CN112237140A - Method for blocking dendrobium officinale tissue culture seedling from being polluted by bacteria - Google Patents
Method for blocking dendrobium officinale tissue culture seedling from being polluted by bacteria Download PDFInfo
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- CN112237140A CN112237140A CN202011112089.2A CN202011112089A CN112237140A CN 112237140 A CN112237140 A CN 112237140A CN 202011112089 A CN202011112089 A CN 202011112089A CN 112237140 A CN112237140 A CN 112237140A
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- 241001076416 Dendrobium tosaense Species 0.000 title claims abstract description 52
- 230000000903 blocking effect Effects 0.000 title claims abstract description 21
- 238000000034 method Methods 0.000 title claims abstract description 18
- 241000894006 Bacteria Species 0.000 title claims abstract description 10
- 229910052802 copper Inorganic materials 0.000 claims abstract description 81
- 239000010949 copper Substances 0.000 claims abstract description 81
- 239000001963 growth medium Substances 0.000 claims abstract description 54
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims abstract description 40
- 238000012258 culturing Methods 0.000 claims abstract description 37
- 239000000645 desinfectant Substances 0.000 claims abstract description 29
- 230000001954 sterilising effect Effects 0.000 claims abstract description 28
- 230000001580 bacterial effect Effects 0.000 claims abstract description 15
- 238000007865 diluting Methods 0.000 claims abstract description 13
- 230000004069 differentiation Effects 0.000 claims abstract description 12
- 230000000694 effects Effects 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 54
- 210000002257 embryonic structure Anatomy 0.000 claims description 42
- 238000002791 soaking Methods 0.000 claims description 30
- 239000012452 mother liquor Substances 0.000 claims description 24
- 239000008223 sterile water Substances 0.000 claims description 24
- 238000005406 washing Methods 0.000 claims description 24
- 239000012153 distilled water Substances 0.000 claims description 18
- 229920001817 Agar Polymers 0.000 claims description 12
- 241000234295 Musa Species 0.000 claims description 12
- 235000018290 Musa x paradisiaca Nutrition 0.000 claims description 12
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 12
- 244000061456 Solanum tuberosum Species 0.000 claims description 12
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 12
- 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 12
- 229930006000 Sucrose Natural products 0.000 claims description 12
- 230000002745 absorbent Effects 0.000 claims description 12
- 239000002250 absorbent Substances 0.000 claims description 12
- 239000008272 agar Substances 0.000 claims description 12
- 238000007796 conventional method Methods 0.000 claims description 12
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims description 11
- 239000002609 medium Substances 0.000 claims description 10
- 238000002156 mixing Methods 0.000 claims description 10
- 235000013399 edible fruits Nutrition 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 229960002523 mercuric chloride Drugs 0.000 claims description 6
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 6
- 238000011218 seed culture Methods 0.000 claims description 6
- 238000002054 transplantation Methods 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims 1
- 238000011109 contamination Methods 0.000 abstract description 6
- 239000000463 material Substances 0.000 abstract description 4
- 239000003242 anti bacterial agent Substances 0.000 abstract description 3
- 229940088710 antibiotic agent Drugs 0.000 abstract description 3
- 210000001161 mammalian embryo Anatomy 0.000 abstract description 3
- 241000196324 Embryophyta Species 0.000 abstract description 2
- 230000007226 seed germination Effects 0.000 abstract description 2
- 230000004083 survival effect Effects 0.000 abstract description 2
- 239000010413 mother solution Substances 0.000 abstract 2
- 239000000243 solution Substances 0.000 abstract 1
- 238000001816 cooling Methods 0.000 description 9
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000005476 size effect Effects 0.000 description 1
- 239000000126 substance Substances 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
- A01H4/008—Methods for regeneration to complete plants
-
- 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
Abstract
The invention provides a method for blocking the bacterial contamination of dendrobium officinale tissue culture seedlings, which is characterized by comprising the following steps: 1) preparing a nano-copper mother solution, 2) diluting the nano-copper mother solution, 3) preparing a disinfectant, 4) preparing a mixed culture medium I, 5) sterilizing the dendrobium officinale seeds, 6) culturing the dendrobium officinale seeds, 7) performing differential culture on the dendrobium officinale seeds, 8) preparing a mixed culture medium II, 9) performing differential embryo culture, and 10) performing bottle seedling culture and transplanting. Will 10‑2% of the nano copper solution is added into the culture medium, and infected bacteria are blocked in two culture processes of seed germination and immature embryo differentiation, so that the bacterial pollution rate of the dendrobium officinale is effectively reduced, the special technical effect is achieved, and the blocking rate is as high as 93%. Can reduce the harm of antibiotics to plants, improve the survival rate of dendrobium officinale seedlings, and can also block even if pollution exists, thereby preserving a large amount of experimental materials.
