robusta tissue culture seedling is as follows: improved MS +2.4mg/L IBA +4mg/L riboflavin, improved MS +3.2mg/L IBA +4mg/L riboflavin, and in the rooting culture media, the rooting rate is as high as 100%, the roots are more hairy, and the roots are developed.
4. The culture conditions of steps (1) to (5) of the present invention are all: controlling the temperature to be (25 +/-2) DEG C; the illumination intensity is 1500-2000lx; the illumination time is 12h/d. The proper temperature, illumination intensity and illumination time can promote the growth of the germinated swamp mahogany seeds, the induction of cluster buds, the test of strong seedlings and the rooting, and create a good environment for the tissue culture seedlings of the swamp mahogany.
Drawings
FIG. 1 shows seed germination of Eucalyptus robusta B4;
FIG. 2 is an induced clumping bud of Eucalyptus robusta C4;
FIG. 3 shows a strong seedling culture experiment of D2;
FIG. 4 shows a strong seedling culture experiment of D3 of Eucalyptus robusta;
FIG. 5 shows a strong seedling culture experiment of D4;
FIG. 6 shows rooting culture experiments for E3;
FIG. 7 shows rooting culture experiments for E4 of Eucalyptus robusta;
FIG. 8 shows rooting culture experiments for E4 of Eucalyptus robusta; .
Detailed Description
In order to describe the present invention in more detail, the present invention is further illustrated by the following examples.
Example 1
(1) Explant treatment: the method selects the plump eucalyptus robusta seeds with high maturity and no impurity as explants. Storing the seeds at-18 deg.C for 24h, taking out, and soaking in a 45 deg.C constant temperature water bath for 6h. The treated explants were sterilized with 0.1% mercuric chloride solution for 2min after 30S with 75% alcohol.
(2) Seed germination: inoculating the sterilized explant into a culture medium, wherein the culture medium comprises: MS +30g/L of sucrose +7g/L of agar is improved, the pH value is 5.8 +/-0.2, and the mixture is cultured under the dark condition until the mixture is planted and germinated.
(3) Inducing cluster buds: when the seeds germinate into tissue culture seedlings with the height of 5cm, cutting off roots and leaves, leaving stem sections with 1-2 bud eyes, inoculating a cluster bud induction culture medium, and inducing and culturing cluster buds. The culture medium for inducing the cluster buds comprises: modified MS +1 mg/L6-BA +0.8mg/L NAA +30g/L sucrose +7g/L agar, and the pH value is 5.8 +/-0.2.
(4) Strong seedling culture: shearing cluster buds, and inoculating the cluster buds into a strong seedling culture medium to culture strong seedlings. Strong seedling culture medium: modified MS +4mg/L6-BA +0.8mg/L riboflavin +30g/L sucrose +7g/L agar, culturing in a pH value of 5.8 +/-0.2, and inoculating 3 seedlings into each bottle.
(5) Rooting culture: and (4) inoculating the well-grown tissue culture seedlings into a rooting culture medium for rooting culture. The rooting culture medium comprises: improved MS +0.8mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, pH value of 5.8 +/-0.2, and 3 tissue culture seedlings are inoculated into each bottle.
The culture conditions of the steps are as follows: controlling the temperature at 25 +/-2 ℃; the illumination intensity is 1500-2000lx; the illumination time is 12h/d.
Example 2
(1) Explant treatment: the plump eucalyptus robusta seeds with high maturity, no impurities are selected as explants. Storing the seeds at-18 deg.C for 32h, taking out, and soaking in a 45 deg.C constant temperature water bath for 7h. The treated explants were disinfected with 0.1% mercuric chloride solution for 4min after 30S in 75% ethanol.
(2) Seed germination: inoculating the sterilized explants into a culture medium, wherein the culture medium comprises: MS +1mg/LIAA +30g/L sucrose +7g/L agar is improved, the pH value is 5.8 +/-0.2, and the mixture is cultured under the dark condition until the mixture is planted and germinated.
(3) Inducing cluster buds: when the seeds germinate into tissue culture seedlings with the height of 5cm, cutting off roots and leaves, leaving stem sections with 1-2 bud eyes, inoculating a cluster bud induction culture medium, and inducing and culturing cluster buds. The cluster bud induction culture medium comprises: modified MS +2.4 mg/L6-BA +0.8mg/L NAA +30g/L sucrose +7g/L agar, and the pH value is 5.8 +/-0.2.
