CN111280062A - Regeneration culture method of Nandina domestica nursery stock with strong adaptability - Google Patents
Regeneration culture method of Nandina domestica nursery stock with strong adaptability Download PDFInfo
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- CN111280062A CN111280062A CN202010235605.4A CN202010235605A CN111280062A CN 111280062 A CN111280062 A CN 111280062A CN 202010235605 A CN202010235605 A CN 202010235605A CN 111280062 A CN111280062 A CN 111280062A
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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
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Abstract
The invention discloses a strong-adaptability regeneration culture method for nandina nursery stocks, which comprises the following steps: tissue culture, adventitious bud induction, embryoid formation, soaking for rooting, transplantation and field planting; the invention collects leaf tissue on the aseptic tissue culture seedling and sterilizes the aseptic chamber, so that the leaf tissue is virus-removed in the culture process, the regeneration culture is carried out through the leaf tissue, the culture material is saved to a certain extent, the culture cost is reduced, a large amount of adventitious buds grow in the culture process through adding the mitogen and the auxin in the culture medium, the regeneration rate of the new buds is increased, the culture efficiency is improved, the generation of embryoid is promoted by adding the reducing nitrogen and the naphthylacetic acid in the culture medium, so that the new nandina seedling keeps the inherent characters and characteristics of the original variety, and the survival rate and the culture success rate of the new nandina seedling are improved by placing the test-tube seedling rooted on the MS culture medium into an artificial substrate for gradual exercise and then planting into soil.
Description
Technical Field
The invention relates to the technical field of seedling culture, in particular to a strong-adaptability regeneration culture method for nandina seedlings.
Background
Nandina domestica is also called as the Carcinia bambusicola, belong to Ranunculaceae, berberidaceae, produce in China Changjiang river basin and provinces such as Shaanxi, Hebei, Shandong, Hubei, Jiangsu, Zhejiang river, Anhui, Jiangxi, Guangdong, Guangxi, Yunnan, Guizhou and Sichuan, it is the common woody flower species in the south of China, because its plant is graceful, the fruit is bright-colored, strong adaptability to the environment, appear in the garden application often, because its form is superior and elegant, also used for making bonsai or potted plant to decorate windowsill, hallway, meeting place, etc., in addition Nandina domestica also has very good medical value, have broad market prospects, therefore cultivated and planted on a large scale;
the prior nandina domestica cultivation method mainly comprises a sowing method and a plant division method, but the two methods have low cultivation success rate, waste land resources and cannot effectively maintain the inherent properties and characteristics of the original variety, and with the continuous development of tissue culture technologies in recent years, particularly seedling propagation by using the plant tissue culture technology, the method is a great revolution of the conventional plant cultivation technology, but the traditional tissue culture technology has long cultivation period, high cost and poor cultivation effect on flame nandina domestica due to the complex genetic characteristics of the nandina domestica, so the invention provides a regeneration cultivation method of nandina domestica seedlings with strong adaptability to solve the problems in the prior art.
Disclosure of Invention
Aiming at the problems, the invention aims to provide a regeneration culture method of nandina nursery stocks with strong adaptability, which uses a lock-independent routing table synchronization algorithm, so that the problem of sequence deadlock cannot be caused when messages are synchronously transmitted among threads, the threads can be reused, and the thread creation and destruction cost is reduced.
In order to achieve the purpose of the invention, the invention is realized by the following technical scheme: a strong adaptability regeneration culture method for nandina nursery stock comprises the following steps:
the method comprises the following steps: tissue culture
Placing the selected sterile tissue culture seedling and a tool required by inoculation into a sterile room, then sterilizing and disinfecting the sterile room, simultaneously wiping hands of operators with alcohol, sequentially burning the inoculation tool with flame of an alcohol lamp, then removing blade tips and blade edges of unfolded new leaves on the sterile tissue culture seedling with a scalpel, then cutting the treated new leaves into leaf tissues and quickly inoculating the leaf tissues into a culture medium in a test tube, after the inoculation is finished, disinfecting the tube orifice of the test tube with the flame of the alcohol lamp, covering a tube cover, and finally placing the test tube with the leaf tissues into a culture room for culture;
step two: inducing adventitious bud
According to the first step, because axillary buds exist in each axillary leaf of the leaf tissue cultured in the culture room, after the axillary buds grow adventitious buds under natural conditions, cytokinin and auxin are added into a culture medium, because of the continuous action of the cytokinin and the auxin, the adventitious buds on the tissue continuously grow and differentiate to form a bud cluster, and then the bud cluster is subjected to subculture by repeatedly cutting and transferring to a new culture medium;
step three: forming embryoid body
According to the second step, after the adventitious bud is obtained, firstly adding reducing nitrogen into the culture medium, then continuously adding auxin containing naphthylacetic acid to induce the generation of embryoid, and timely transferring the embryoid to the culture medium without the auxin for continuous culture after the embryoid is generated so as to promote the maturation and the growth of the embryoid;
step four: soaking for rooting
Cutting off the test-tube plantlet after the bud generated by the adventitious bud proliferation way grows into a tender and dendritic test-tube plantlet, soaking the cut rootless test-tube plantlet in a high-concentration auxin solution for twenty-four hours, taking out the test-tube plantlet and inoculating the test-tube plantlet to an MS culture medium without hormone for seventy-two hours, and enabling the soaked test-tube plantlet to take roots on the MS culture medium;
step five: transplanting
According to the fourth step, when the root primordium of the test-tube plantlet protrudes on the MS culture medium and forms a short root, taking out the test-tube plantlet and washing off agar on the surface of the test-tube plantlet, then transplanting the cleaned test-tube plantlet into an artificial substrate containing nutrients, keeping the temperature around the test-tube plantlet unchanged after transplanting and avoiding direct sunlight;
step six: planting
According to the fifth step, after the test-tube plantlets are cultured for forty-eight hours and are gradually exercised in the artificial matrix, the test-tube plantlets are taken out of the artificial matrix and planted in sandy soil with fertile soil and good drainage, and field planting is completed.
