CN111642402B - Open tissue culture rooting method for actinidia arguta - Google Patents
Open tissue culture rooting method for actinidia arguta Download PDFInfo
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- CN111642402B CN111642402B CN202010698173.0A CN202010698173A CN111642402B CN 111642402 B CN111642402 B CN 111642402B CN 202010698173 A CN202010698173 A CN 202010698173A CN 111642402 B CN111642402 B CN 111642402B
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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
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention discloses an open tissue culture rooting method for actinidia arguta, which comprises the following steps: adding a bacteriostatic agent into the culture medium for later use; and inoculating the actinidia arguta tissue culture seedlings into the culture medium containing the bacteriostatic agent for culture. According to the method, the bacteriostat tetrachloroisophthalonitrile is added into the culture medium, and the auxin concentration required by the actinidia arguta open type tissue culture rooting and the adding concentration of tetrachloroisophthalonitrile in the culture medium are limited, so that the technical purpose of the actinidia arguta open type tissue culture rooting is realized, and when the actinidia arguta is subjected to tissue culture rooting, a strict and sterile operation environment, an autoclave and an ultra-clean workbench are not required, and a good rooting effect can be realized, so that the operation can be simplified, the workload is reduced, and the cost of tissue culture industrial seedling culture is obviously reduced.
Description
Technical Field
The invention belongs to the technical field of plant culture, and particularly relates to an open tissue culture rooting method for actinidia arguta.
Background
At present, the tissue culture technology of actinidia arguta is mature day by day, but the traditional tissue culture requires strict environmental conditions, the operation is complex, and the cost is increased undoubtedly. The open tissue culture is to add bacteriostatic agent into the culture medium, so that the plant tissue culture is separated from a strict aseptic operation environment, and the plant tissue culture is carried out in a natural and open sterile environment without autoclaving and an ultra-clean workbench, thereby reducing the environmental requirements, reducing the operation links, and remarkably reducing the cost in the aspects of equipment, field, energy and the like. Open tissue culture in Japan and other countries has been widely applied to tissue culture of ornamental plants such as chrysanthemum and orchid, and China has studied open tissue culture of banana, sugarcane, butterfly orchid, potato and the like. At present, the tissue culture and rapid propagation of actinidia arguta mainly comprises proliferation, rooting and seedling strengthening, and particularly in the rooting and seedling strengthening stage, the production materials and the manual demand are high, so that the operation links can be reduced, the cost is reduced, and the efficiency is improved by researching the open culture in the rooting stage. At present, relevant reports about open culture of actinidia arguta at the rooting stage are rarely seen.
Disclosure of Invention
In order to solve the technical problems in the prior art, the invention provides an open tissue culture rooting method for actinidia arguta, which can realize a good rooting effect of actinidia arguta without strict aseptic operating environment, high-pressure sterilization and an ultra-clean workbench.
In order to achieve the technical purpose, the invention provides the following technical scheme: an open tissue culture rooting method for actinidia arguta comprises the following steps:
(1) Adding a bacteriostatic agent into the culture medium for later use;
(2) Adding auxin into the culture medium containing the bacteriostatic agent obtained in the step (1) for later use;
(3) And (3) inoculating the actinidia arguta tissue culture seedling into the culture medium containing the bacteriostatic agent and the auxin, which is obtained in the step (2), and culturing.
Preferably, the culture medium in the step (1) is a sugar-free solid culture medium of 1/4MS, the bacteriostatic agent is tetrachloroisophthalonitrile, and the concentration in the culture medium is 0.2-0.5g/L.
Tetrachloroisophthalonitrile is a protective fungicide. The action mechanism is that the enzyme can act with the glyceraldehyde triphosphate dehydrogenase in the fungal cells, and the enzyme is combined with protein containing cysteine in the enzyme, so that the activity of the enzyme is damaged, and the metabolism of the fungal cells is damaged and the vitality of the fungal cells is lost. Tetrachloroisophthalonitrile has good adhesion, is stable in alkali and acid aqueous solutions and is irradiated by ultraviolet light, and has a long drug action period.
Preferably, the auxin is indolebutyric acid (IBA) and the concentration in the culture medium is 0.01-0.05mg/L.
