CN109006472B - Method for tissue culture and rapid propagation of ornamental plants by using carbon nano tubes with specific concentrations - Google Patents
Method for tissue culture and rapid propagation of ornamental plants by using carbon nano tubes with specific concentrations Download PDFInfo
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- CN109006472B CN109006472B CN201810601662.2A CN201810601662A CN109006472B CN 109006472 B CN109006472 B CN 109006472B CN 201810601662 A CN201810601662 A CN 201810601662A CN 109006472 B CN109006472 B CN 109006472B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
Abstract
The invention discloses a method for tissue culture and rapid propagation of ornamental plants by using carbon nanotubes with specific concentrations. After the cluster buds are obtained by using the plant growth regulator, the plant growth regulator is not used, but the promotion effect of the carbon nano tube on the plant growth is used for carrying out the plant tissue culture and rapid propagation. According to the invention, the carbon nano tubes are used for replacing a plant growth regulator in the stages of strong bud, strong seedling and rooting, so that the seedling revival period of aseptic seedling transplantation is effectively shortened, the survival rate of transplantation is improved, and the propagation efficiency of tissue culture rapid propagation is greatly improved; the invention adopts the synergistic effect of the plant growth regulator and the carbon nano tube to induce the germination and proliferation of the cluster buds, and can greatly improve the propagation coefficient of tissue culture rapid propagation; meanwhile, the carbon nano tube replaces a plant growth regulator, so that strong seedling culture and rooting culture can be carried out simultaneously, the process of plant tissue culture and rapid propagation is greatly accelerated, and the tissue culture period is shortened.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a method for carrying out plant tissue culture and rapid propagation by using carbon nanotubes.
Background
Carbon Nanotubes (CNTs) are a novel carbon crystal found by electron microscopy experts ii jima in basic research laboratories of NEC corporation, japan in 1991 from carbon fibers produced by the arc process. Since the discovery of carbon nanotubes, due to their special nanostructures and unique mechanical, electrical, thermal and chemical properties, and the application prospects in various fields such as electricity, optics, biomedicine, energy storage, catalysis and sensing, the carbon nanotubes have attracted high attention in the scientific and industrial circles of the world. Researches show that the carbon nano tube can pierce through the seed coat and promote water absorption, thereby promoting seed germination and seedling growth and rooting.
In the current technical system of plant tissue culture and rapid propagation, growth regulators with different concentrations are added to induce explants to dedifferentiate and redifferentiate, so as to obtain aseptic seedlings. The obtained aseptic seedlings grow slowly or even stop after losing the stimulation of the high-concentration plant growth regulator, so that a long seedling revival period exists after the aseptic seedlings are transplanted, and part of the aseptic seedlings even die. Therefore, the establishment of a tissue culture rapid propagation method for effectively shortening the seedling revival stage of the rooted plant tissue culture seedling has important significance for the further development of the plant tissue culture technology.
Disclosure of Invention
In view of the above, the present invention provides a method for tissue culture and rapid propagation of plants by using the promoting effect of carbon nanotubes on plant growth without using plant growth regulators after obtaining cluster buds by using the plant growth regulators.
The method specifically comprises the following steps:
step (1), sterilizing and disinfecting explants;
step (2), inducing cluster buds;
the bud induction culture medium adopts a common bud induction culture medium, but must contain a certain concentration of plant growth regulator and 0.5-5mg/L of carbon nano-tubes; the selection of the concentration of the plant growth regulator is well known to those skilled in the art.
Step (3), proliferating the adventitious buds;
the bud multiplication culture medium adopts a common bud multiplication culture medium, but must contain a certain concentration of plant growth regulator and 0.5-4.5mg/L of carbon nano-tubes; the selection of the concentration of the plant growth regulator is well known to those skilled in the art.
Step (4), strong bud culture;
the bud-strengthening culture medium comprises 0.5-4 mg/L of carbon nano-tube, 35-45 mg/L of cane sugar and other components, but must not contain a plant growth regulator. The specific selection and concentration of carbon nanotubes, sucrose and other components are adjusted according to the specific species.
Step (5), strong seedling and rooting co-culture;
the culture medium comprises 0.5-3 mg/L of carbon nano tubes, 35-45 mg/L of cane sugar and other components, but the culture medium cannot contain a plant growth regulator. The specific selection and concentration of carbon nanotubes, sucrose and other components are adjusted according to the specific species.
And (6) hardening and transplanting seedlings.