Description
Technical Field
The invention relates to the field of new materials and plant tissue culture, in particular to a method for blocking bacterial pollution of dendrobium officinale tissue culture seedlings by using nano-copper.
Background
The nano material is prepared by advanced physical, chemical, biological and other technologies, has one dimension, two dimensions or three dimensions, has the dimension of only a few nanometers or dozens of nanometers, and has a structure which is greatly different from that of a common material, such as large atomic distance of a nano grain boundary, low density, random atomic arrangement and the like. In recent years, studies related to antibacterial effects using nano copper have been attracting attention. The nano copper has the characteristics of quantum effect, small size effect, large surface area and the like, and shows better safety, antibacterial property and antibacterial long-acting property.
After the seeds exposed after the dendrobium officinale fruit pods are cracked are subjected to aseptic treatment, bacterial pollution is still easily caused.
Therefore, there is a need to research a new method capable of blocking the bacterial contamination of the dendrobium officinale tissue culture seedling.
Disclosure of Invention
The invention aims to solve the technical problem of providing a method capable of effectively blocking the bacterial pollution of the dendrobium officinale tissue culture seedlings.
The invention is completed by the following technical scheme: a method for blocking dendrobium officinale tissue culture seedlings from being polluted by bacteria is characterized by comprising the following steps:
1) preparing nano-copper mother liquor, namely putting nano-copper into sterilized distilled water, carrying out ice bath for 20-40 minutes at the temperature of 40-60 ℃, and then putting the nano-copper into an ultrasonic cell disruption instrument for treatment for 20-40 minutes under the ice bath condition to obtain the nano-copper mother liquor with the mass concentration of 0.1%;
2) diluting the nano-copper mother liquor, adding sterilized distilled water into the nano-copper mother liquor obtained in the step 1), and diluting to obtain a solution with the mass concentration of 10-2 % of the nano copper solution is sent into an oven, heated to 60-70 ℃ and then kept warm, and the activity of the nano copper is maintained;
3) preparing a disinfectant, namely adding the Youjie 1003 disinfectant into sterile distilled water until the mass concentration is 0.2%, and performing high-pressure sterilization for 20-30min at the temperature of 110-;
4) preparing mixed culture medium I, namely adding the following culture medium MS + NAA 0.05ml/L + 6-BA 0.1ml/L + banana puree 50g/L + potato 50g/L + white sugar 30g/L + agar 7g/L into 1L culture medium under the conditions of 110-125 ℃ and 0.1Mpa, sterilizing at high pressure for 20-30min, cooling to 50-60 ℃, and adding 20ml culture medium with the mass concentration of 10 ml under the aseptic condition-2 % of the nano copper solution, the mass concentration is 10-2% of the nano copper solution is added into the culture medium and stirred and mixed evenly to obtain a mixed culture medium I;
5) sterilizing Dendrobium officinale seeds, namely breaking the Dendrobium officinale fruit pods, taking out the seeds, putting the seeds into a gauze bag subjected to high-pressure sterilization treatment, and soaking the seeds in alcohol with the mass concentration of 70% for 40-60 seconds; then sequentially washing with sterile water for 2-4 times, sterilizing with 0.1% mercuric chloride for 6-10min, washing with sterile water for 2-4 times, soaking with 10% sodium hypochlorite for 6-10min, and washing with sterile water for 2-4 times; soaking for 1-2min by using the disinfectant in the step 3), taking out, putting on a sterile operating platform, and drying by using sterile absorbent paper to obtain sterilized seeds;