The further improvement lies in that: in the first step, the sterile room is sterilized and disinfected by irradiating the sterile room with an ultraviolet sterilizing lamp, wherein the irradiation time of the ultraviolet sterilizing lamp is thirty minutes.
The further improvement lies in that: in the first step, the culture medium consists of inorganic nutrients, carbohydrate, B vitamins, agar and gibberellin, wherein the inorganic nutrients comprise nitrate nitrogen, phosphate, sulfate, potassium, calcium salt, phosphate, chelated iron, iodine, manganese, zinc, molybdenum, copper, cobalt and iron.
The further improvement lies in that: in the first step, the culture of leaf tissue in the test tube is carried out by combining dark culture for preventing the aging of rapidly dividing callus cells and light culture for inducing differentiation, and the time of the dark culture is four times that of the light culture.
The further improvement lies in that: in the second step, the cytokinin added to the medium is 6-benzylpurine with a concentration of 5 mg/l, and the auxin added to the medium is naphthylacetic acid with a concentration of 0.5 mg/l.
The further improvement lies in that: in the fourth step, after the test-tube plantlet is replaced with the MS culture medium for twenty-four hours, the bottleneck of the test-tube plantlet is opened, two layers of abrasive cloth are covered on the bottleneck, the test-tube plantlet is used for ventilating, and meanwhile, the illumination is properly enhanced, so that the autotrophic capacity of the test-tube plantlet is improved.
The invention has the beneficial effects that: the invention collects leaf tissue on the aseptic tissue culture seedling and carries out thirty minutes ultraviolet irradiation disinfection on an aseptic room, so that the leaf tissue of Nandina domestica can be virus-removed in the culture process to become a nontoxic seedling, thereby being convenient for viewing and exporting, simultaneously saving culture materials to a certain extent by carrying out regeneration culture through the leaf tissues, reducing the culture cost, the addition of the mitogen and the auxin in the culture medium enables the leaf tissue to grow a large number of adventitious buds in the culture process, increases the regeneration rate of new buds and improves the culture efficiency, the addition of the reducing nitrogen and the naphthylacetic acid in the culture medium promotes the generation of embryoid, therefore, the new nandina domestica seedling keeps the inherent properties and characteristics of the original variety, and the survival rate and the culture success rate of the new nandina domestica seedling are improved by placing the test-tube seedling rooted on the MS culture medium into the artificial substrate for gradual exercise and then planting the test-tube seedling into soil.
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FIG. 1 is a flow chart of the culture of the present invention.
Detailed Description
In order to further understand the present invention, the following detailed description will be made with reference to the following examples, which are only used for explaining the present invention and are not to be construed as limiting the scope of the present invention.
