CN117084173A - Method for culturing dendrobium candidum tissue culture seedlings by using tissue culture bags - Google Patents
Method for culturing dendrobium candidum tissue culture seedlings by using tissue culture bags Download PDFInfo
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- CN117084173A CN117084173A CN202311206819.9A CN202311206819A CN117084173A CN 117084173 A CN117084173 A CN 117084173A CN 202311206819 A CN202311206819 A CN 202311206819A CN 117084173 A CN117084173 A CN 117084173A
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- tissue culture
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- seedlings
- dendrobium candidum
- culturing
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Links
- 241000026010 Dendrobium candidum Species 0.000 title claims abstract description 37
- 238000012258 culturing Methods 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 title claims abstract description 24
- 230000035755 proliferation Effects 0.000 claims abstract description 21
- 239000012883 rooting culture medium Substances 0.000 claims abstract description 19
- 230000006698 induction Effects 0.000 claims abstract description 17
- 239000001963 growth medium Substances 0.000 claims abstract description 9
- 239000002609 medium Substances 0.000 claims abstract description 9
- 239000000843 powder Substances 0.000 claims description 15
- 240000004638 Dendrobium nobile Species 0.000 claims description 12
- 244000061456 Solanum tuberosum Species 0.000 claims description 10
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 10
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 10
- 229930006000 Sucrose Natural products 0.000 claims description 10
- 239000005720 sucrose Substances 0.000 claims description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 8
- 239000000203 mixture Substances 0.000 claims description 7
- 229920001817 Agar Polymers 0.000 claims description 6
- 239000008272 agar Substances 0.000 claims description 6
- 238000005286 illumination Methods 0.000 claims description 5
- 239000000679 carrageenan Substances 0.000 claims description 4
- 229920001525 carrageenan Polymers 0.000 claims description 4
- 229940113118 carrageenan Drugs 0.000 claims description 4
- 235000010418 carrageenan Nutrition 0.000 claims description 4
- 235000015193 tomato juice Nutrition 0.000 claims description 4
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 claims description 4
- 241000196324 Embryophyta Species 0.000 abstract description 15
- 210000004027 cell Anatomy 0.000 description 5
- 239000012882 rooting medium Substances 0.000 description 5
- 239000008223 sterile water Substances 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 2
- 240000008790 Musa x paradisiaca Species 0.000 description 2
- 235000018290 Musa x paradisiaca Nutrition 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000004161 plant tissue culture Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 241001523681 Dendrobium Species 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229960002523 mercuric chloride Drugs 0.000 description 1
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 210000001938 protoplast Anatomy 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012353 t test Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Classifications
-
- 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
- A01H4/002—Culture media for tissue culture
-
- 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 provides a method for culturing dendrobium candidum tissue culture seedlings by using tissue culture bags, and belongs to the technical field of tissue culture. The invention provides a method for culturing dendrobium candidum tissue culture seedlings by using tissue culture bags, which comprises the following steps: (1) Selecting stem tips of sprouting of dendrobium candidum as explants, inoculating the explants into a cluster bud induction medium, and performing induction culture to obtain dendrobium candidum cluster buds; (2) Dividing the clustered shoots of the dendrobium candidum, inoculating the segmented shoots into a clustered shoot proliferation culture medium, and carrying out proliferation culture to obtain strong dendrobium candidum seedlings; (3) And (3) after the strong seedlings of the dendrobium candidum are segmented, inoculating the strong seedlings of the dendrobium candidum into a tissue culture bag filled with a rooting culture medium, and carrying out rooting culture to obtain complete dendrobium candidum plants. The invention provides a method for culturing dendrobium candidum tissue culture seedlings by using tissue culture bags, which reduces the pollution rate of dendrobium candidum tissue culture plants and improves the rooting rate of dendrobium candidum tissue culture plants.
Description
Technical Field
The invention relates to the technical field of tissue culture, in particular to a method for culturing dendrobium candidum tissue culture seedlings by using tissue culture bags.
Background
Plant tissue culture is a technique in which living isolated organs (such as roots, stems, leaves, stem segments, protoplasts, etc.), tissues or cells are placed in a medium under artificially created sterile conditions and placed in a suitable environment to perform continuous culture to obtain cells, tissues or individuals, and this technique has been widely used in agricultural, biological and medical research. Plant tissue culture exploits the totipotency of cells, meaning that cells that have been differentiated, still have the potential to develop into a complete organism. Each plant cell contains a complete set of genetic material, but only under specific conditions. Based on the principle, the tissue culture can dedifferentiate plant tissues which are already at differentiation terminal or are differentiating to induce the formation of callus, and then induce the formation of new cluster buds on the callus.