Preferably, in the step (3), the culture temperature is 20-24 ℃, the illumination intensity is 1600-2000 lx, the illumination time per day is 10-14h, and the culture time is 18-22 days.
Preferably, in step (3), the culture is performed in a tissue culture flask or a culture box.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention adds the bacteriostatic agent tetrachloroisophthalonitrile into the culture medium, which can effectively inhibit the growth of bacteria and is superior to the bacteriostatic agent carbendazim in the aspects of bacteriostasis and root growth promotion. The technical purpose of the open tissue culture rooting of actinidia arguta is achieved by adding auxin into a 1/4MS sugar-free solid culture medium, so that when actinidia arguta is subjected to tissue culture rooting, a strict sterile operating environment, an autoclave and an ultra-clean workbench are not needed, a good rooting effect can be achieved, the operation can be simplified, the workload is reduced, and meanwhile, the cost of tissue culture industrial seedling culture is remarkably reduced.
2. The method limits the auxin concentration required by the open tissue culture rooting of actinidia arguta and the addition concentration of tetrachloroisophthalonitrile in a culture medium, can obtain higher survival rate, and has the advantages of good plant growth state, white and clean roots, uniform thickness, good appearance and shape, a large number of roots, better plant height and good rooting effect.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.
FIG. 1 is a morphological diagram of the appearance of plants cultured in examples 1-7.
FIG. 2 is the appearance and morphology of the plants obtained from the culture of examples 9-12.
FIG. 3 is a statistical chart of the survival rates of plants cultured in examples 9-12.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but rather as a more detailed description of certain aspects, features and embodiments of the invention. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference herein for the purpose of disclosing and describing the methods and/or materials associated with the documents. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including but not limited to.
In the following examples, the actinidia arguta tissue culture seedling is a tissue culture seedling cultured by a single-bud stem section of a 'kuilu' green branch in the institute of specialty of academy of agricultural sciences of china.
Example 1
(1) Adding 30mL of 1/4MS sugar-free solid culture medium with the thickness of 2cm into a culture bottle, and adding 9mg of tetrachloroisophthalonitrile and 0.3 mu g of auxin IBA for later use;
(2) And (2) inoculating the 5 actinidia arguta tissue culture seedlings into the culture medium containing the bacteriostatic agent in the step (1) for culture, wherein the culture temperature is 22 ℃, the illumination intensity is 1800lx, the illumination time is 12 hours per day, and the culture is carried out for 20 days.
Example 2
(1) Adding 30mL of 1/4MS sugar-free solid culture medium with the thickness of 2cm into a culture bottle, and adding 6mg of tetrachloroisophthalonitrile and 1.5 mu g of auxin IBA for later use;
(2) And (2) inoculating 6 actinidia arguta tissue culture seedlings into the culture medium containing the bacteriostatic agent in the step (1) for culture, wherein the culture temperature is 20 ℃, the illumination intensity is 1600lx, the illumination time is 14h per day, and the culture is carried out for 22 days.
Example 3
(1) Adding 30mL of 1/4MS sugar-free solid culture medium with the thickness of 2.5cm into a culture bottle, and adding 15mg of tetrachloroisophthalonitrile and 3 mu g of auxin IBA for later use;
(2) And (2) inoculating 4 actinidia arguta tissue culture seedlings into the culture medium containing the bacteriostatic agent in the step (1) for culture, wherein the culture temperature is 24 ℃, the illumination is given, the illumination intensity is 2000lx, the illumination time is 10h per day, and the culture is carried out for 18 days.
Example 4
(1) Adding 30mL of 1/4MS sugar-free solid culture medium with the thickness of 2cm into a culture bottle, and adding 9mg of tetrachloroisophthalonitrile and 6 mu g of auxin IBA for later use;
(2) And (2) inoculating the 5 actinidia arguta tissue culture seedlings into the culture medium containing the bacteriostatic agent in the step (1) for culture, wherein the culture temperature is 22 ℃, the illumination is given, the illumination intensity is 2000lx, the illumination time is 12h per day, and the culture is carried out for 20 days.