6) performing dendrobium officinale seed culture, uniformly placing the sterilized seeds in the step 5) in the mixed culture medium I in the step 4), performing dark culture until the seeds germinate, transferring the seeds to 1000lux of weak light, and culturing for 25-30 days to obtain young embryos;
7) performing differentiation culture on dendrobium officinale seeds, transferring the immature embryos obtained in the step 6) to a sterile environment with the temperature of 22-26 ℃ and the volume of 1000lux, culturing for 6-8 days, transferring to a light condition, and continuously culturing until the immature embryos of the dendrobium officinale are differentiated;
8) mixed Medium II was prepared by mixing the following media: MS + NAA 0.1ml/L + 6-BA 1ml/L + banana puree 50g/L + potato 50g/L + white sugar 30g/L + agar 7g/L, sterilizing at 110--2 % of the nano copper solution, the mass concentration is10-2% of the nano copper solution is added into the culture medium and stirred and mixed evenly to obtain a mixed culture medium II;
9) differentiating and culturing the young embryos, selecting the differentiated young embryos which are not polluted in the step 7), soaking the young embryos in sodium hypochlorite with the mass concentration of 10% for 6-10min, and washing the young embryos for 2-4 times by using sterile water; soaking for 1-2min by using the disinfectant in the step 3), taking out, putting on a sterile operating platform, sucking dry the solution by using sterile absorbent paper, inoculating the solution into the mixed culture medium II in the step 8), and culturing for 6-8 days at 23-26 ℃ in a sterile environment of 1000lux to obtain a pollution-free bottle seedling;
10) bottle seedling culture and transplantation, namely culturing and differentiating the pollution-free bottle seedling obtained in the step 9) according to a conventional method, and then rooting, hardening off, transplanting and planting according to the conventional method to achieve the purpose of blocking bacterial pollution.
The invention has the advantages and effects that: the invention adopts nano-copper solution to block seed bacterial pollution caused by dendrobium officinale fruit pod rupture, which is 10 times-2% of the nano copper solution is added into the culture medium, so that infected bacteria are blocked in two culture processes of seed germination and immature embryo differentiation, and the bacterial pollution rate of the dendrobium officinale is effectively reduced. Compared with the traditional method for adding antibiotics, the method has unique technical effect and the blocking rate is up to 93%. The method can reduce the damage of antibiotics to plants, improve the survival rate of the dendrobium officinale seedlings, and block even if pollution exists, so that a large amount of experimental materials are stored. The method adds the nano-copper solution into the sterile culture medium, blocks bacterial pollution of the dendrobium officinale seeds, and has the characteristics of good blocking effect, simple and convenient operation, no toxicity, low cost and the like.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
A method for blocking dendrobium officinale tissue culture seedlings from being polluted by bacteria is characterized by comprising the following steps:
1) preparing nano-copper mother liquor, namely putting nano-copper into sterilized distilled water, carrying out ice bath for 40 minutes at the temperature of 40 ℃, and then putting the nano-copper into an ultrasonic cell disruption instrument to treat for 40 minutes under the ice bath condition to obtain the nano-copper mother liquor with the mass concentration of 0.1%;