The plant growth regulator is 6-benzylamino adenine (6-BA) and Naphthalene Acetic Acid (NAA).
The carbon nano-tube is a multi-wall carbon nano-tube, a single-wall carbon nano-tube or other carbon nano-tubes (such as multi-wall carboxylation carbon nano-tubes) which are simply modified, and the like.
The invention has the beneficial effects that:
1. according to the invention, the carbon nano tubes are used for replacing a plant growth regulator in the stages of strong bud, strong seedling and rooting, so that the seedling revival period of aseptic seedling transplantation is effectively shortened, the survival rate of transplantation is improved, and the propagation efficiency of tissue culture rapid propagation is greatly improved;
2. the carbon nano tube has obvious promotion effect on the growth and rooting of plants. The invention adopts the synergistic effect of the plant growth regulator and the carbon nano tube to induce the germination and proliferation of the cluster buds, and can greatly improve the propagation coefficient of tissue culture rapid propagation; meanwhile, the carbon nano tube replaces a plant growth regulator, so that strong seedling culture and rooting culture can be carried out simultaneously, the process of plant tissue culture and rapid propagation is greatly accelerated, and the tissue culture period is shortened.
Detailed Description
The present invention is further analyzed with reference to the following specific examples.
Example 1 establishment of tissue culture and Rapid propagation System of Amarylris
The method comprises the following specific steps: 1) sterilizing underground bulb of Lycoris radiata, inducing cluster bud, proliferating adventitious bud, and culturing strong bud to obtain new bulb; 2) inoculating the new bulb into a callus induction culture medium for dark culture to obtain a callus with the diameter of 1.5-2.5 cm; 3) cutting the callus into blocks with the diameter of 1-2 cm, inoculating the blocks into a bud induction culture medium, and differentiating the callus for 15-20 days to obtain cluster buds; 4) and inoculating the cluster buds into a strong bud rooting culture medium for strong bud rooting culture to obtain the tissue culture seedlings.
The invention obtains the new bulb by sterilizing the underground bulb of the lycoris radiata, inducing cluster buds, proliferating adventitious buds and culturing strong buds. The lycoris radiata variety is preferably lycoris radiata; the preferred lycoris radiata underground bulb is more than 3 years old, more preferably 3-5 years old and 1.5-4 cm in diameter. Before underground bulbs are sterilized, the underground bulbs are preferably pretreated to obtain blocky bulbs; the pretreatment comprises the steps of obtaining a bulb dish, cleaning the bulb dish, cutting into blocks and washing.
Preferably removing outer-layer scale leaves of the underground bulbs, cutting off all roots and 1/2-2/3 scale leaves of the underground bulbs, and reserving a bulb dish; the preferable washing uses a hairbrush and clear water to wash off soil on the surface of the bulb tray; the size of the blocky bulb dish obtained by cutting is preferably 1.0-1.5 cm3(ii) a The washing is preferably shaking washing, and the washing liquid used for the washing is preferably 84 disinfectant; the washing time is preferably 15-50 min, and more preferably 25-40 min. After washing, the washed bulb tray is preferably washed by flowing clear water for 25-35 min.
After the bulb dish is obtained, the bulb dish is sterilized. The sterilization operation is preferably carried out in a clean bench; the sterilization preferably comprises alcohol rinsing, HgCl2Sterilized and washed with sterile water. In the invention, the volume fraction of the alcohol used for alcohol rinsing is preferably 70-80%, and more preferably 75%; the time for rinsing with the alcohol is preferably 25-35 s, and more preferably 30 s. In the present invention, the HgCl2HgCl for sterilization2The preferable mass fraction of the solution is HgCl of 0.05-0.15%2A solution; the HgCl2The time for sterilizing the solution is preferably 35-45 min, and more preferably 40 min. In the present invention, the number of times of the sterile water washing is preferably 4 to 6 times, and more preferably 5 times. After the sterile water washing is finished, the invention preferably adopts sterile paper to absorb the water on the surface of the bulb dish.
After the sterilization, the invention induces cluster buds on the sterilized bulbs. In the invention, the sterilized blocky bulbs are preferably directly inoculated to a bud induction culture medium in a manner that a basal disc is downward for culture; the bud induction culture medium is preferably an MS culture medium comprising 25-35 g/L of sucrose, 5-10 g/L of agar, 1.0-3.0mg/L of multi-wall carbon nano-tubes, 5.5-10.0mg/L of 6-benzylamino adenine and 1-2.0 mg/L of naphthylacetic acid; more preferably, the MS culture medium comprises 30g/L of sucrose, 7.5g/L of agar, 6.0mg/L of 6-benzylamino adenine and 1.5mg/L of naphthalene acetic acid, and 3.0mg/L of multi-wall carbon nano-tubes.