Example 5
The same as example 1 except that tetrachloroisophthalonitrile in step (1) was replaced with carbendazim.
Example 6
(1) Adding 30mL of 1/4MS sugar-free solid culture medium with the thickness of 2.5cm into a culture bottle, and adding 6mg of tetrachloroisophthalonitrile for later use;
(2) Dipping a stem section of 0.5cm of a stem base part of 6 actinidia arguta tissue culture seedlings in 0.5mg/L auxin IBA for 35s at a medium speed, then inoculating the stem section into the culture medium containing the bacteriostatic agent in the step (1), and culturing for 20 days, wherein the culture temperature is 22 ℃, the illumination intensity is 2000lx, and the illumination time is 14h per day.
Example 7
(1) Adding 30mL of 1/4MS sugar-free solid culture medium with the thickness of 2cm into a culture bottle, and adding 15mg of tetrachloroisophthalonitrile for later use;
(2) Dipping stem sections of 0.5cm of stem base parts of 5 actinidia arguta tissue culture seedlings in 1.0mg/L auxin IBA for 25s at medium speed, then inoculating the stem sections into the culture medium containing the bacteriostatic agent in the step (1) for culture, wherein the culture temperature is 24 ℃, the illumination is given, the illumination intensity is 1600lx, the illumination time is 14h per day, and the culture lasts for 22 days.
Example 8
(1) Adding 30mL of 1/4MS sugar-free solid culture medium with the thickness of 2cm into a culture bottle, and adding 9mg of tetrachloroisophthalonitrile for later use;
(2) And (2) inoculating 5 actinidia arguta tissue culture seedlings into the culture medium containing the bacteriostatic agent in the step (1) for culture, wherein the culture temperature is 22 ℃, the illumination is given, the illumination intensity is 1800lx, the illumination time is 12 hours per day, and the culture is carried out for 20 days.
Example 9
The difference from example 2 is that the culture medium is placed in the culture dish, and the vent hole of the culture dish cover is closed.
Example 10
The difference from example 2 is that the culture medium was placed in the culture dish, and the vent hole of the lid of the culture dish was set to 1/3 of the size.
Example 11
The difference from example 2 is that the culture medium was placed in the culture dish, and the vent hole of the culture dish cover was half-opened.
Example 12
The difference from example 2 is that the culture medium was placed in the culture dish, and the vent hole of the culture dish cover was fully opened.
Example 13
The same as example 2, except that tetrachloroisophthalonitrile was not added to the medium in the step (1).
Example 14
The same as example 2 except that tetrachloroisophthalonitrile was added in an amount of 4mg in the step (1).
Example of Effect verification
The morphology of the plants obtained by culturing in examples 1-14 was observed, and the results are shown in Table 1; the average plant height and the average root number of the plants obtained in each of examples 1 to 14 were counted, and the results are shown in Table 2; the appearance of the plants of examples 1-7 is shown in FIG. 1.
TABLE 1
TABLE 2
| Group of | Average plant height (cm) | Average root number (strip) |
| Example 1 | 4.63 | 8.39 |
| Example 2 | 4.88 | 9.28 |
| Example 3 | 4.48 | 9.69 |
| Example 4 | 4.86 | 10.82 |
| Example 5 | 4.12 | - |
| Example 6 | 4.35 | 8.67 |
| Example 7 | 4.86 | 9.78 |
| Example 8 | 4.07 | 7.24 |
| Example 9 | 4.38 | 8.87 |
| Example 10 | 4.22 | 8.40 |
| Example 11 | 4.72 | 7.98 |
| Example 12 | 4.87 | 8.21 |
| Example 13 | 4.33 | - |
| Example 14 | 4.48 | - |
As can be seen from Table 1, the culture medium can not effectively inhibit the growth of bacteria when the tetrachloroisophthalonitrile is not added or added but the addition amount is less than 0.2g/L or the tetrachloroisophthalonitrile is replaced by carbendazim, and the actinidia arguta tissue culture seedlings can not effectively root because the culture medium can not be sterilized and a large amount of bacteria can breed when the tetrachloroisophthalonitrile is not added; when the addition amount of tetrachloroisophthalonitrile is less than 0.2g/L, or tetrachloroisophthalonitrile is replaced by carbendazim, the bacteriostatic effect is poor, and bacteria are bred in the rooting process, so that the rooting and growing effects of the tissue culture seedlings are poor.