According to the illustration in fig. 1, the present embodiment provides a method for regeneration and cultivation of nandina nursery stock with strong adaptability, which includes the following steps:
the method comprises the following steps: tissue culture
Placing the selected sterile tissue culture seedling and the tools required by inoculation into a sterile room, irradiating and disinfecting the sterile room for thirty minutes by an ultraviolet sterilizing lamp, wiping hands of operators with alcohol, sequentially burning the inoculation tools by using the flame of the alcohol lamp, removing leaf tips and leaf edges of unfolded new leaves on the sterile tissue culture seedling by using a scalpel, cutting the treated new leaves into leaf tissues, quickly inoculating the leaf tissues into a culture medium consisting of inorganic nutrients, carbohydrates, B vitamins, agar and gibberellin in a test tube, wherein the inorganic nutrients comprise nitrate nitrogen, phosphate, sulfate, potassium, calcium salt, phosphate, chelated iron, iodine, manganese, zinc, molybdenum, copper, cobalt and iron, disinfecting the tube orifice of the test tube by using the flame of the alcohol lamp after inoculation is finished, covering a tube cover, and finally placing the test tube with the leaf tissues into the culture room for dark culture and light culture, the dark culture time is four times of that of the light culture, wherein the dark culture is used for preventing the aging of the rapidly dividing callus cells, and the light culture is used for inducing differentiation;
step two: inducing adventitious bud
According to the first step, because axillary buds exist in each axillary leaf of the leaf tissue cultured in the culture room, after the axillary buds grow adventitious buds under natural conditions, cytokinin and auxin are added into a culture medium firstly, wherein the cytokinin is 6-benzylpurine, the concentration is 5 mg/L, the auxin is naphthylacetic acid, the concentration is 0.5 mg/L, and due to the continuous action of the cytokinin and the auxin, the adventitious buds on the tissue continuously grow and differentiate to form a bud cluster, and then the bud cluster is repeatedly cut and transferred to a new culture medium for subculture;
step three: forming embryoid body
According to the second step, after the adventitious bud is obtained, firstly adding reducing nitrogen into the culture medium, then continuously adding auxin containing naphthylacetic acid to induce the generation of embryoid, and timely transferring the embryoid to the culture medium without the auxin for continuous culture after the embryoid is generated so as to promote the maturation and the growth of the embryoid;
step four: soaking for rooting
Cutting off the test-tube plantlet after the bud generated by the adventitious bud hyperplasia way grows into a tender and dendritic test-tube plantlet, soaking the cut rootless test-tube plantlet in a high-concentration auxin solution for twenty-four hours, taking out the test-tube plantlet and inoculating the test-tube plantlet to an MS culture medium without hormone for seventy-two hours, so that the soaked test-tube plantlet takes root on the MS culture medium, opening the bottleneck of the test-tube plantlet and covering the bottleneck with two layers of abrasive cloths for ventilating the test-tube plantlet and properly enhancing illumination to improve the autotrophic capacity of the test-tube plantlet after the test-tube plantlet is replaced to the MS culture medium for twenty-four hours;
step five: transplanting
According to the fourth step, when the root primordium of the test-tube plantlet protrudes on the MS culture medium and forms a short root, taking out the test-tube plantlet and washing off agar on the surface of the test-tube plantlet, then transplanting the cleaned test-tube plantlet into an artificial substrate containing nutrients, keeping the temperature around the test-tube plantlet unchanged after transplanting and avoiding direct sunlight;
step six: planting
According to the fifth step, after the test-tube plantlets are cultured for forty-eight hours and are gradually exercised in the artificial matrix, the test-tube plantlets are taken out of the artificial matrix and planted in sandy soil with fertile soil and good drainage, and field planting is completed.
The regeneration culture method of the Nandina domestica seedling with strong adaptability is characterized in that leaf tissue is collected on a sterile tissue culture seedling and is irradiated by ultraviolet rays for thirty minutes to kill virus in a sterile room, so that the leaf tissue of the Nandina domestica is virus-removed in the culture process to form a nontoxic seedling, so that the Nandina domestica is convenient to view and export, meanwhile, the regeneration culture is carried out through the leaf tissue, so that culture materials are saved to a certain extent, the culture cost is reduced, a large number of adventitious buds grow in the culture process of the leaf tissue by adding mitogen and auxin in a culture medium, the culture efficiency is improved while the regeneration rate of new buds is increased, the occurrence of embryoids is promoted by adding reducing nitrogen and naphthylacetic acid in the culture medium, so that the new Nandina domestica seedling keeps the inherent characters and characteristics of the original variety, and the test-tube seedling rooted on the MS culture medium is planted in soil after being gradually exercised, the survival rate and the culture success rate of the nandina new seedling are improved.