The process of inducing the cluster buds preferably comprises primary culture and secondary culture, and specifically comprises the following steps: and sequentially carrying out primary culture and secondary culture on the sterilized bulbs on a bud induction culture medium. In the invention, the time of the primary culture is preferably 35-45 d, and more preferably 40 d; the time of subculture is preferably 35-45 d, and more preferably 40 d; the culture temperature of the induced cluster buds is preferably 24-26 ℃, and more preferably 25 ℃; the illumination intensity of the induced cluster buds is preferably 100-300 mu mol.m-2·s-1More preferably 200. mu. mol. m-2·s-1(ii) a The illumination time is preferably 10-14 h/d, and more preferably 12 h/d.
After the cluster buds are induced, the obtained bulb cluster buds are proliferated to obtain adventitious buds, and specifically, the cluster buds induced are transplanted to a bud proliferation culture medium to be proliferated to obtain the adventitious buds. In the invention, the bud multiplication culture medium is preferably an MS culture medium comprising 25-35 g/L of sucrose, 5-10 g/L of agar, 1.0-2.5mg/L of multi-walled carbon nanotubes, 2.5-3.5 mg/L of 6-benzylaminopurine and 0.5-1.5mg/L of naphthylacetic acid, and is more preferably an MS culture medium comprising 30g/L of sucrose, 7.5g/L of agar, 3.0mg/L of 6-benzylaminopurine and 1.0mg/L of naphthylacetic acid, and 2.5mg/L of multi-walled carbon nanotubes.
In the invention, the culture temperature of the proliferation adventitious bud is preferably 24-26 ℃, and more preferably 25 ℃; the light intensity of the propagated adventitious bud is preferably 100 to 300 [ mu ] mol.m-2·s-1More preferably 200. mu. mol. m-2·s-1(ii) a The illumination time is preferably 14-18 h/d, and more preferably 16 h/d; the time for culturing the propagation adventitious buds is preferably 35-45 d, and more preferably 40 d.
After the adventitious buds are proliferated, the obtained adventitious buds of the bulbs are subjected to strong bud culture to obtain new bulbs. In the invention, the strong bud culture preferably comprises 1-2 times of subculture, and the time of each subculture is preferably 25-35 d; more preferably 30 d. In the present invention, the temperature for culturing the strong bud is preferably selected24-26 ℃, more preferably 25 ℃; the illumination intensity of the strong bud culture is preferably 100-300 mu mol.m-2·s-1More preferably 200. mu. mol. m-2·s-1(ii) a The illumination time for the strong bud culture is preferably 14-18 h/d, and more preferably 16 h/d.
In the invention, the strong bud culture specifically comprises the following steps: transplanting the adventitious bud of the bulb obtained by proliferating the adventitious bud to a strong bud culture medium for strong bud culture. In the invention, the culture medium for bud-strengthening culture is preferably an MS culture medium comprising 35-45 g/L of sucrose, 5-10 g/L of agar and 1.0-2.5mg/L of multi-wall carbon nano-tubes; more preferably, the MS culture medium comprises 40g/L of sucrose, 7.5g/L of agar and 2.5mg/L of multi-wall carbon nano-tubes. After the bud-strengthening culture is completed, the new bulblet with the diameter of 2-3 mm is obtained.
After the bud strengthening culture is completed, the obtained new bulblets are subjected to seedling strengthening and rooting co-culture to obtain new bulblets. In the invention, the co-culture of the strong bud and the rooting preferably comprises 1-2 times of subculture, and the time of each subculture is preferably 25-35 d; more preferably 30 d. In the invention, the temperature for culturing the strong buds is preferably 24-26 ℃, and more preferably 25 ℃; the illumination intensity of the strong bud culture is preferably 100-300 mu mol.m-2·s-1More preferably 200. mu. mol. m-2·s-1(ii) a The illumination time for the strong bud culture is preferably 14-18 h/d, and more preferably 16 h/d.
