CN111316919A - Method for improving regeneration efficiency in cinnamomum camphora tissue culture process - Google Patents
Method for improving regeneration efficiency in cinnamomum camphora tissue culture process Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
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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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
Abstract
The invention provides a method for improving regeneration efficiency in a cinnamomum camphora tissue culture process, which comprises the steps of firstly disinfecting cinnamomum camphora seeds and germinating the disinfected seeds to obtain cinnamomum camphora aseptic seedlings; then, taking tender stem segments of the camphor tree aseptic seedlings as explants, and establishing a corresponding camphor tree tissue culture method through the steps of bud induction, bud multiplication and bud rooting; wherein activated carbon is added as an additional component to the "multiplication medium" used in the "multiplication of buds" step and the "rooting medium" used in the "rooting of buds" step, respectively. The invention has the advantages that: (1) activated carbon is added into part of the culture medium to absorb metabolic substances, so that the increment coefficient of the tissue culture seedling is improved, the browning rate is reduced, and the rooting rate is improved, thereby improving the regeneration efficiency of the cinnamomum camphora tissue culture; (2) the seeds are sterilized, so that the damage of the sterilization process to the tender stem segments of the required materials in the subsequent test is effectively avoided.
Description
Technical Field
The invention relates to the technical field of plant culture, in particular to a method for improving regeneration efficiency in a cinnamomum camphora tissue culture process.
Background
Cinnamomum camphora (Cinnamomum camphora), Lauraceae Cinnamomum, tree-shaped ambidextrous spectabilis, tall and straight trunk, evergreen four seasons, heavy and dark crowns, beautiful branches and leaves and fragrant smell, is a very beautiful and excellent street tree in southern cities of China, and is one of the indispensable tree species for forming plant gardening landscape in landscape gardens.
At present, the cinnamomum camphora is mainly cultivated in three ways. Firstly, seedling raising is carried out, which is the main mode for culturing camphor tree seedlings at present; but seedling culture has the defects of strong seasonality, difficult preservation of excellent characters and the like. Secondly, cutting seedling raising needs more scion stock plants, and meanwhile, the cutting is greatly influenced by environmental conditions and the rooting effect is unstable; thirdly, tissue culture technology, which is the most suitable technology for rapid propagation of the female parent material when the female parent material is limited.
However, during the tissue culture process of cinnamomum camphora, since various metabolic substances are accumulated during the growth of cinnamomum camphora, the plant often has the phenomena of low proliferation coefficient, difficult rooting, high browning rate and the like, so the normal growth of cinnamomum camphora plants is affected by different degrees, and the plant regeneration efficiency during the tissue culture process of cinnamomum camphora cannot be guaranteed.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for improving the regeneration efficiency in the camphor tree tissue culture process; the method greatly improves the regeneration efficiency of camphor plants by effectively improving the proliferation coefficient of camphor trees, reducing the browning rate of bud seedlings and improving the rooting rate of plants.
The invention adopts the following technical scheme to solve the technical problems:
a method for improving regeneration efficiency in Cinnamomum camphora tissue culture process comprises sterilizing Cinnamomum camphora seeds, and germinating the sterilized seeds to obtain aseptic Cinnamomum camphora seedlings; then, taking tender stem segments of the camphor tree aseptic seedlings as explants, and establishing a corresponding camphor tree tissue culture method through the steps of bud induction, bud multiplication and bud rooting; wherein activated carbon is added as an additional component to the "multiplication medium" used in the "multiplication of buds" step and the "rooting medium" used in the "rooting of buds" step, respectively.
