TWI675102B - Use of composition as insulating layer for plant tissue culture - Google Patents

Abstract

The invention provides the use of a composition as an insulation layer for plant tissue culture. The aforementioned composition is composed of a polyvinyl acetate emulsion resin and a polar solvent, thereby effectively isolating the medium from contact with a source of pollution, which includes microorganisms to prevent the microorganisms from growing in the medium of plant tissue culture.

Description

Use of composition as insulation layer of plant tissue culture

The invention relates to the use of a composition, in particular to the use of a composition as a plant tissue culture barrier.

Plant tissue culture is a technology that separates part of the cells or tissues of the plant body from the mother body and cultivates them under appropriate conditions so that they can grow, develop, differentiate and proliferate. The principle is derived from the totipotent differentiation ability of plant cells, that is, a certain type of cells in a plant can develop independently and differentiate into a complete plant adult. Plant tissue culture can produce a large number of plants with a small number of mothers, which makes plant tissue culture have many uses, such as basic botany and genetics research, and agricultural breeding and variety retention.

Infection with microorganisms is the most common cause of culture failure. Generally, plant tissue culture needs to be performed on a sterile operation table, and all equipment, such as tweezers and culture bottles, need to be sterilized or sterilized to prevent the tissues to be cultured from being affected. Bacterial and fungal infections. In addition to equipment, if the plant tissue to be cultivated is from a plant that is generally grown outdoors, then These plant tissues also need to be disinfected with disinfecting water. The water used in the operation is also sterilized distilled water.

In order to ensure that the tissue culture plants in the tissue culture flask are not infected by microorganisms, and maintain the tissue culture flask in a sterile state, the tissue culture plants after the operation are usually sealed in the tissue culture flask to isolate external microorganisms. However, plants need oxygen and carbon dioxide to improve their growth. It is difficult to make tissue culture plants grow healthy in a limited confined space. A small amount of ethylene is also released during the growth of the plant. When ethylene accumulates to a certain concentration, it will have a negative effect on the growth of the plant. Therefore, ventilation in a limited space is very important for tissue culture plants. In addition, the tissue culture bottle easily accumulates water vapor or water droplets in a closed environment to maintain a certain humidity, which becomes an appropriate environment for microbial growth. Therefore, incompletely sterilized microorganisms can grow in the tissue culture bottle and cause contamination of tissue culture plants.

In view of this, one aspect of the present invention is to provide a use of a composition as an insulation layer for plant tissue culture, wherein the composition is composed of a polyvinyl acetate emulsion resin and a polar solvent.

According to the use of the aforementioned composition as an insulating layer for plant tissue culture, wherein the polar solvent may be water, ethanol, or a combination thereof.

According to the use of the aforementioned composition as an insulating layer for plant tissue culture, when the polar solvent is water, the polyvinyl acetate emulsion resin and water may be Mix in a volume ratio of 1: 2 to 3: 1. Preferably, the polyvinyl acetate emulsion resin and water may be mixed in a volume ratio of 2: 1.

According to the use of the aforementioned composition as an insulation layer for plant tissue culture, when the polar solvents are water and ethanol, the polyvinyl acetate emulsion resin, ethanol and water can be mixed in a volume ratio of 5: 12: 3 to 5: 4: 1. . Preferably, the polyvinyl acetate emulsion resin, ethanol and water may be mixed in a volume ratio of 4: 3: 1. Or polyvinyl acetate emulsion resin, ethanol and water can be mixed in a volume ratio of 2: 5: 1.

According to the use of the aforementioned composition as an insulation layer for plant tissue culture, the insulation layer can be used to isolate the medium from contact with a pollution source. The pollution source can be a microorganism. Preferably, the microorganism can be a fungus or a bacterium.

In this way, the composition of the present invention can be used as an insulation layer for plant tissue culture, which is covered on the medium for plant tissue culture and isolates the medium from contact with microorganisms, so that tissue culture plants can be cultivated in open spaces without being affected by bacteria Or fungal contamination.