(4) Seedling strengthening experiment: shearing the cluster buds, inoculating the cluster buds into a strong seedling culture medium, and culturing the strong seedlings. Strong seedling culture medium: improved MS +4mg/L6-BA +2mg/L IBA +0.8mg/L riboflavin +30g/L sucrose +7g/L agar, pH value is 5.8 +/-0.2, and 3 seedlings are inoculated into each bottle.
(5) Rooting experiments: and (4) inoculating the well-grown tissue culture seedlings into a rooting culture medium for rooting culture. The rooting culture medium comprises: improved MS +1.6mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, pH value of 5.8 +/-0.2, and 3 tissue culture seedlings are inoculated into each bottle.
The culture conditions of the steps are as follows: controlling the temperature to be 25 +/-2 ℃; the illumination intensity is 1500-2000lx; the illumination time is 12h/d.
Example 3
(1) Explant treatment: the plump eucalyptus robusta seeds with high maturity, no impurities are selected as explants. Storing the seeds at-18 ℃ for 48h, taking out the seeds, and soaking the seeds in a constant-temperature water bath kettle at 45 ℃ for 8h. The treated explants were sterilized with 0.1% mercuric chloride solution for 6min after 30S in 75% ethanol.
(2) Seed germination: inoculating the sterilized explant into a culture medium, wherein the culture medium comprises: MS +2mg/LIAA +30g/L sucrose +7g/L agar is improved, the pH value is 5.8 (+ -0.2), and the mixture is cultured under the dark condition until the mixture is planted and germinated.
(3) Inducing cluster buds: and (3) cutting off roots and leaves after the seeds germinate into tissue culture seedlings with the height of 5cm, leaving stem sections with 1-2 bud eyes, inoculating a cluster bud induction culture medium, and performing induction culture on the cluster buds. The cluster bud induction culture medium comprises: improved MS +2.8 mg/L6-BA +0.8mg/L NAA +30g/L sucrose +7g/L agar, and pH value is 5.8 +/-0.2.
(4) Strong seedling culture: shearing the cluster buds, inoculating the cluster buds into a strong seedling culture medium, and culturing the strong seedlings. Strong seedling culture medium: improved MS +2mg/L6-BA +2mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, pH value is 5.8 +/-0.2, and 3 seedlings are inoculated into each bottle.
(5) Rooting culture: and (4) inoculating the well-grown tissue culture seedlings into a rooting culture medium for rooting culture. The rooting culture medium comprises: improved MS +2.4mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, pH value of 5.8 +/-0.2, and 3 tissue culture seedlings are inoculated into each bottle.
Example 4
(1) Explant treatment: the method selects the plump eucalyptus robusta seeds with high maturity and no impurity as explants. Storing the seeds at-18 deg.C for 48h, taking out, and soaking in a 45 deg.C constant temperature water bath for 8h. The treated explants were sterilized with 0.1% mercuric chloride solution for 8min after 30S in 75% ethanol.
(3) Seed germination: inoculating the sterilized explants into a culture medium, wherein the culture medium comprises: improved MS +1mg/LGA 3 +30g/L of sucrose +7g/L of agar, the pH value is 5.8 +/-0.2, and the mixture is cultured under the dark condition until the plants germinate.
(3) Inducing cluster buds: when the seeds germinate into tissue culture seedlings with the height of 5cm, cutting off roots and leaves, leaving stem sections with 1-2 bud eyes, inoculating a cluster bud induction culture medium, and inducing and culturing cluster buds. The culture medium for inducing the cluster buds comprises: modified MS +3.2mg/L6-BA +0.8mg/L NAA +30g/L sucrose +7g/L agar, and the pH value is 5.8 +/-0.2.
(4) Strong seedling culture: shearing the cluster buds, inoculating the cluster buds into a strong seedling culture medium, and culturing the strong seedlings. Strong seedling culture medium: improved MS +4mg/L6-BA +0.8mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, pH value of 5.8 +/-0.2, and 3 seedlings are inoculated into each bottle.
(5) Rooting culture: and (4) inoculating the well-grown tissue culture seedlings into a rooting culture medium for rooting culture. The rooting culture medium comprises: improved MS +3.2mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, pH value of 5.8 +/-0.2, and 3 tissue culture seedlings are inoculated into each bottle.
The culture conditions of the steps are as follows: controlling the temperature to be 25 +/-2 ℃; the illumination intensity is 1500-2000lx; the illumination time is 12h/d.