In a preferred embodiment of the present invention, the method for culturing cinnamomum camphora tissue is specifically as follows:
(1) obtaining and disinfecting explants:
① selecting full Cinnamomum camphora seeds, soaking in sterile water for 3-5h, rinsing with 75% alcohol on a superclean bench for 10-20s, repeating for 2-4 times, soaking with 1% sodium hypochlorite for 5min, and washing with sterile water for 4-6 times;
② inoculating the Cinnamomum camphora seed with water to aseptic seedling culture medium (MS, pH 5.8) for culturing to obtain Cinnamomum camphora aseptic seedling;
(2) and (3) induction of buds:
taking tender stem section of 25-35 days cultured Cinnamomum camphora aseptic seedling as explant, and cutting into size of 0.5-0.6 cm; then inoculating the bud-inducing culture medium into a culture bottle containing an inducing culture medium to induce adventitious buds; wherein the formula of the induction culture medium is as follows: MS +1 mg/L6-BA +0.1mg/L NAA, pH 5.8;
(3) and (3) bud multiplication:
dividing the buds obtained in the step (2) into individual plants, and respectively inoculating the individual plants into a proliferation culture medium for proliferation culture; wherein the formula of the proliferation culture medium is as follows: MS +3 mg/L6-BA +0.5mg/L NAA +2g/L active carbon, pH 5.8;
(4) rooting of the buds:
when the camphor bud seedlings grow to 3cm high, inoculating the camphor bud seedlings into a rooting culture medium for rooting culture; wherein, the formula of the rooting culture is as follows: 1/2MS +0.5mg/L IBA +0.3mg/L NAA +2g/L activated carbon, pH 5.8.
As one of the preferable modes of the invention, in the step (1), the cinnamomum camphora seeds with full particles are selected, soaked in sterile water for 4 hours, rinsed for 15 seconds by using 75% alcohol on a super-clean workbench, repeated for 3 times, soaked for 5min by using 1% sodium hypochlorite, and finally washed for 5 times by using sterile water; after washing, the seeds are dried by using sterile absorbent paper for standby.
In a preferred embodiment of the present invention, the cinnamomum camphora seeds are selected from cinnamomum camphora plants which are strong in growth, straight in dry shape, erect in crown, free from plant diseases and insect pests, strong in age, and resistant to flooding.
In a preferred embodiment of the present invention, in the step (1), the cinnamomum camphora seeds with moisture absorbed are inoculated into culture bottles containing sterile seedling culture medium for culture, 2 seeds are inoculated into each bottle, after 3 weeks of observation, 50 bottles are inoculated, the test is repeated three times, and the germination rate and the contamination rate of the cinnamomum camphora seeds are counted.
In a preferred mode of the present invention, in the step (2), young stem segments of aseptic seedlings of cinnamomum camphora cultured for 30 days are used as explants and cut into 0.55cm size; then, the culture medium was inoculated into a culture flask containing an induction medium to induce adventitious buds.
In a preferred embodiment of the present invention, in the step (1), the step (2), the step (3) and the step (4), the specific culture conditions for the sterile shoot culture, the shoot induction culture, the shoot proliferation culture and the shoot rooting culture are as follows: the culture temperature is 25 +/-1 ℃, the illumination time is 14-16h/d, and the illumination intensity is 1500-.
As one of the preferable modes of the invention, the camphor tree tissue culture method further comprises a seedling exercising and transplanting step after the bud rooting step:
placing the test-tube plantlet obtained in the step of rooting buds in a sterile tissue culture room for acclimatization for a week in a mode of gradually opening a cover, and pouring sterile water into corresponding culture bottles to keep water; taking out the test-tube plantlet after one week, cleaning the culture medium attached to the root of the test-tube plantlet by flowing water, and then transplanting the test-tube plantlet into a sterilized culture medium; placing the container in a room, watering thoroughly, covering the periphery of the container with a plastic film, watering regularly, and reducing the watering frequency after ensuring one week; after 4d, the plastic film is removed until the plastic film is completely removed, and after 7d, the plastic film is removed from the culture chamber and is grown under natural light; wherein the culture medium comprises the following components: perlite is 1: 3.
Compared with the prior art, the invention has the advantages that:
(1) according to the invention, the activated carbon is respectively added into the proliferation culture medium and the rooting culture medium, and the activated carbon can absorb various metabolic substances generated by cinnamomum camphora in the tissue culture process, so that the growth environment of the tissue culture seedling is better improved, the increment coefficient of the tissue culture seedling is improved, the browning rate is reduced, and the rooting rate is improved; based on the method, the regeneration efficiency of the tissue culture of the cinnamomum camphora can be further improved, the cinnamomum camphora tissue culture seedlings can be obtained more massively and efficiently, and the market demand is met;
(2) the invention improves the disinfection mode of the cinnamomum camphora explant, and the explant 'cinnamomum camphora stem section' is obtained by 'disinfecting cinnamomum camphora seeds, germinating the disinfected seeds to obtain cinnamomum camphora aseptic seedlings, and then taking tender stem sections of the cinnamomum camphora aseptic seedlings as explants'; compared with a mode of directly disinfecting the tender stem sections of the cinnamomum camphora, the method can effectively avoid the damage of the tender stem sections of the materials required in the subsequent test caused by direct disinfection, thereby improving the test efficiency.