The above summary is intended to provide a simplified summary of the present disclosure so that the reader may have a basic understanding of the present disclosure. This summary is not a comprehensive overview of the disclosure, and it is not intended to indicate important / critical elements of the embodiments of the invention or to define the scope of the invention.

110‧‧‧ Example 1 Composition

111, 112, 113, 114, 115‧‧‧ Example 1 Composition Test Group

120‧‧‧ Example 2 Composition

130‧‧‧ Example 3 Composition

140‧‧‧Example 4 Composition

150‧‧‧ Example 5 Composition

210‧‧‧ Example 6 Composition

211, 212, 213‧‧‧ Example 6 composition test group

310‧‧‧Example 7 Composition

311, 312, 313, 314, 315‧‧‧ Example 7 composition test group

410, 420, 430‧‧‧ control group

510‧‧‧Comparative example insulation layer

511, 512, 513, 514, 515, 516, 517, 518, 519‧‧‧ Comparative example 3 test group

In order to make the above and other objects, features, advantages, and embodiments of the present invention more comprehensible, the description of the drawings is as follows: Figure 1A is a graph showing the results of the composition of the first embodiment of the present invention after being formulated on the first day; Figure 1B is a graph showing the results of the solidification state of the composition of Example 1 of the first embodiment of the present invention on the third day Figure 1C shows the result of solidification of the composition of Example 2 of the first embodiment of the present invention on the third day; Figure 1D shows the composition of the composition of Example 3 of the first embodiment of the present invention on the third day The result chart of the condition; FIG. 1E is a result chart showing the solidification status of the composition of Example 4 of Embodiment 1 of the present invention on the third day; FIG. 1F is a view showing the composition of Example 5 of Embodiment 1 of the present invention on Results of solidification on the third day; Figures 2A and 2B show the test results of the control group to isolate microbial growth; Figures 3A, 3B, and 3C are examples of the first embodiment of the present invention 1 Composition test results for isolation of microbial growth; Figures 4A and 4B show test results for composition 6 for blocking microorganism growth in Embodiment 6 of the present invention; Figures 5A and 5B show the test results. Implementation of the invention The results of the second embodiment of the composition of Example 7 were tested against microbial growth; and FIGS. 6A and 6B show the results of the test of Comparative Example 3 from microbial growth.

The invention provides the use of a composition as an insulation layer for plant tissue culture, wherein the composition is composed of a polyvinyl acetate emulsion resin and a polar solvent.

Polyvinyl acetate resin (PVAc) is a thermoplastic long-chain polymer resin obtained by the addition reaction of vinyl acetate (VAc) monomer. Its polymerization reaction is a free Radical polymerization (Free-radical polymerization). However, since the vinyl acetate monomer is only slightly soluble in water, the polyvinyl acetate resin formed by further polymerization is insoluble in water. Therefore, if you want to obtain a water-based adhesive, you must use emulsion polymerization to form a resin emulsion. This reaction system It contains an oil-soluble polyvinyl acetate resin monomer, an emulsifier (surfactant; protective colloid), a reaction initiator, and water (dispersed phase) to obtain a polyvinyl acetate emulsion resin. Its curing is the drying and hardening of the adhesive by evaporation of the moisture of the adhesive, so it can be cured at room temperature without the need of adding a curing agent.

The polar solvent may be water, ethanol, or a combination thereof. When the polar solvent is water, the polyvinyl acetate emulsion resin and water may be mixed in a volume ratio of 1: 2 to 3: 1. Preferably, the polyvinyl acetate emulsion resin and water may be mixed in a volume ratio of 2: 1. When the polar solvents are water and ethanol, the polyvinyl acetate emulsion resin, ethanol and water may be mixed in a volume ratio of 5: 12: 3 to 5: 4: 1. Preferably, the polyvinyl acetate emulsion resin, ethanol and water may be mixed in a volume ratio of 4: 3: 1. Or polyvinyl acetate emulsion resin, ethanol and water can be mixed in a volume ratio of 2: 5: 1.