As can be seen from tables 1 and 2, the plant heights of the plants cultured by the culture medium and subjected to the quick dipping are slightly different, but the differences among different treatments are not obvious, which indicates that the different rooting modes have small influence on the plant heights of the cultured seedlings. However, in examples 6 and 7, which adopt the quick-dipping rooting method, the stem base expands obviously, the stem is 0.6cm-1cm thick, the root is thick, short, dense and black, the method is mainly concentrated on the stem base by 1-1.5cm, aerial roots are arranged at positions above 2cm of the stem base, the analysis reason is probably that the concentration of dipped IBA is too high, the dedifferentiation of the stem base is promoted seriously, the root system elongation is inhibited, and the aerial roots above 2cm of the stem base show that the concentration of IBA absorbed by the upper stem is proper, so that the generation of the root system is promoted. In the rooting culture mode by adopting the culture medium, the rooting number of the plants is gradually increased along with the increase of the IBA concentration, the rooting number is higher by 0.2mg/L of IBA in example 4, and 10.82 plants are obtained on average. The growth state of the root systems of the embodiment 1 and the embodiment 2 is good, the root systems are white and clean, the growth is normal, the thicknesses are uniform, the root systems are mainly concentrated at the base part of 0.3-0.5cm, and the growth state of the stem base part is normal; the root system of example 3 is slightly coarse and short, the root system is white and clean, but the stem base is slightly thickened; the stem base of example 4 was significantly enlarged, the root system was dense and coarse, and the effect was poor, indicating that higher concentrations of IBA inhibited root elongation. In conclusion, the two treatment modes can achieve the purpose of rooting the actinidia arguta through open tissue culture, and the treatment with the best effect is shown in example 1 and example 2, namely the rooting effect is best when 0.01-0.05mg/L IBA is added into a sugar-free culture medium of 1/4MS containing tetrachloroisophthalonitrile with the concentration higher than 0.2 g/L.
The appearance and the form of the plants obtained in examples 9-12 are shown in fig. 2, and the survival rate of the plants is shown in fig. 3, which shows that the rooting effect of the plants is better in different treatments, the different opening and closing modes of the vent holes of the cover of the culture tray can affect the growth state of the plants, the vent holes are completely closed, the growth state of the plants is best, the leaves of the other treated plants are wilted in different degrees, and the analysis reason is that the water content in the culture tray is reduced due to the opening of the vent holes, so that the change range is too large compared with the original culture medium environment; and from the survival rate, the survival rate that the vent hole is completely closed is the highest. Therefore, in the process of cultivation, the vent hole should be closed to maintain a transparent cultivation environment with a certain humidity.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (4)
1. An open tissue culture rooting method for actinidia arguta is characterized by comprising the following steps:
(1) Adding a bacteriostatic agent into the culture medium;
(2) Adding auxin into the culture medium containing the bacteriostatic agent obtained in the step (1) for later use;
(3) Inoculating the actinidia arguta tissue culture seedlings into the culture medium containing the bacteriostatic agent and the auxin obtained in the step (2) for culture;
the culture medium in the step (1) is a 1/4MS sugar-free culture medium, the bacteriostatic agent is tetrachloroisophthalonitrile, and the concentration of the bacteriostatic agent in the culture medium is 0.2-0.5g/L.
2. The method for open tissue culture rooting of actinidia arguta according to claim 1, wherein in step (2) the auxin is indolebutyric acid and the concentration in the culture medium is 0.01-0.05mg/L.
3. The method for open tissue culture rooting of actinidia arguta according to claim 1, wherein in step (3), the culture temperature is 20-24 ℃, the illumination intensity is 1600-2000 lx, the illumination time per day is 10-14h, and the culture time is 18-22 days.
4. The method for open tissue culture rooting of actinidia arguta according to claim 1, wherein in step (3), the culture is performed in a tissue culture bottle or a culture box.
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