Test examples
In order to verify that the method can realize rapid propagation in a short period, the inventor develops a series of experimental researches according to the method recorded in the embodiment 1-4 of the invention in 9-2022-2020, and screens out the phytohormones which are the optimal method for treating seeds, the optimal disinfection time, the optimal method for germinating the seeds, inducing the cluster buds, strengthening the seedlings and rooting in the early stage of the experiment. Wherein, the prophase treatment of the explants is totally 3 treatments, each treatment comprises 7 bottles, each bottle comprises a proper amount of explants, and the numbers of the explants are 1, 2 and 3. Counting the pollution rate 7 days after inoculation, counting the germination number and the browning rate 14 days after inoculation, respectively recording the germination conditions of 14 days and 30 days, and repeating each test for 3 times; 4 treatments are carried out on the explants in a disinfection mode, 7 bottles of the explants are treated respectively, the numbers of the bottles are A1, A2, A3 and A4, the germination condition and the pollution rate of the seeds are recorded 7 days after inoculation, and each test is repeated for 3 times; the germination experiment of the seeds comprises 4 treatments, wherein each treatment comprises 7 bottles, each bottle comprises a proper amount of explants numbered as B1, B2, B3 and B4, the pollution rate is counted after 7 days of inoculation, the germination number and the browning rate are counted after 14 days, the germination conditions of 14 days and 30 days are respectively recorded, and each experiment is repeated for 3 times; 4 treatments are carried out on the cluster bud induction test, wherein each treatment comprises 7 bottles, 1 stem segment is numbered as C1, C2, C3 and C4, the number of the cluster buds is recorded after 30 days, the induction conditions of the cluster buds in 60 days and 90 days are recorded respectively, and the experiment is repeated for 3 times for each treatment; 4 treatments are carried out on strong seedling culture, each treatment comprises 7 bottles, each bottle comprises 3 tissue culture seedlings, the numbers of the 3 tissue culture seedlings are D1, D2, D3 and D4, the growth conditions of the tissue culture seedlings are recorded 7 days after inoculation, and each treatment is carried out for 3 times; and (3) treating the rooting culture for 4 times, wherein 7 bottles are treated, 3 tissue culture seedlings are treated in each bottle, the numbers of the tissue culture seedlings are E1, E2, E3 and E4, the growth vigor of the seedlings and the growth condition of roots are observed for 7 days, the growth conditions of the roots after 15 days and 30 days are recorded respectively, and the experiment is repeated for 3 times for each treatment. The germination condition of the seeds is shown in table 1 and figure 1, the induction condition of the cluster buds is shown in table 2 and figure 2, the growth condition of the strong seedlings is shown in table 3 and figure 3, figure 4 and figure 5, and the rooting condition is shown in table 4 and figure 6, figure 7 and figure 8.
TABLE 1 Eucalyptus robusta seed Germination
TABLE 2 Eucalyptus robusta cluster bud Induction
TABLE 3 Eucalyptus robusta seedling strengthening situation
TABLE 4 Eucalyptus robusta rooting conditions
Note: root length was measured after 1 month; rooting rate = (number of rooted bud seedlings/total number of inoculated tissue culture seedlings) × 100%
The foregoing is only a partial embodiment of this invention and all changes, equivalents and modifications that come within the spirit and scope of the invention are desired to be protected.
Claims (10)
1. The eucalyptus grandis tissue culture method is characterized by comprising the following steps:
(1) And (3) processing the explants: taking the eucalyptus robusta seeds as explants, and sequentially carrying out cold storage dark treatment, heat treatment and disinfection;
(2) Seed germination: inoculating the treated eucalyptus robusta seeds into an MS minimal medium, and culturing under a dark condition until the seeds are planted and germinated;
(3) Inducing cluster buds: when the seeds germinate to 4-5cm high tissue culture seedlings, taking stems with 1-2 axillary buds, and placing the stems in a cluster bud induction culture medium for cluster bud induction;
(4) Strong seedling culture: when the cluster buds grow to be 2-3 cm high, taking out the cluster buds, and inoculating the cluster buds into a culture medium containing 6-BA and NAA for strong seedling culture;
(5) Rooting culture: when the tissue culture seedling grows to 4-5cm, transferring the tissue culture seedling into a rooting culture medium containing rooting phytohormone and riboflavin for rooting culture.
2. The tissue culture method of Eucalyptus robusta of claim 1, wherein in step (1), the dark treatment conditions comprise placing Eucalyptus robusta seeds in-20-3 deg.C for dark treatment for 24-48h; the heat treatment conditions are as follows: immersing the eucalyptus robusta seeds in water, and carrying out constant-temperature water bath at 45 ℃ for 6-8h; in the disinfection treatment, the eucalyptus robusta seeds are soaked in 75% alcohol for 30s, vibrated and washed in sterile water for 3 times, soaked in 0.1% mercuric chloride for disinfection for 2-8min, and vibrated and washed in sterile water for 5 times.