The insulation layer covers the culture medium of the plant tissue, and can be used to isolate the contact between the culture medium and the pollution source, and the pollution source can be a microbiotic. Preferably, the microorganism may be a fungus or a bacterium. Thereby, the tissue culture plant can be cultivated in an open space without being contaminated by bacteria or fungi.

The following specific examples are provided to further illustrate the present invention, and are intended to benefit those of ordinary skill in the technical field to which the present invention pertains, and can make full use and practice of the present invention without excessive interpretation, and these test examples should not be considered The scope of the present invention is limited, but is used to explain how to implement the materials and methods of the present invention.

[Examples and Comparative Examples] Embodiment 1

As mentioned above, the present invention aims to provide a composition that can be used as a barrier layer for plant tissue culture. The composition is based on a polyvinyl acetate emulsion resin, and the polyvinyl acetate emulsion resin is dissolved in a polar material. Derived from a solvent. In the first embodiment, the polar solvent is water, especially sterilized sterile water. The polyvinyl acetate emulsion resin and water may be mixed in a volume ratio of 1: 2 to 3: 1. Specifically, the polyvinyl acetate emulsion resin and water were respectively prepared in the mixing ratio shown in Table 1 to prepare the composition 110 of Example 1, the composition 120 of Example 2, the composition 130 of Example 3, the composition 140 of Example 4, and implementation. Example 5 Composition 150.

Please refer to FIG. 1A to FIG. 1F, where FIG. 1A is a graph showing the results of the composition of Embodiment 1 on the first day, and from left to right are the composition 1 of Example 1 of Embodiment 1, 110, Example 2 composition 120, Example 3 composition 130, Example 4 composition 140, and Example 5 composition 150. FIG. 1B is a graph showing the result of the solidification of the composition 110 of Example 1 on the third day, FIG. 1C is a graph showing the result of the solidification of the composition 120 of Example 2 on the third day, and FIG. 1D is a graph showing Example 3 The result chart of the solidification state of the composition 130 on the third day. FIG. 1E shows the result chart of the solidification state of the composition 140 of Example 4 on the third day. The first figure shows the composition of the composition 150 of Example 5 on the third day. Result graph for condition.

After the preparation on the first day, the largest viscosity was found to be the composition 120 of Example 2, and the viscosity of the remaining examples was the composition of Example 110 and the composition of Example 3 in order from large to small. 130. Example 5 composition 150 and Example 4 composition 140. The easiest to pour into the glass tube is the composition 110 of Example 1. On the third day after the preparation, the examples were observed in a solidified state during an oblique observation period. From the results of FIG. 1B and FIG. 1C, it is shown that the composition 110 of Example 1 and the composition 120 of Example 2 have been cured on the third day. The results in FIG. 1D show that the composition 130 of Example 3 moved slightly on the third day. However, the results of FIG. 1E and FIG. 1F show that the composition of Example 4 and the composition of Example 5 150 remained liquid on the third day. Subsequently, the composition 110 of Example 1 will be used to conduct an isolation test for microbial growth.