Drawings
FIG. 1 is a diagram showing the growth and proliferation status of individual seedlings of Cinnamomum camphora of example 3;
FIG. 2 is a diagram showing the overall growth state of tissue culture seedlings of Cinnamomum camphora in example 3;
FIG. 3 is a root display of tissue culture seedlings of Cinnamomum camphora in example 3.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
According to the method for improving the regeneration efficiency in the cinnamomum camphora tissue culture process, cinnamomum camphora seeds are firstly sterilized, and the sterilized seeds germinate to obtain cinnamomum camphora aseptic seedlings; then, the tender stem section of the camphor aseptic seedling is used as an explant, and the corresponding camphor tissue culture method is established through the steps of bud induction, bud multiplication, bud rooting and hardening and transplanting. Wherein activated carbon is added as an additional component to the "multiplication medium" used in the "multiplication of a bud" step and the "rooting medium" used in the "rooting of a bud" step, respectively.
Further, in this embodiment, the cinnamomum camphora tissue culture method established based on the above method is specifically as follows:
(1) obtaining and disinfecting explants:
① selecting strong, dry, straight, and strong-crown tree, no plant diseases and insect pests, and water-logging resistant Cinnamomum camphora plants, selecting Cinnamomum camphora seeds with plump particles, soaking in sterile water for 3h, rinsing with 75% alcohol on a superclean bench for 10s, repeating for 2 times, soaking with 1% sodium hypochlorite for 5min, and washing with sterile water for 4 times;
② inoculating the Cinnamomum camphora seeds with water to a culture bottle containing sterile seedling culture medium, culturing to obtain Cinnamomum camphora sterile seedlings, inoculating 2 seeds to each bottle, observing after 3 weeks, inoculating 50 bottles, repeating three times of test, and counting germination rate and contamination rate of Cinnamomum camphora seeds, wherein the sterile seedling culture medium is prepared from MS, pH5.8, culture temperature 24 deg.C, illumination time 14h/d, illumination intensity 1500Lx, seed contamination rate (%) (number of contaminated seeds/total number of inoculated seeds) × 100%, and seed germination rate (%) (number of germinated seeds/total number of inoculated seeds) × 100%.
(2) And (3) induction of buds:
taking tender stem segments of 25-day-cultured camphor tree aseptic seedlings as explants, and cutting the tender stem segments into 0.5 cm; then inoculating the bud-inducing culture medium into a culture bottle containing an inducing culture medium to perform induction culture of adventitious buds; wherein, the formula of the induction culture medium is as follows: MS +1 mg/L6-BA +0.1mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 24 ℃, the illumination time is 14h/d, and the illumination intensity is 1500 Lx.
(3) And (3) bud multiplication:
dividing the buds obtained in the step (2) into individual plants, and respectively inoculating the individual plants into a proliferation culture medium for proliferation culture; wherein, the formula of the proliferation culture medium is as follows: MS +3 mg/L6-BA +0.5mg/L NAA +2g/L active carbon, pH 5.8; the culture conditions were: the culture temperature is 24 ℃, the illumination time is 14h/d, and the illumination intensity is 1500 Lx.
(4) Rooting of the buds:
when the camphor bud seedlings grow to 3cm high, inoculating the camphor bud seedlings into a rooting culture medium for rooting culture; wherein, the formula of rooting culture is as follows: 1/2MS +0.5mg/L IBA +0.3mg/L NAA +2g/L activated carbon, pH 5.8; the culture conditions were: the culture temperature is 24 ℃, the illumination time is 14h/d, and the illumination intensity is 1500 Lx.