2) diluting the nano-copper mother liquor, adding sterilized distilled water into the nano-copper mother liquor obtained in the step 1), and diluting to obtain a solution with the mass concentration of 10-2 % of the nano copper solution is sent into an oven, heated to 70 ℃ and then kept warm, and the activity of the nano copper is maintained;
3) preparing a disinfectant, namely adding Youjie 1003 disinfectant into sterile distilled water until the mass concentration is 0.2%, and performing high-pressure sterilization for 20min at 125 ℃ under the condition of 0.1Mpa to obtain the disinfectant;
4) mixed medium i was prepared by mixing the following media: MS + NAA 0.05ml/L + 6-BA 0.1ml/L + banana puree 50g/L + potato 50g/L + white sugar 30g/L + agar 7g/L, autoclaving at 125 deg.C and 0.1Mpa for 20min, cooling to 60 deg.C, and adding 20ml culture medium with mass concentration of 10-2 % of the nano copper solution, the mass concentration is 10-2% of the nano copper solution is added into the culture medium and stirred and mixed evenly to obtain a mixed culture medium I;
5) sterilizing Dendrobium officinale seeds, namely breaking the Dendrobium officinale fruit pods, taking out the seeds, putting the seeds into a gauze bag subjected to high-pressure sterilization treatment, and soaking the seeds in alcohol with the mass concentration of 70% for 50 seconds; then washing with sterile water for 2 times, sterilizing with 0.1% mercuric chloride for 10min, washing with sterile water for 4 times, soaking with 10% sodium hypochlorite for 6min, and washing with sterile water for 2 times; soaking for 1min by using the disinfectant in the step 3), taking out, putting on a sterile operating platform, and drying by using sterile absorbent paper to obtain sterilized seeds;
6) performing dendrobium officinale seed culture, uniformly placing the sterilized seeds in the step 5) in the mixed culture medium I in the step 4), performing dark culture until the seeds germinate, transferring the seeds to 1000lux of weak light, and culturing for 25 days to obtain young embryos;
7) performing differentiation culture on dendrobium officinale seeds, transferring the immature embryos obtained in the step 6) to a sterile environment with the temperature of 26 ℃ and the lux of 1000, culturing for 6-8 days, transferring to a light condition, and continuously culturing until the immature embryos of the dendrobium officinale are differentiated;
8) preparation of mixed culture medium IIThe following media were prepared: MS + NAA 0.1ml/L + 6-BA 1ml/L + banana puree 50g/L + potato 50g/L + white sugar 30g/L + agar 7g/L, autoclaving at 125 deg.C and 0.1Mpa for 20min, cooling to 60 deg.C, adding 20ml culture medium with mass concentration of 10-2 % of the nano copper solution, the mass concentration is 10-2% of the nano copper solution is added into the culture medium and stirred and mixed evenly to obtain a mixed culture medium II;
9) differentiating and culturing the young embryos, selecting the differentiated young embryos which are not polluted in the step 7), soaking the young embryos in sodium hypochlorite with the mass concentration of 10% for 10min, and washing the young embryos for 2 times by using sterile water; soaking for 2min by using the disinfectant in the step 3), taking out, putting on an aseptic operation table, sucking the solution by using aseptic absorbent paper, inoculating the solution into the mixed culture medium II in the step 8), and culturing for 6 days in an aseptic environment of 1000lux at 26 ℃ to obtain a pollution-free bottle seedling;
10) bottle seedling culture and transplantation, namely culturing the pollution-free bottle seedlings obtained in the step 9) according to a conventional method, and after differentiation is completed, rooting, seedling hardening, transplanting and planting according to the conventional method.
The efficiency of blocking bacterial contamination of example 1 was 75%.