The medium used for the isolated microorganism growth test is 1/2 MS semi-solid medium, which uses the basic salt formula of MS (Murashige and Skoog 1962), but the nitrogen content is 1/2, and a large number of elements contain 950mg / l KNO ₃ , 440mg / l CaCl ₂ . 2H ₂ O, 185 mg / l of MgSO ₄ . 7H ₂ O and 85 mg / l KH ₂ PO ₄ . Trace elements include KI of 0.265 mg / l, H ₃ BO _{3 of} 3.1 mg / l, and MnSO _{4 of} 11.15 mg / l. 4H ₂ O, 4.3 mg / l of ZnSO ₄ . 7H ₂ O, 0.125 mg / l Na ₂ MoO ₄ . 2H ₂ O, 0.0125 mg / l CuSO ₄ . 5 H ₂ O and 0.0125 mg / l CoCl ₂ . 6H ₂ O. Iron salt has 13.9mg / l FeSO ₄ . 7H ₂ O and 18.65 mg / l NaEDTA. 2 H ₂ O. Other additives include 50mg / l of Inositol, 0.25mg / l of Nicotinic acid, 0.25mg / l of Pyridoxin, and 0.05mg / l of Thiamine-HCL , 1 mg / l of glycine (Glycine), etc., and 3% agar (agarose) was added. The plant material is a sterile seedling of the succulent plant of Haworthia, all operations are performed in a sterile operation table. The test procedure is as follows: plant material is implanted into the culture medium for later use, a glass tube is implanted into a plant material, and a total of 9 tubes in the test group. Then, the polyvinyl acetate emulsion resin and water are mixed uniformly in a volume ratio of 2: 1 to prepare a composition of the present invention (composition 110 of Example 1). Then, the composition 110 of Example 1 is poured into a test tube with a thickness of about 0.2-0.3 cm, so that the composition 110 of Example 1 sufficiently covers the surface of the culture medium to form an insulating layer. Then, the tissue culture plant covered with the composition 110 of Example 1 was placed in an open space to conduct an isolation test for microbial growth, and photographs were taken on the 1st, 28th, and 34th days to observe the time when the microorganisms started to breed. The experiment also includes a control group, which is a 1/2 MS semi-solid medium without any antibacterial conditions. The control group consists of 3 tubes (control group 410, control group 420, and control group 430). The control group is also placed open. The space is tested for microbial growth, and the time at which microbes begin to breed is observed.

Please refer to FIGS. 2A to 3C again. Figures 2A and 2B show the test results of the control group to isolate microbial growth. Figures 2A and 2B are from left to right for the control group 410, the control group 420, and the control group 430, respectively. The test results on day 1 and Figure 2B are the test results on day 4. Figures 3A, 3B, and 3C show the results of the test for isolating microbial growth in the test group of the composition of Example 1 (5 of 9 tubes). Figures 3A, 3B, and 3C are from left to right. On the right are the composition test group 111 of embodiment 1, the composition test group 112 of embodiment 1, the composition test group 113 of embodiment 1, the composition test group 114 of embodiment 1, and the composition test group 115 of embodiment 1, respectively. Figure 3A shows the test results on day 1, Figure 3B shows the test results on day 28, and Figure 3C shows the test results on day 34.

The results in Figure 2B show that the moldy condition can be observed on control group 410, control group 420, and control group 430 without any antibacterial condition on the fourth day, and according to the moldy condition, it can be pushed back on the second day. By the third day, mold growth began to occur. The results of FIGS. 3B and 3C show that the composition test group 111, the test composition 112, the test composition 113, the test composition 113, and the test composition of Example 1 are provided with an insulating layer. The group 114 and the composition test group 115 of Example 1 showed no microbial growth on the 28th and 34th days, showing that the composition according to the first embodiment of the present invention can be used as an insulation layer for plant tissue culture, and can make tissues Cultivated plants grow in open spaces and effectively avoid bacterial or fungal contamination.

Embodiment 2

In the second embodiment, the polar solvents are water and ethanol, especially sterilized sterile water. The polyvinyl acetate emulsion resin, ethanol and water may be mixed in a volume ratio of 5: 12: 3 to 5: 4: 1.

In an example of the second embodiment, specifically, a polyvinyl acetate emulsion resin, ethanol and water are mixed in a volume ratio of 4: 3: 1 to prepare a composition 210 of Example 6. The prepared composition 6 of Example 6 is poured into a test tube containing a culture medium and a plant material, and the thickness is about 0.2 to 0.3 cm, so that the composition 210 of Example 6 sufficiently covers the surface of the culture medium to form an insulation layer. 9 tubes. Then, the tissue culture plant covered with the composition 210 of Example 6 was placed in an open space to conduct an isolation test for microbial growth, and photographs were taken on the 1st and 28th days to observe the time when the microorganisms started to breed.