(5) Hardening and transplanting seedlings:
placing the test-tube plantlet obtained in the step of rooting buds in a sterile tissue culture room for acclimatization for a week in a mode of gradually opening a cover, and pouring sterile water into corresponding culture bottles to keep water; taking out the test-tube plantlet after one week, cleaning the culture medium attached to the root of the test-tube plantlet by flowing water, and then transplanting the test-tube plantlet into a sterilized culture medium; placing the container in a room, watering thoroughly, covering the periphery of the container with a plastic film, watering regularly, and reducing the watering frequency after ensuring one week; after 4d, the plastic film is removed until the plastic film is completely removed, and after 7d, the plastic film is removed from the culture chamber and is grown under natural light; wherein, the specific composition of the culture medium is black soil: perlite is 1: 3.
Example 2
According to the method for improving the regeneration efficiency in the cinnamomum camphora tissue culture process, cinnamomum camphora seeds are firstly sterilized, and the sterilized seeds germinate to obtain cinnamomum camphora aseptic seedlings; then, the tender stem section of the camphor aseptic seedling is used as an explant, and the corresponding camphor tissue culture method is established through the steps of bud induction, bud multiplication, bud rooting and hardening and transplanting. Wherein activated carbon is added as an additional component to the "multiplication medium" used in the "multiplication of a bud" step and the "rooting medium" used in the "rooting of a bud" step, respectively.
Further, in this embodiment, the cinnamomum camphora tissue culture method established based on the above method is specifically as follows:
(1) obtaining and disinfecting explants:
① selecting strong, dry, straight, and strong-crown, no plant diseases and insect pests, and water-logging resistant Cinnamomum camphora plants, selecting Cinnamomum camphora seeds with plump particles, soaking in sterile water for 3-5h, rinsing with 75% alcohol on a superclean bench for 20s, repeating for 4 times, soaking with 1% sodium hypochlorite for 5min, and washing with sterile water for 6 times;
② inoculating the Cinnamomum camphora seeds with water to a culture bottle containing sterile seedling culture medium, culturing to obtain Cinnamomum camphora sterile seedlings, inoculating 2 seeds to each bottle, observing after 3 weeks, inoculating 50 bottles, repeating three times of test, and counting germination rate and contamination rate of Cinnamomum camphora seeds, wherein the sterile seedling culture medium is prepared from MS, pH5.8, culture temperature 26 deg.C, illumination time 16h/d, illumination intensity 2000Lx, contamination rate (%) of seeds (contaminated seeds/total inoculated seeds) × 100%, and germination rate (%) of seeds (germinated seeds/total inoculated seeds) × 100%.
(2) And (3) induction of buds:
taking tender stem segments of 35-day-cultured camphor aseptic seedlings as explants, and cutting the tender stem segments into 0.6 cm; then inoculating the bud-inducing culture medium into a culture bottle containing an inducing culture medium to perform induction culture of adventitious buds; wherein, the formula of the induction culture medium is as follows: MS +1 mg/L6-BA +0.1mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 26 ℃, the illumination time is 16h/d, and the illumination intensity is 2000 Lx.
(3) And (3) bud multiplication:
dividing the buds obtained in the step (2) into individual plants, and respectively inoculating the individual plants into a proliferation culture medium for proliferation culture; wherein, the formula of the proliferation culture medium is as follows: MS +3 mg/L6-BA +0.5mg/L NAA +2g/L active carbon, pH 5.8; the culture conditions were: the culture temperature is 26 ℃, the illumination time is 16h/d, and the illumination intensity is 2000 Lx.
(4) Rooting of the buds:
when the camphor bud seedlings grow to 3cm high, inoculating the camphor bud seedlings into a rooting culture medium for rooting culture; wherein, the formula of rooting culture is as follows: 1/2MS +0.5mg/L IBA +0.3mg/L NAA +2g/L activated carbon, pH 5.8; the culture conditions were: the culture temperature is 26 ℃, the illumination time is 16h/d, and the illumination intensity is 2000 Lx.