In the invention, the strong seedling and rooting co-culture specifically comprises the following steps: and (4) carrying the new bulblets and the bands on a culture medium for the bud-strengthening and rooting co-culture. In the invention, the culture medium for the co-culture of the strong buds and the rooting is preferably an MS culture medium comprising 35-45 g/L of sucrose, 5-10 g/L of agar and 0.5-2.0mg/L of multi-wall carbon nano tubes; more preferably, the MS culture medium comprises 40g/L of sucrose, 7.5g/L of agar and 2.0mg/L of multi-wall carbon nano-tubes. After the bud-strengthening culture is completed, the new bulbs with the diameter of 3-5 mm and 3-5 adventitious roots are obtained.
After the new bulb with root is obtained, the obtained bulblet is hardened and transplanted. Hardening seedlings are carried out indoors. And opening the tissue culture bottle, and placing the tissue culture bottle under natural scattered light for 2-3 days. After the hardening-off, the test-tube plantlet is taken out by tweezers, the culture medium at the root is washed off, and the test-tube plantlet is transplanted into a 10X 10 plug tray. The culture medium is peat soil: perlite: the garden soil is 1:1:1, and the culture medium is disinfected by 800-fold carbendazim and 1000-fold. And then performing normal water and fertilizer management.
Example 2 establishment of tissue culture and rapid propagation System of cymbidium
The method comprises the following specific steps: 1) sterilizing explants; 2) inducing cluster buds; 3) multiplication of cluster buds; 4) strong bud culture; 5) strong seedling and rooting culture; 6) hardening and transplanting the seedlings.
In the step 1), the cymbidium hybridum protocorm is taken as an explant, the surface of the protocorm is cleaned by a detergent, and then the protocorm is washed by running water for 1 hour. Sterilizing with 75% alcohol for 30s on an ultraclean workbench, washing with sterile water, sterilizing with 5% NaClO solution for 15min, shaking while shaking, washing with sterile water for 3-4 times, and drying with sterile filter paper.
Inoculating the sterilized protocorm into a cluster bud induction culture medium to obtain cluster buds through induction; the used minimal medium is 1/2MS, and sucrose is added at 30 g.L-1And agar powder 7-8 g.L-10.5-2.5mg/L single-walled carbon nanotube, and 0.15-0.25 mg/L culture medium added before sterilization-1NAA and 1.0-2.0 mg.L-16-BA, culture conditions: the illumination time is 12 hours, the light intensity is 2000-2500 lx, and the culture temperature is 25 +/-1 ℃;
subculturing cluster buds obtained by inducing protocorms into a cluster bud multiplication culture medium, wherein the used basic culture medium is 1/2 MS; sucrose 30 g.L is added into the culture medium-1And agar powder 7 g.L-1(ii) a Adding 0.05-0.1 mg.L into the culture medium before sterilization-1NAA and 0.5-1.5 mg.L-16-BA, 0.5-2.0mg/L single-walled carbon nanotube, the illumination time is 16h, the light intensity is 2000-2500 lx, the culture temperature is 25 +/-1 ℃, and the proliferation of cluster buds is carried out;
the cluster buds obtained by induction are transplanted into a bud multiplication culture medium for adventitious bud multiplication. In the invention, the bud multiplication culture medium is preferably 1/2MS culture medium containing 25-35 g/L of sucrose, 7-8g/L of agar, 0.5-1.5mg/L of 6-benzylaminopurine and 0.1-0.3mg/L of naphthylacetic acid, and 0.5-2.0mg/L of single-walled carbon nanotube, and is more preferably 1/2MS culture medium containing 30g/L of sucrose, 7.5g/L of agar, 1.0mg/L of 6-benzylaminopurine and 0.2mg/L of naphthylacetic acid, and 1.0mg/L of single-walled carbon nanotube.
After the adventitious bud proliferation is finished, the invention carries out strong bud culture to obtain the new protocorm. In the invention, the strong bud culture preferably comprises 1-2 times of subculture, and the time of each subculture is preferably 25-35 d; more preferably 30 d. In the invention, the temperature for culturing the strong buds is preferably 24-26 ℃, and more preferably 25 ℃; the illumination intensity of the strong bud culture is preferably 100-300 mu mol.m-2·s-1More preferably 200. mu. mol. m-2·s-1(ii) a The illumination time for the strong bud culture is preferably 14-18 h/d, and more preferably 16 h/d.