Example 2
A method for blocking dendrobium officinale tissue culture seedlings from being polluted by bacteria is characterized by comprising the following steps:
1) preparing nano-copper mother liquor, namely putting nano-copper into sterilized distilled water, carrying out ice bath for 20 minutes at the temperature of 60 ℃, and then putting the nano-copper into an ultrasonic cell disruption instrument to treat for 20 minutes under the ice bath condition to obtain the nano-copper mother liquor with the mass concentration of 0.1%;
2) diluting the nano-copper mother liquor, adding sterilized distilled water into the nano-copper mother liquor obtained in the step 1), and diluting to obtain a solution with the mass concentration of 10-2 % of the nano copper solution is sent into an oven, heated to 60 ℃ and then kept warm, and the activity of the nano copper is maintained;
3) preparing a disinfectant, namely adding Youjie 1003 disinfectant into sterile distilled water until the mass concentration is 0.2%, and performing high-pressure sterilization for 30min at 110 ℃ and 0.1Mpa to obtain the disinfectant;
4) mixed medium i was prepared by mixing the following media: MS + NAA 0.05ml/L + 6-BA 0.1ml/L + banana puree 50g/L + potato 50g/L + white sugar 30g/L + agar 7g/L, autoclaving at 110 deg.C and 0.1Mpa for 30min, cooling to 60 deg.C, and adding 20ml culture medium with mass concentration of 10 under sterile condition-2 % of the nano copper solution, the mass concentration is 10-2% of the nano copper solution is added into the culture medium and stirred and mixed evenly to obtain a mixed culture medium I;
5) sterilizing Dendrobium officinale seeds, namely breaking the Dendrobium officinale fruit pods, taking out the seeds, putting the seeds into a gauze bag subjected to high-pressure sterilization treatment, and soaking the seeds in alcohol with the mass concentration of 70% for 40 seconds; then washing with sterile water for 4 times, sterilizing with 0.1% mercuric chloride for 10min, washing with sterile water for 2 times, soaking with 10% sodium hypochlorite for 10min, and washing with sterile water for 2 times; soaking for 2min by using the disinfectant in the step 3), taking out, putting on a sterile operating platform, and drying by using sterile absorbent paper to obtain sterilized seeds;
6) performing dendrobium officinale seed culture, uniformly placing the sterilized seeds in the step 5) in the mixed culture medium I in the step 4), performing dark culture until the seeds germinate, transferring the seeds to 1000lux of weak light, and culturing for 25 days to obtain young embryos;
7) performing differentiation culture on dendrobium officinale seeds, transferring the immature embryos obtained in the step 6) to a sterile environment with the temperature of 22 ℃ and the lux, culturing for 8 days, transferring to a light condition, and continuously culturing until the immature embryos of the dendrobium officinale are differentiated;
8) mixed Medium II was prepared by mixing the following media: MS + NAA 0.1ml/L + 6-BA 1ml/L + banana puree 50g/L + potato 50g/L + white sugar 30g/L + agar 7g/L, sterilizing at 110 deg.C and 0.1Mpa under high pressure for 20min, cooling to 50 deg.C, adding 20ml culture medium with mass concentration of 10-2 % of the nano copper solution, the mass concentration is 10-2% of the nano copper solution is added into the culture medium and stirred and mixed evenly to obtain a mixed culture medium II;
9) differentiating and culturing the young embryos, selecting the differentiated young embryos which are not polluted in the step 7), soaking the young embryos in sodium hypochlorite with the mass concentration of 10% for 6min, and washing the young embryos for 2 times by using sterile water; soaking for 1min by using the disinfectant in the step 3), taking out, putting on an aseptic operation table, sucking the solution by using aseptic absorbent paper, inoculating the solution into the mixed culture medium II in the step 8), and culturing for 6 days in an aseptic environment of 1000lux at the temperature of 23 ℃ to obtain a pollution-free bottle seedling;
10) bottle seedling culture and transplantation, namely culturing the pollution-free bottle seedlings obtained in the step 9) according to a conventional method, and after differentiation is completed, rooting, seedling hardening, transplanting and planting according to the conventional method.
The efficiency of blocking bacterial contamination of example 2 was 85%.