(5) Hardening and transplanting seedlings:
placing the test-tube plantlet obtained in the step of rooting buds in a sterile tissue culture room for acclimatization for a week in a mode of gradually opening a cover, and pouring sterile water into corresponding culture bottles to keep water; taking out the test-tube plantlet after one week, cleaning the culture medium attached to the root of the test-tube plantlet by flowing water, and then transplanting the test-tube plantlet into a sterilized culture medium; placing the container in a room, watering thoroughly, covering the periphery of the container with a plastic film, watering regularly, and reducing the watering frequency after ensuring one week; after 4d, the plastic film is removed until the plastic film is completely removed, and after 7d, the plastic film is removed from the culture chamber and is grown under natural light; wherein, the specific composition of the culture medium is black soil: perlite is 1: 3.
Example 3
According to the method for improving the regeneration efficiency in the cinnamomum camphora tissue culture process, cinnamomum camphora seeds are firstly sterilized, and the sterilized seeds germinate to obtain cinnamomum camphora aseptic seedlings; then, the tender stem section of the camphor aseptic seedling is used as an explant, and the corresponding camphor tissue culture method is established through the steps of bud induction, bud multiplication, bud rooting and hardening and transplanting. Wherein activated carbon is added as an additional component to the "multiplication medium" used in the "multiplication of a bud" step and the "rooting medium" used in the "rooting of a bud" step, respectively.
Further, in this embodiment, the cinnamomum camphora tissue culture method established based on the above method is specifically as follows:
(1) obtaining and disinfecting explants:
① selecting strong, dry, straight, and strong-crown tree, no plant diseases and insect pests, and water-logging resistant Cinnamomum camphora plants, selecting Cinnamomum camphora seeds with plump particles, soaking in sterile water for 4h, rinsing with 75% alcohol on a superclean bench for 15s, repeating for 3 times, soaking with 1% sodium hypochlorite for 5min, and washing with sterile water for 5 times;
② inoculating the Cinnamomum camphora seeds with water to a culture bottle containing sterile seedling culture medium, culturing to obtain Cinnamomum camphora sterile seedlings, inoculating 2 seeds to each bottle, observing after 3 weeks, inoculating 50 bottles, repeating three times of test, and counting germination rate and contamination rate of Cinnamomum camphora seeds, wherein the sterile seedling culture medium is prepared from MS, pH5.8, culture temperature 25 deg.C, illumination time 15h/d, illumination intensity 1750Lx, seed contamination rate (%) (number of contaminated seeds/total number of inoculated seeds) × 100% and seed germination rate (%) (number of germinated seeds/total number of inoculated seeds) × 100%.
(2) And (3) induction of buds:
taking tender stem segments of 30-day-cultured camphor aseptic seedlings as explants, and cutting the tender stem segments into 0.55 cm; then inoculating the bud-inducing culture medium into a culture bottle containing an inducing culture medium to perform induction culture of adventitious buds; wherein, the formula of the induction culture medium is as follows: MS +1 mg/L6-BA +0.1mg/L NAA, pH 5.8; the culture conditions were: the culture temperature is 25 ℃, the illumination time is 15h/d, and the illumination intensity is 1750 Lx.
(3) And (3) bud multiplication:
dividing the buds obtained in the step (2) into individual plants, and respectively inoculating the individual plants into a proliferation culture medium for proliferation culture; wherein, the formula of the proliferation culture medium is as follows: MS +3 mg/L6-BA +0.5mg/L NAA +2g/L active carbon, pH 5.8; the culture conditions were: the culture temperature is 25 ℃, the illumination time is 15h/d, and the illumination intensity is 1750 Lx; after a period of culture, the growth and proliferation states of the individual seedlings of cinnamomum camphora are shown in figure 1.
(4) Rooting of the buds:
when the camphor bud seedlings grow to 3cm high, inoculating the camphor bud seedlings into a rooting culture medium for rooting culture; wherein, the formula of rooting culture is as follows: 1/2MS +0.5mg/L IBA +0.3mg/L NAA +2g/L activated carbon, pH 5.8; the culture conditions were: the culture temperature is 25 ℃, the illumination time is 15h/d, and the illumination intensity is 1750 Lx; after the culture was matured, the overall growth state of the tissue culture plantlets is shown in FIG. 2, and the root display is shown in FIG. 3.