Please refer to FIG. 4A and FIG. 4B, which show the test results of microbial growth isolation of the composition 210 of Example 6 (3 of 9 tubes), and FIGS. 4A and 4B are Example 6 from left to right. The composition test group 211, the composition test group 212 of Example 6, and the composition test group 213 of Example 6, wherein FIG. 4A is the test result on the first day, and FIG. 4B is the test result on the 28th day. The results in FIG. 4B show that in the test group 211, the composition test group 212, and the composition test group 213 of Example 6, there was no microbial growth on the 28th day, indicating the implementation of the present invention. The composition of the second method can be used as an insulation layer for plant tissue culture, which can enable tissue culture plants to grow in open spaces, and effectively avoid bacterial or fungal pollution.

In another example of the second embodiment, specifically, a polyvinyl acetate emulsion resin, ethanol and water are mixed and prepared in a volume ratio of 2: 5: 1. Example 7 Composition 310. The prepared composition 7 of Example 7 is poured into a test tube containing a culture medium and a plant material, and the thickness is about 0.2-0.3 cm, so that the composition 310 of Example 7 sufficiently covers the surface of the culture medium to form an insulation layer. 9 tubes. Then, the tissue culture plant covered with the composition 310 of Example 7 was placed in an open space to conduct an isolation test for microbial growth, and photographs were taken on the first day and the 28th day to observe the time when the microbial growth began.

Please refer to FIG. 5A and FIG. 5B, which show the test results (3 tubes out of 9 tubes) of the composition 310 of Example 7 for isolating microbial growth. Among them, FIG. 5A is the test result on the first day, and FIG. From left to right are a composition test group 311, a composition test group 312, a composition test group 313, a composition test group 314, and a composition test group 315. FIG. 5B is the test result on the 28th day, and FIG. 5B is from left to right of the Example 7 composition test group 313, the Example 7 composition test group 314, and the Example 7 composition test group 315, respectively. The results in FIG. 5B show that the microbial growth condition was still not observed on the 28th day of the 7th composition test group 313, the 7th composition test group 314, and the 7th composition test group 315 with the barrier layer. It is shown that the composition of the second embodiment of the present invention can be used as an insulation layer for plant tissue culture, which can enable tissue culture plants to grow in an open space, and effectively avoid bacterial or fungal pollution.

Comparative example

In this test, Clorox colloid was used as a comparison layer in plant tissue culture as a comparative example, and a test for microbial growth isolation was further performed. Clorox colloids are prepared by preparing 5%, 10%, and 15% Clorox (NaOCl) solutions in sterile water, and then adding 1% agar to form 5%. Clorox colloid (comparative example 1), 10% Clorox colloid (comparative example 2), and 15% Clorox colloid (comparative example 3). The medium used for the isolation test for microbial growth was a 1/2 MS semi-solid medium, and the plant material was a sterile seedling of a succulent plant of Haworthia. All operations were completed in a sterile operation table. The test procedure is as follows: first pour 1/2 MS semi-solid medium into the sterilized test tube, wait for the 1/2 MS semi-solid medium to solidify, and then pour into Comparative Example 1, Comparative Example 2 and Comparative Example 3 to test tubes, The thickness of the pouring is about 1 to 1.5 cm, so that Comparative Example 1, Comparative Example 2 and Comparative Example 3 sufficiently cover the surface of the culture medium to form a comparative example isolation layer 510. The plant material was implanted 2 days after Clorox colloids were solidified, one glass tube and one plant material. There were 9 tubes in each test group. Then the tissue culture plants covering Comparative Example 1, Comparative Example 2 and Comparative Example 3 were placed in an open space to conduct an isolation test for microbial growth, and photos were taken on the 1st and 28th days to observe the time when the microorganisms started to breed.