When cutting the plant stem and leaf, the forceps, scissors and dissecting knife are needed to be used for operation by exchanging hands for many times, and the process is easy to cause pollution; meanwhile, due to the limitation of a culture bottle mouth, plant materials are inconvenient to put in a designated position in a culture medium; in addition, the difficulty in grasping the tissue of the palm when the sample is contacted with forceps is likely to destroy the tissue of the sample, and plant dysplasia is caused. The tissue culture bag is beneficial to saving space and the bag mouth is larger to facilitate the placement of seedlings, but the problem is that the plant pollution rate of the tissue culture bag is higher.
Disclosure of Invention
The invention aims to provide a method for culturing dendrobium candidum tissue culture seedlings by using a tissue culture bag, which reduces the problem of high plant pollution rate caused by using the tissue culture bag as a rooting culture container.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a method for culturing dendrobium candidum tissue culture seedlings by using tissue culture bags, which comprises the following steps:
(1) Selecting stem tips of sprouting of dendrobium candidum as explants, inoculating the explants into a cluster bud induction medium, and performing induction culture to obtain dendrobium candidum cluster buds;
(2) Dividing the clustered shoots of the dendrobium candidum, inoculating the segmented shoots into a clustered shoot proliferation culture medium, and carrying out proliferation culture to obtain strong dendrobium candidum seedlings;
(3) Dividing the strong seedlings of the dendrobium candidum, inoculating the strong seedlings of the dendrobium candidum into a tissue culture bag filled with a rooting culture medium, and carrying out rooting culture to obtain complete dendrobium candidum plants, wherein the rooting culture medium is put into the tissue culture bag and then is arranged by a scraper so as to gather the rooting culture medium adhered to the side wall of the tissue culture bag to the bottom;
the rooting culture medium comprises the following components: 1/2 MS+15-20 g/L of potato powder+4-6 ml/L of mature tomato juice+0.8-1.2 g/L of Huabao No. 1+0.3-0.7 g/L of activated carbon+4-6 mg/L of NAA+8-12 g/L of sucrose+5-7 g/L of carrageenan.
Preferably, the composition of the cluster bud induction medium is as follows: MS+6-BA 2.0-4.0 mg/L+NAA 0.1-0.3 mg/L+GA 3 0.3-0.5 mg/L+25-35 g/L sucrose+5-6 g/L agar powder.
Preferably, the conditions of the induction culture are: culturing for 18-22 d at 23-27 ℃, illuminating for 14-16 h each day, and darkening for 8-10 h, wherein the illumination intensity is 1500-2000 lx.
Preferably, the composition of the cluster bud proliferation medium is as follows: MS+6-BA 1.0-3.0 mg/L+NAA 0.08-0.12 mg/L+sucrose 25-35 g/L+agar 5-7 g/L.
Preferably, the conditions of the proliferation culture are: culturing for 30-35 d at 23-27 ℃, illuminating for 14-16 h each day, and darkening for 8-10 h, wherein the illumination intensity is 1500-2000 lx.
Preferably, the proliferation culture is further followed by a secondary culture to obtain strong seedlings of dendrobium.
Preferably, the rooting culture conditions are as follows: after culturing for 50-60 d at 23-27 ℃, the light is irradiated for 12-13 h each day, the darkness is carried out for 11-12 h, and the light intensity is 1500-1800 lx.
The invention provides a method for culturing dendrobium candidum tissue culture seedlings by using a tissue culture bag, and provides a rooting culture medium suitable for the tissue culture bag culture.
Drawings
FIG. 1 is a comparative graph of the growth states of the dendrobium nobile plants in the experimental group and the control group in example 1, wherein the left graph is the control group, and the right graph is the experimental group.
Detailed Description
The technical solutions provided by the present invention are described in detail below with reference to examples, but they should not be construed as limiting the scope of the present invention.
Example 1
A method for culturing dendrobium nobile tissue culture seedlings by using tissue culture bags comprises the following steps:
(1) Acquisition of explants: taking germinated buds (1.5 cm in length) on healthy parent plant branches of dendrobium candidum, carrying out sterile water washing for 3 times, soaking in 75% alcohol for 5s, taking out sterile water for 3 times, soaking in 0.1% mercuric chloride solution for sterilization for 1min, taking out sterile water for 3 times, and cutting stem tips with a sterilized scalpel to obtain explants.