3. The method for tissue culture of eucalyptus robusta as claimed in claim 1, wherein the explant sterilization: sterilizing with 0.1% mercuric chloride solution 30s after 75% alcohol for 4min, with moderate seed pollution rate and highest seed germination amount.
4. The method for tissue culture of eucalyptus robusta as claimed in claim 1, wherein the explant treatment in step (1): storing the seeds at-18 ℃ for 48h, taking out the seeds, and soaking the seeds in a constant-temperature water bath kettle at 45 ℃ for 8h.
5. The method for tissue culture of Eucalyptus robusta as claimed in claim 1, wherein in step (2), the MS minimal medium is modified MS minimal medium comprising modified MS +1mg/L GA 3 +30g/L sucrose +7g/L agar, pH 5.8. + -. 0.2.
6. The tissue culture method of Eucalyptus robusta according to claim 1, wherein in the step (3), the culture medium for inducing the multiple shoots comprises modified MS +3.2mg/L6-BA +0.8mg/L NAA +30g/L sucrose +7g/L agar, pH is 5.8 ± 0.2, and the average number of the multiple shoots per bottle is 3.
7. The tissue culture method of eucalyptus robusta as claimed in claim 1, wherein the strong seedling culture medium in step (4) is: modified MS +4mg/L6-BA +2mg/L IBA +0.8mg/L riboflavin +30g/L sucrose +7g/L agar, modified MS +2mg/L6-BA +2mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, or modified MS +4mg/L6-BA +0.8mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, and the pH value is 5.8 +/-0.2.
8. The method for tissue culture of eucalyptus robusta as claimed in claim 1, wherein in step (4), the strong seedling culture conditions are as follows: 3 cluster buds are inoculated into each bottle of culture medium, the culture temperature is controlled at 25 +/-2 ℃, the illumination intensity is 1500-2000lx, and the illumination time is 12h/d.
9. The method for tissue culture of Eucalyptus robusta of claim 1, wherein the rooting medium of step (5) is: modified MS +2.4mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, modified MS +3.2mg/L IBA +4mg/L riboflavin +30g/L sucrose +7g/L agar, and pH value is 5.8 +/-0.2.
10. The method for tissue culture of Eucalyptus robusta as claimed in claim 1, wherein in the step (5), rooting culture conditions are as follows: 2-3 seedlings are inoculated into each bottle of culture medium, the culture temperature is controlled at 25 +/-2 ℃, the illumination intensity is 1500-2000lx, and the illumination time is 12h/d.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
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| CN202211041380.4A CN115250919A (en) | 2022-08-29 | 2022-08-29 | Eucalyptus robusta tissue culture method |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101331853A (en) * | 2008-08-05 | 2008-12-31 | 山东省林业科学研究院 | Rooting method of tissue culture and rapid propagation of eucalyptus dunni |
| CN107517854A (en) * | 2017-10-10 | 2017-12-29 | 江苏凤谷生物有限公司 | A kind of tissue culture and rapid propagation method of roundleaf eucalyptus |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101331853A (en) * | 2008-08-05 | 2008-12-31 | 山东省林业科学研究院 | Rooting method of tissue culture and rapid propagation of eucalyptus dunni |
| CN107517854A (en) * | 2017-10-10 | 2017-12-29 | 江苏凤谷生物有限公司 | A kind of tissue culture and rapid propagation method of roundleaf eucalyptus |
Non-Patent Citations (8)
| Title |
|---|
| SILVA ET AL.: "MICROPROPAGATION OF EUCALYPTUS SALIGNA SM. FROM COTYLEDONARY NODES", 《PAK. J. BOT.》 * |
| 宋艳祥等: "热处理和抗生素对桉树固有内生细菌去除的研究", 《河北农业大学学报》 * |
| 易敏等: "邓恩桉的组织培养技术研究", 《广东林业科技》 * |
| 杨卫星等: "桉树无性系雷11 组织培养技术研究", 《安徽农业科学》 * |
| 杨艳等: "红冠桉茎段离体培养再生植株的研究", 《中南林业科技大学学报》 * |
| 蔡玲等: "尾圆桉离体芽组培快繁技术研究", 《广西林业科学》 * |
| 陈剑勇等: "尾赤桉组织培养技术研究", 《西南林业学院学报》 * |
| 高丽琼等: "EC21号桉树无性系的组织培养技术研究", 《桉树科技》 * |
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