(5) Hardening and transplanting seedlings:
placing the test-tube plantlet obtained in the step of rooting buds in a sterile tissue culture room for acclimatization for a week in a mode of gradually opening a cover, and pouring sterile water into corresponding culture bottles to keep water; taking out the test-tube plantlet after one week, cleaning the culture medium attached to the root of the test-tube plantlet by flowing water, and then transplanting the test-tube plantlet into a sterilized culture medium; placing the container in a room, watering thoroughly, covering the periphery of the container with a plastic film, watering regularly, and reducing the watering frequency after ensuring one week; after 4d, the plastic film is removed until the plastic film is completely removed, and after 7d, the plastic film is removed from the culture chamber and is grown under natural light; wherein the culture medium comprises the following specific components: perlite is 1: 3.
Example 4
This example illustrates the parameter selection in examples 1-3 above:
influence of different disinfection combinations on seed pollution and germination rate
In the experiment, 0.5% of sodium hypochlorite and 1% of sodium hypochlorite are selected as disinfectants, and 6 treatment combinations are selected, wherein the treatment combinations are respectively as follows: a: treating with 0.5% sodium hypochlorite for 3 min; b: treating with 0.5% sodium hypochlorite for 5 min; c: treating with 0.5% sodium hypochlorite for 7 min; d: treating with 1% sodium hypochlorite for 3 min; e: treating with 1% sodium hypochlorite for 5 min; f: treating with 1% sodium hypochlorite for 7 min.
After 3 weeks, the contamination rate and germination rate of the seeds after different disinfection treatments were counted as shown in tables 1 and 2; as can be seen from Table 1, the average contamination ratio of "combination of E treatments" was 0, with the best results; as can be seen from Table 2, the germination percentage of the "E-treated combination" was the highest, 95%. Accordingly, the "E-treatment combination" is the optimal sterilization combination for this embodiment.
TABLE 1 Effect of different combinations of disinfectants on seed contamination Rate
| Treatment combination | Number of inoculated leaves/ | Number of infected seeds/number | Average contamination ratio/%) |
| A | 50 | 22 | 44 |
| B | 50 | 19 | 38 |
| C | 50 | 12 | 24 |
| D | 50 | 8 | 16 |
| E | 50 | 0 | 0 |
| F | 50 | 5 | 10 |
TABLE 2 Effect of different combinations of disinfectants on seed germination
| Treatment combination | Number of uncontaminated seeds/piece | The germination number of seeds/seed | Average germination/degree |
| A | 40 | 29 | 72 |
| B | 40 | 31 | 77 |
| C | 40 | 34 | 85 |
| D | 40 | 35 | 87 |
| E | 40 | 38 | 95 |
| F | 40 | 36 | 90 |
Second, the induction effect of different hormone ratios on shoots
And (3) taking tender stems with axillary buds on the camphor aseptic seedlings, inoculating the stems into culture media with different combinations, culturing for 10-20 days, and observing the budding condition.
The results are shown in Table 3. As shown in Table 3, the highest induction rate of the sprouts was 81% when the hormone combination was MS +1 mg/L6-BA +0.1mg/L NAA.
TABLE 3 Induction Effect of different hormone combinations on shoots
| 6-BA(mg/L) | NAA(mg/L) | Inductivity (%) |
| 0.5 | 0.05 | 10 |
| 0.5 | 0.10 | 21 |
| 0.5 | 0.15 | 43 |
| 0.5 | 0.20 | 51 |
| 1 | 0.05 | 68 |
| 1 | 0.10 | 81 |
| 1 | 0.15 | 79 |
| 1 | 0.20 | 68 |
| 1.5 | 0.05 | 52 |
| 1.5 | 0.10 | 52 |
| 1.5 | 0.15 | 43 |
| 1.5 | 0.20 | 40 |
Influence of different hormone formulations on bud proliferation
The results are shown in Table 4. As is clear from Table 4, the hormone combination MS +3 mg/L6-BA +0.5mg/L NAA showed the best effect of proliferation, and the proliferation factor was 6.7.