The tissue culture plants using Comparative Example 1 and Comparative Example 2 as insulation layers began to show microbial growth on the 3rd and 4th day of the test, respectively. For the tissue culture plant using Comparative Example 3 as an insulation layer, the microbiological growth of three tissue culture plants was found on the 10th day of the test, and the microbiological growth of 6 tissue culture plants was found on the 12th day of the test. On the 15th day of the experiment, it was found that microbial growth occurred in 8 tissue culture plants. Please refer to Fig. 6A and Fig. 6B, which show the test results of isolating microbial growth in Comparative Example 3 (6 of 9 tubes), of which Fig. 6A is the test result on the first day, and Fig. 6A is from left to On the right are Comparative Example 3 Test Group 511, Comparative Example 3 Test Group 512, Comparative Example 3 Test Group 513, Comparative Example 3 Test Group 514, Comparative Example 3 Test Group 515, Comparative Example 3 Test Group 516, Comparative Example 3 test group 517, Comparative Example 3 test group 518, and Comparative Example 3 test group 519. Figure 6B shows the test results on the 28th day, and Figure 6B shows from left to right Comparative Example 3 Test Group 515, Comparative Example 3 Test Group 516, Comparative Example 3 Test Group 517, Comparative Example 3 Test Group 513, and Comparative Example. 3 test groups 514 and Comparative Example 3 test groups 519. On the 28th day of the test, Comparative Example 3 test group 515, Comparative Example 3 test group 516, Comparative Example 3 test group 517, Comparative Example 3 test group 513, Comparative Example 3 test group 514, and Comparative Example 3 test group 519 had serious problems. Microbial contamination, it can be seen that using Clorox, a commonly used disinfectant for plant tissue culture, as an insulation layer still cannot effectively inhibit microbial growth.

In summary, the composition of the present invention is obtained by using a polyvinyl acetate emulsion resin as the main component, and then dissolving the polyvinyl acetate emulsion resin in a polar solvent, which can be used as an insulation layer for plant tissue culture. Covered on the plant tissue culture medium, it can effectively isolate the contact between the medium and the microorganisms, so that the tissue culture plants can be cultivated in the open space without being contaminated by bacteria or fungi, and the time without being contaminated by microorganisms can be up to 34 Therefore, the composition disclosed in the present invention has the potential for application in plant tissue culture.

However, the present invention has been disclosed in the above embodiments, but it is not intended to limit the present invention. Any person skilled in the art can make various modifications and retouches without departing from the spirit and scope of the present invention. Therefore, the protection of the present invention The scope shall be determined by the scope of the attached patent application.

Claims (6)

Use of a composition as an insulation layer for plant tissue culture, wherein the composition is composed of polyvinyl acetate emulsion resin and a polar solvent, and a plant tissue in plant tissue culture is a succulent plant Sterile vaccine; where the polar solvent is water, ethanol, or a combination thereof; when the polar solvent is water, the polyvinyl acetate emulsion resin and the water system are mixed in a volume ratio of 2: 1; when the polar solvent is water and In the case of ethanol, the polyvinyl acetate emulsion resin, the ethanol and the water system are mixed in a volume ratio of 4: 3: 1 to 2: 5: 1. The use of the composition described in item 1 of the scope of patent application as an insulation layer for plant tissue culture, wherein when the polar solvent is water and ethanol, the polyvinyl acetate emulsion resin, the ethanol and the water system are 4: Mix in a volume ratio of 3: 1. The use of the composition described in item 1 of the scope of patent application as an insulation layer for plant tissue culture, wherein when the polar solvent is water and ethanol, the polyvinyl acetate emulsion resin, the ethanol and the water system have a 2: 5: 1 volume ratio mixing. Use of the composition according to item 1 of the scope of patent application as an insulation layer for plant tissue culture, wherein the insulation layer is used to isolate a medium from contact with a pollution source. The use of the composition according to item 4 of the patent application as an insulation layer for plant tissue culture, wherein the source of pollution is a microorganism. The use of the composition according to item 5 of the patent application as an insulation layer for plant tissue culture, wherein the microorganism is a fungus or a bacterium.