(2) Induction of cluster buds: the stem tip explants were inoculated into a clustered shoot induction medium (200 mL flask) and subjected to induction culture. The composition of the cluster bud induction culture medium is as follows: MS+6-BA 3.0mg/L+NAA0.2mg/L+GA 3 0.4 mg/L+30 g/L of sucrose+5 mg/L of agar powder. The induction culture conditions are as follows: culturing at 25deg.C for 20d, 16 hr light, 8 hr darkness, and light intensity of 2000lx.
(3) Proliferation of clumped buds: cutting the induced cluster buds into small buds by using a sterile knife, and respectively inoculating the small buds into a cluster bud proliferation culture medium (a container is a culture bottle) for proliferation culture to obtain cluster buds with a proliferation coefficient of 7.6. The composition of the cluster bud proliferation culture medium is as follows: MS+6-BA2.0mg/L+NAA0.1mg/L+sucrose 30 g/L+agar 6g/L. The proliferation culture conditions are as follows: culturing at 25deg.C for 35d, and illuminating for 16 hr each day for 8 hr in dark, wherein the illumination intensity is 2000lx. Then replacing the proliferation culture medium once, and carrying out secondary culture for 1 time to obtain robust cluster buds.
(4) Rooting culture: cutting robust cluster buds into single buds (2 cm in height) by using a sterile guide, inoculating the single buds to a rooting culture medium (a container is a culture bag, the length is multiplied by 20cm multiplied by 15 cm), arranging the rooting culture medium into a tissue culture bag by using a scraper, gathering the rooting culture medium adhered to the side wall of the tissue culture bag to the bottom, reducing the pollution rate to a certain extent, performing rooting culture on the rooting culture medium, and culturing at 25 ℃ for 60 days, wherein the rooting culture medium is irradiated for 12 hours per day, is dark for 12 hours, and has the irradiation intensity of 1500lx. Whole plants with a plant height of 8cm were obtained, and the rooting rate was 100%. The rooting culture medium comprises the following components: 1/2 MS+15 g/L of potato powder+5 ml/L of mature tomato juice+1 g/L of Huabao No. 1+0.5 g/L of activated carbon+5 mg/L of NAA+10 g/L of sucrose+6 g/L of carrageenan. The potato powder and Huabao No. one were purchased from Beijing Xindacheng Chengdi technology Co.
In the present invention, since the explant and the cluster buds are small in volume and are easy to operate in the culture flask due to the induction culture and proliferation culture process of the cluster buds, the use of the culture flask is a better choice, but when rooting culture is performed, the height of the cluster buds reaches 2cm, so that the operation is inconvenient and plant tissues are easily injured when the cluster buds are inoculated in the culture flask, and the risk of pollution is increased. Thus, the present invention uses a tissue culture bag as a culture vessel during rooting culture. The culture medium used in the invention is subjected to high-temperature sterilization treatment at 121 ℃ for 15min before use.
Meanwhile, the robust cluster buds obtained in the step (3) are cut into single buds (2 cm in height) by a sterile knife and inoculated into a control rooting medium, the container is still a culture bag, and the length is multiplied by 20cm multiplied by 15cm. The formula of the control rooting medium is as follows: 1/2MS, 15g/L of potato powder, 1g/L of Huabao No. 1, 0.5g/L of activated carbon, 5mg/L of NAA, 10g/L of sucrose and 6g/L of carrageenan. The culture conditions are unchanged.
The contamination ratios of the two were counted as shown in Table 1. As shown in Table 1, compared with the control rooting medium (30.75% of the control group), the rooting medium formula (11.50% of the experimental group) provided by the invention can remarkably reduce the pollution rate of plants in the rooting stage of the dendrobium candidum. The invention shows that the addition of the mature tomato juice into the rooting culture medium has the function of reducing the plant pollution rate in the tissue culture bag culture process. A comparison graph of typical growth states of the experimental group and the control group is shown in fig. 1.
TABLE 1 statistical results of contamination Rate
Example 2
The influence of the potato powder consumption in rooting culture on the rooting rate of dendrobium candidum is studied, the potato powder consumption in the rooting culture medium in the step (3) of the example 1 is changed to 13g/L, induction, proliferation and rooting culture are carried out according to the method and the steps of the example 1, and the rooting rate is counted. Meanwhile, the potato powder is replaced by banana powder with the concentration of 10g/L and 13g/L, the experiment is also carried out, and the rooting rate is counted. The results are shown in Table 2.