TABLE 4 Effect of different hormone combinations on the proliferation of shoots
However, as can be seen from the test results, the browning rate is relatively high during the subculture process, and the sprouts are browned and dead, which seriously affects the regeneration efficiency. Then, the test was conducted by adding activated carbon to the hormone combinations having the highest growth factors in table 4, and the effects of different concentrations of activated carbon on the browning rate and the growth factor were observed, and the results are shown in table 5.
As is clear from Table 5, when the activated carbon concentration was 2g/L, the growth factor was 6.9, which was improved as compared with the previous 6.7; meanwhile, the browning rate is 3.1% at least, and compared with the browning rate without adding active carbon, the browning rate is reduced by 23.1%, and the regeneration efficiency of the tissue culture seedlings is greatly improved.
TABLE 5 Effect of different concentrations of activated carbon on browning rate and proliferation
| Activated carbon concentration (g/L) | Browning rate (%) | Coefficient of proliferation | Growth vigor of plants |
| 0.5 | 23.1 | 6.8 | Weak (weak) |
| 1.0 | 14.3 | 6.7 | Is weaker |
| 1.5 | 8.7 | 6.7 | Is weaker |
| 2.0 | 3.1 | 6.9 | Zhuang |
| 2.5 | 4.3 | 6.6 | Is stronger |
| 3.0 | 4.5 | 6.7 | Is stronger |
Influence of different hormone proportions on seedling rooting
The results are shown in Table 6. As shown in Table 6, the combination of the hormones 1/2MS +0.5mg/LIBA +0.3mg/LNAA showed the best rooting effect, with a rooting rate of 93%.
TABLE 6 Effect of different hormone combinations on shoot rooting
However, the test results show that the browning rate is higher in the rooting process, the bud seedlings do not root or even die due to browning, and the regeneration efficiency is seriously influenced. Then, the test was conducted by adding activated carbon to the hormone combinations having the highest rooting rates in Table 6, and the effects of different concentrations of activated carbon on the browning rate and the rooting rate were observed, and the results are shown in Table 7.
As shown in Table 7, when the activated carbon concentration was 2g/L, the rooting percentage was 98%, which was higher than that of the previous 93%, and the browning percentage was 3.4% at the lowest. Compared with the method without adding active carbon, the browning rate is reduced by 21.6%, and the regeneration efficiency of the tissue culture seedling is greatly improved.
TABLE 7 influence of different concentrations of activated carbon on browning rate and rooting rate
Fifthly, hardening and transplanting test-tube plantlets
When the test-tube plantlet is acclimatized in an aseptic tissue culture room at the initial stage, the loss of water is found to be very quick, so that aseptic water is gradually added into a tissue culture bottle with a cover opened to keep the water required by the normal growth of the test-tube plantlet. When the test-tube plantlet is taken out of the culture medium, the damage to the root is reduced as much as possible, the residual culture medium at the base part of the test-tube plantlet is washed completely by running water and then transferred into the culture medium. The test-tube plantlet of Cinnamomum camphora grows slowly in the early stage and has developed root system.
In addition, it should be noted that the MS culture medium and 1/2MS used in the present invention are conventional culture media in the art, and the formulation of MS culture medium is shown in Table 8.
TABLE 8 MS Medium formulation
Note: 1/2 the MS culture medium is obtained by halving macroelements of MS culture medium.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (8)
1. A method for improving regeneration efficiency in a camphor tree tissue culture process is characterized in that camphor tree seeds are firstly disinfected, and the disinfected seeds germinate to obtain camphor tree aseptic seedlings; then, taking tender stem segments of the camphor tree aseptic seedlings as explants, and establishing a corresponding camphor tree tissue culture method through the steps of bud induction, bud multiplication and bud rooting; wherein activated carbon is added as an additional component to the "multiplication medium" used in the "multiplication of buds" step and the "rooting medium" used in the "rooting of buds" step, respectively.