In the invention, the strong bud culture specifically comprises the following steps: inoculating adventitious buds obtained by adventitious bud proliferation onto a strong bud culture medium for strong bud culture. In the invention, the culture medium for bud-strengthening culture is preferably 1/2MS culture medium containing 35-45 g/L of sucrose, 5-10 g/L of agar and 0.5-2.5mg/L of single-walled carbon nanotubes; more preferably 1/2MS culture medium containing 40g/L of sucrose, 7.5g/L of agar and 1.5mg/L of single-walled carbon nanotubes. After the strong bud culture is completed, protocorms with the diameter of 2-3 mm are obtained.
After strong seedling culture is completed, the obtained new protocorm is subjected to strong seedling and rooting co-culture, and the aseptic seedling corm of the cymbidium hybridum is obtained. In the invention, the strong seedling and rooting co-culture preferably comprises 1-2 times of subculture, and the time of each subculture is preferably 25-35 d; more preferably 30 d. In the invention, the temperature for culturing the strong buds is preferably 24-26 ℃, and more preferably 25 ℃; the illumination intensity of the strong bud culture is preferably 100-300 mu mol.m-2·s-1More preferably 200. mu. mol. m-2·s-1(ii) a The illumination time for the strong bud culture is preferably 14-18 h/d, and more preferably 16 h/d.
In the invention, the strong seedling and rooting co-culture specifically comprises the following steps: and subculturing the new protocorm to a culture medium for strong seedling and rooting co-culture. In the invention, the culture medium for the co-culture of the strong buds and the rooting is preferably an MS culture medium comprising 35-45 g/L of sucrose, 7-8g/L of agar and 0.5-3.0mg/L of single-walled carbon nanotubes; more preferably 1/2MS culture medium containing 40g/L of sucrose, 7.5g/L of agar and 1.5mg/L of single-walled carbon nanotubes. After the strong bud culture is completed, the new protocorm with the diameter of 3-5 mm and 3-5 adventitious roots is obtained.
After the invention obtains the new protocorm with root, the obtained protocorm is hardened and transplanted. Hardening seedlings are carried out indoors. And opening the tissue culture bottle, and placing the tissue culture bottle under natural scattered light for 2-3 days. After the hardening-off, the test-tube plantlet is taken out by tweezers, the culture medium at the root is washed off, and the test-tube plantlet is transplanted into a 10X 10 plug tray. The culture medium is moss, coco coir and perlite which are 1: 9: 12, and the culture medium is subjected to 800-fold carbendazim disinfection treatment. And then performing normal water and fertilizer management.
Three different culture schemes are performed on lycoris radiata and cymbidium hybridum respectively, and the experimental results are compared, which are shown in table 1.
TABLE 1
Note: 7.5mg/L agar was added to the formulation. MWCNTs: a multi-walled carbon nanotube; SWCNTs: single-walled carbon nanotubes.
The above embodiments are not intended to limit the present invention, and the present invention is not limited to the above embodiments, and all embodiments are within the scope of the present invention as long as the requirements of the present invention are met.
Claims (3)
1. The method for tissue culture and rapid propagation of lycoris radiata and cymbidium hybridum by using the carbon nano tube with specific concentration is characterized by comprising the following steps of:
step (1), sterilizing and disinfecting explants;
step (2), inducing cluster buds;
the bud induction culture medium adopts a common bud induction culture medium, but must contain a certain concentration of plant growth regulator and 0.5-5mg/L of carbon nano-tubes;
step (3), proliferating the adventitious buds;
the bud multiplication culture medium adopts a common bud multiplication culture medium, but must contain a certain concentration of plant growth regulator and 0.5-4.5mg/L of carbon nano-tubes;
step (4), strong bud culture;
the bud-strengthening culture medium comprises 0.5-4 mg/L of carbon nano tube, 35-45 mg/L of cane sugar and other components, but must not contain a plant growth regulator;
step (5), strong seedling and rooting co-culture;
the culture medium comprises 0.5-3 mg/L of carbon nano tubes, 35-45 mg/L of cane sugar and other components, but the culture medium cannot contain a plant growth regulator;
and (6) hardening and transplanting seedlings.
2. The method for tissue culture and rapid propagation of ornamental plants by using carbon nanotubes with specific concentration as claimed in claim 1, wherein the plant growth regulator is 6-benzylamino adenine or naphthylacetic acid.
3. The method for tissue culture and rapid propagation of ornamental plants by using carbon nanotubes with specific concentration as claimed in claim 1, wherein the carbon nanotubes are multi-walled carbon nanotubes, single-walled carbon nanotubes or other simply modified carbon nanotubes.
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