TABLE 2 statistical results of rooting rate
As shown in table 2, when the amount of potato powder is reduced from 15g/L to 13g/L, the rooting rate of dendrobium nobile is greatly affected, the rooting rate is extremely reduced (t test, p=0.001), and after potato powder is replaced by banana powder with different concentrations, the rooting rate of dendrobium nobile is also greatly affected. The special composition and the proportion in the rooting culture medium provided by the invention have obvious effect on improving the rooting rate of the dendrobium candidum.
As can be seen from the above examples, the present invention provides a method for culturing dendrobium candidum tissue culture seedlings by using tissue culture bags, and provides a rooting medium suitable for tissue culture bag culture.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (7)
1. The method for culturing the dendrobium nobile tissue culture seedlings by using the tissue culture bags is characterized by comprising the following steps:
(1) Selecting stem tips of sprouting of dendrobium candidum as explants, inoculating the explants into a cluster bud induction medium, and performing induction culture to obtain dendrobium candidum cluster buds;
(2) Dividing the clustered shoots of the dendrobium candidum, inoculating the segmented shoots into a clustered shoot proliferation culture medium, and carrying out proliferation culture to obtain strong dendrobium candidum seedlings;
(3) Dividing the strong seedlings of the dendrobium candidum, inoculating the strong seedlings of the dendrobium candidum into a tissue culture bag filled with a rooting culture medium, and carrying out rooting culture to obtain complete dendrobium candidum plants, wherein the rooting culture medium is put into the tissue culture bag and then is arranged by a scraper so as to gather the rooting culture medium adhered to the side wall of the tissue culture bag to the bottom;
the rooting culture medium comprises the following components: 1/2 MS+15-20 g/L of potato powder+4-6 ml/L of mature tomato juice+0.8-1.2 g/L of Huabao No. 1+0.3-0.7 g/L of activated carbon+4-6 mg/L of NAA+8-12 g/L of sucrose+5-7 g/L of carrageenan.
2. The method for culturing dendrobium nobile tissue culture seedlings by using tissue culture bags according to claim 1, wherein the composition of the cluster bud induction medium is as follows: MS+6-BA 2.0-4.0 mg/L+NAA 0.1-0.3 mg/L+GA 3 0.3-0.5 mg/L+25-35 g/L sucrose+5-6 g/L agar powder.
3. The method for culturing dendrobium nobile tissue culture seedlings by using a tissue culture bag according to claim 2, wherein the conditions of the induction culture are as follows: culturing for 18-22 d at 23-27 ℃, illuminating for 14-16 h each day, and darkening for 8-10 h, wherein the illumination intensity is 1500-2000 lx.
4. The method for culturing dendrobium nobile tissue culture seedlings by using tissue culture bags according to claim 1, wherein the composition of the cluster bud proliferation medium is as follows: MS+6-BA 1.0-3.0 mg/L+NAA 0.08-0.12 mg/L+sucrose 25-35 g/L+agar 5-7 g/L.
5. The method for culturing dendrobium nobile tissue culture seedlings by using a tissue culture bag as claimed in claim 4, wherein the proliferation culture conditions are as follows: culturing for 30-35 d at 23-27 ℃, illuminating for 14-16 h each day, and darkening for 8-10 h, wherein the illumination intensity is 1500-2000 lx.
6. The method for culturing dendrobium nobile tissue culture seedlings by using a tissue culture bag as claimed in claim 5, wherein the proliferation culture is further followed by a secondary culture to obtain dendrobium nobile strong seedlings.
7. The method for culturing dendrobium nobile tissue culture seedlings by using a tissue culture bag according to claim 1, wherein the rooting culture conditions are as follows: after culturing for 50-60 d at 23-27 ℃, the light is irradiated for 12-13 h each day, the darkness is carried out for 11-12 h, and the light intensity is 1500-1800 lx.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202311206819.9A CN117084173A (en) | 2023-09-19 | 2023-09-19 | Method for culturing dendrobium candidum tissue culture seedlings by using tissue culture bags |
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| CN202311206819.9A CN117084173A (en) | 2023-09-19 | 2023-09-19 | Method for culturing dendrobium candidum tissue culture seedlings by using tissue culture bags |
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