2. The method for improving the regeneration efficiency in the cinnamomum camphora tissue culture process according to claim 1, wherein the established cinnamomum camphora tissue culture method is as follows:
(1) obtaining and disinfecting explants:
① selecting full Cinnamomum camphora seeds, soaking in sterile water for 3-5h, rinsing with 75% alcohol on a superclean bench for 10-20s, repeating for 2-4 times, soaking with 1% sodium hypochlorite for 5min, and washing with sterile water for 4-6 times;
② inoculating the Cinnamomum camphora seed with water to aseptic seedling culture medium (MS, pH 5.8) for culturing to obtain Cinnamomum camphora aseptic seedling;
(2) and (3) induction of buds:
taking tender stem section of 25-35 days cultured Cinnamomum camphora aseptic seedling as explant, and cutting into size of 0.5-0.6 cm; then inoculating the bud-inducing culture medium into a culture bottle containing an inducing culture medium to induce adventitious buds; wherein the formula of the induction culture medium is as follows: MS +1 mg/L6-BA +0.1mg/L NAA, pH 5.8;
(3) and (3) bud multiplication:
dividing the buds obtained in the step (2) into individual plants, and respectively inoculating the individual plants into a proliferation culture medium for proliferation culture; wherein the formula of the proliferation culture medium is as follows: MS +3 mg/L6-BA +0.5mg/L NAA +2g/L active carbon, pH 5.8;
(4) rooting of the buds:
when the camphor bud seedlings grow to 3cm high, inoculating the camphor bud seedlings into a rooting culture medium for rooting culture; wherein, the formula of the rooting culture is as follows: 1/2MS +0.5mg/L IBA +0.3mg/L NAA +2g/L activated carbon, pH5.8.
3. The method for improving regeneration efficiency during the tissue culture process of cinnamomum camphora according to claim 2, wherein in step (1), full cinnamomum camphora seeds are selected, soaked in sterile water for 4h, rinsed on an ultra-clean bench with 75% alcohol for 15s, repeated for 3 times, soaked in 1% sodium hypochlorite for 5min, and finally rinsed with sterile water for 5 times; after washing, the seeds are dried by using sterile absorbent paper for standby.
4. The method for improving regeneration efficiency during tissue culture of cinnamomum camphora according to claim 3, wherein the cinnamomum camphora seeds are selected from cinnamomum camphora plants that are strong in growth, straight in dry shape, erect in crown, free from plant diseases and insect pests, strong in age, and resistant to flooding.
5. The method for improving regeneration efficiency during tissue culture of cinnamomum camphora according to claim 2, wherein in step (1), cinnamomum camphora seeds with moisture absorbed are inoculated into culture bottles containing sterile seedling culture medium for culture, 2 seeds are inoculated into each bottle, after 3 weeks, the culture is carried out, 50 bottles are inoculated, the test is repeated for three times, and the germination rate and the pollution rate of cinnamomum camphora seeds are counted.
6. The method for improving regeneration efficiency during tissue culture of cinnamomum camphora according to claim 2, wherein in step (2), young stem segments of aseptic cinnamomum camphora seedlings cultured for 30 days are used as explants and cut into 0.55cm size; then, the culture medium was inoculated into a culture flask containing an induction medium to induce adventitious buds.
7. The method for improving regeneration efficiency during the tissue culture process of cinnamomum camphora according to claim 2, wherein the specific culture conditions of aseptic seedling culture, bud induction culture, bud proliferation culture and bud rooting culture in the steps (1), (2), (3) and (4) are as follows: the culture temperature is 25 +/-1 ℃, the illumination time is 14-16h/d, and the illumination intensity is 1500-.
8. The method for improving regeneration efficiency in the tissue culture process of cinnamomum camphora according to any one of claims 1-7, wherein the step of rooting buds in the tissue culture method of cinnamomum camphora further comprises the step of seedling exercising and transplanting:
placing the test-tube plantlet obtained in the step of rooting buds in a sterile tissue culture room for acclimatization for a week in a mode of gradually opening a cover, and pouring sterile water into corresponding culture bottles to keep water; taking out the test-tube plantlet after one week, cleaning the culture medium attached to the root of the test-tube plantlet by flowing water, and then transplanting the test-tube plantlet into a sterilized culture medium; placing the container in a room, watering thoroughly, covering the periphery of the container with a plastic film, watering regularly, and reducing the watering frequency after ensuring one week; after 4d, the plastic film is removed until the plastic film is completely removed, and after 7d, the plastic film is removed from the culture chamber and is grown under natural light; wherein the culture medium comprises the following components: perlite is 1: 3.
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