The foregoing illustrates and describes the principles, general features, and advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. A strong adaptability regeneration culture method of Nandina domestica nursery stock is characterized in that: the method comprises the following steps:
the method comprises the following steps: tissue culture
Placing the selected sterile tissue culture seedling and a tool required by inoculation into a sterile room, then sterilizing and disinfecting the sterile room, simultaneously wiping hands of operators with alcohol, sequentially burning the inoculation tool with flame of an alcohol lamp, then removing blade tips and blade edges of unfolded new leaves on the sterile tissue culture seedling with a scalpel, then cutting the treated new leaves into leaf tissues and quickly inoculating the leaf tissues into a culture medium in a test tube, after the inoculation is finished, disinfecting the tube orifice of the test tube with the flame of the alcohol lamp, covering a tube cover, and finally placing the test tube with the leaf tissues into a culture room for culture;
step two: inducing adventitious bud
According to the first step, because axillary buds exist in each axillary leaf of the leaf tissue cultured in the culture room, after the axillary buds grow adventitious buds under natural conditions, cytokinin and auxin are added into a culture medium, because of the continuous action of the cytokinin and the auxin, the adventitious buds on the tissue continuously grow and differentiate to form a bud cluster, and then the bud cluster is subjected to subculture by repeatedly cutting and transferring to a new culture medium;
step three: forming embryoid body
According to the second step, after the adventitious bud is obtained, firstly adding reducing nitrogen into the culture medium, then continuously adding auxin containing naphthylacetic acid to induce the generation of embryoid, and timely transferring the embryoid to the culture medium without the auxin for continuous culture after the embryoid is generated so as to promote the maturation and the growth of the embryoid;
step four: soaking for rooting
Cutting off the test-tube plantlet after the bud generated by the adventitious bud proliferation way grows into a tender and dendritic test-tube plantlet, soaking the cut rootless test-tube plantlet in a high-concentration auxin solution for twenty-four hours, taking out the test-tube plantlet and inoculating the test-tube plantlet to an MS culture medium without hormone for seventy-two hours, and enabling the soaked test-tube plantlet to take roots on the MS culture medium;
step five: transplanting
According to the fourth step, when the root primordium of the test-tube plantlet protrudes on the MS culture medium and forms a short root, taking out the test-tube plantlet and washing off agar on the surface of the test-tube plantlet, then transplanting the cleaned test-tube plantlet into an artificial substrate containing nutrients, keeping the temperature around the test-tube plantlet unchanged after transplanting and avoiding direct sunlight;
step six: planting
According to the fifth step, after the test-tube plantlets are cultured for forty-eight hours and are gradually exercised in the artificial matrix, the test-tube plantlets are taken out of the artificial matrix and planted in sandy soil with fertile soil and good drainage, and field planting is completed.
2. The method for regenerating Nandina domestica seedlings with strong adaptability according to claim 1, wherein the method comprises the following steps: in the first step, the sterile room is sterilized and disinfected by irradiating the sterile room with an ultraviolet sterilizing lamp, wherein the irradiation time of the ultraviolet sterilizing lamp is thirty minutes.
3. The method for regenerating Nandina domestica seedlings with strong adaptability according to claim 1, wherein the method comprises the following steps: in the first step, the culture medium consists of inorganic nutrients, carbohydrate, B vitamins, agar and gibberellin, wherein the inorganic nutrients comprise nitrate nitrogen, phosphate, sulfate, potassium, calcium salt, phosphate, chelated iron, iodine, manganese, zinc, molybdenum, copper, cobalt and iron.
4. The method for regenerating Nandina domestica seedlings with strong adaptability according to claim 1, wherein the method comprises the following steps: in the first step, the culture of leaf tissue in the test tube is carried out by combining dark culture for preventing the aging of rapidly dividing callus cells and light culture for inducing differentiation, and the time of the dark culture is four times that of the light culture.
5. The method for regenerating Nandina domestica seedlings with strong adaptability according to claim 1, wherein the method comprises the following steps: in the second step, the cytokinin added to the medium is 6-benzylpurine with a concentration of 5 mg/l, and the auxin added to the medium is naphthylacetic acid with a concentration of 0.5 mg/l.
6. The method for regenerating Nandina domestica seedlings with strong adaptability according to claim 1, wherein the method comprises the following steps: in the fourth step, after the test-tube plantlet is replaced with the MS culture medium for twenty-four hours, the bottleneck of the test-tube plantlet is opened, two layers of abrasive cloth are covered on the bottleneck, the test-tube plantlet is used for ventilating, and meanwhile, the illumination is properly enhanced, so that the autotrophic capacity of the test-tube plantlet is improved.
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| CN202010235605.4A CN111280062A (en) | 2020-03-30 | 2020-03-30 | Regeneration culture method of Nandina domestica nursery stock with strong adaptability |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113875593A (en) * | 2021-10-22 | 2022-01-04 | 杭州木木生物科技有限公司 | Sugar-free tissue culture method and device for improving rooting rate of nandina domestica |
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2020
- 2020-03-30 CN CN202010235605.4A patent/CN111280062A/en not_active Withdrawn
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113875593A (en) * | 2021-10-22 | 2022-01-04 | 杭州木木生物科技有限公司 | Sugar-free tissue culture method and device for improving rooting rate of nandina domestica |
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