Example 3
A method for blocking dendrobium officinale tissue culture seedlings from being polluted by bacteria is characterized by comprising the following steps:
1) preparing nano-copper mother liquor, namely putting nano-copper into sterilized distilled water, carrying out ice bath for 30 minutes at the temperature of 50 ℃, and then putting the nano-copper into an ultrasonic cell disruption instrument for treatment for 30 minutes under the ice bath condition to obtain the nano-copper mother liquor with the mass concentration of 0.1%;
2) diluting the nano-copper mother liquor, adding sterilized distilled water into the nano-copper mother liquor obtained in the step 1), and diluting to obtain a solution with the mass concentration of 10-2 % of the nano copper solution is sent into an oven, heated to 65 ℃ and then kept warm, and the activity of the nano copper is maintained;
3) preparing a disinfectant, namely adding Youjie 1003 disinfectant into sterile distilled water until the mass concentration is 0.2%, and performing autoclaving for 25min at the temperature of 120 ℃ and the pressure of 0.1Mpa to obtain the disinfectant;
4) mixed medium i was prepared by mixing the following media: MS + NAA 0.05ml/L + 6-BA 0.1ml/L + banana puree 50g/L + potato 50g/L + white sugar 30g/L + agar 7g/L, autoclaving at 120 deg.C and 0.1Mpa for 25min, cooling to 55 deg.C, and adding 20ml culture medium with mass concentration of 10 under aseptic condition-2 % of the nano copper solution, the mass concentration is 10-2% of the nano copper solution is added into the culture medium and stirred and mixed evenly to obtain a mixed culture medium I;
5) sterilizing Dendrobium officinale seeds, namely breaking the Dendrobium officinale fruit pods, taking out the seeds, putting the seeds into a gauze bag subjected to high-pressure sterilization treatment, and soaking the seeds in alcohol with the mass concentration of 70% for 60 seconds; then washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 8min, washing with sterile water for 3 times, soaking with 10% sodium hypochlorite for 8min, and washing with sterile water for 3 times; soaking for 2min by using the disinfectant in the step 3), taking out, putting on a sterile operating platform, and drying by using sterile absorbent paper to obtain sterilized seeds;
6) performing dendrobium officinale seed culture, uniformly placing the sterilized seeds in the step 5) in the mixed culture medium I in the step 4), performing dark culture until the seeds germinate, transferring the seeds to 1000lux of weak light, and culturing for 28 days to obtain young embryos;
7) performing differentiation culture on dendrobium officinale seeds, transferring the immature embryos obtained in the step 6) to a sterile environment with 24 ℃ and 1000lux, culturing for 6-8 days, transferring to a light condition, and continuously culturing until the immature embryos of the dendrobium officinale are differentiated;
8) mixed Medium II was prepared by mixing the following media: MS + NAA 0.1ml/L + 6-BA 1ml/L + banana puree 50g/L + potato 50g/L + white sugar 30g/L + agar 7g/L, autoclaving at 120 deg.C and 0.1Mpa for 25min, cooling to 55 deg.C, adding 20ml culture medium with mass concentration of 10 under aseptic condition-2 % of the nano copper solution, the mass concentration is 10-2% of the nano copper solution is added into the culture medium and stirred and mixed evenly to obtain a mixed culture medium II;
9) differentiating and culturing the young embryos, selecting the differentiated young embryos which are not polluted in the step 7), soaking the young embryos in sodium hypochlorite with the mass concentration of 10% for 8min, and washing the young embryos for 3 times by using sterile water; soaking for 1min by using the disinfectant in the step 3), taking out, putting on an aseptic operation table, sucking the solution by using aseptic absorbent paper, inoculating the solution into the mixed culture medium II in the step 8), and culturing for 6-8 days in an aseptic environment of 1000lux at 24 ℃ to obtain a pollution-free bottle seedling;
10) bottle seedling culture and transplantation, namely culturing the pollution-free bottle seedlings obtained in the step 9) according to a conventional method, and after differentiation is completed, rooting, seedling hardening, transplanting and planting according to the conventional method.
The efficiency of blocking bacterial contamination of example 3 was 93%.
Example 4
A method for blocking dendrobium officinale tissue culture seedlings from being polluted by bacteria is characterized by comprising the following steps:
1) preparing nano-copper mother liquor, namely putting nano-copper into sterilized distilled water, carrying out ice bath for 35 minutes at the temperature of 55 ℃, and then putting the nano-copper into an ultrasonic cell disruption instrument for treatment for 35 minutes under the ice bath condition to obtain the nano-copper mother liquor with the mass concentration of 0.1%;
2) diluting the nano-copper mother liquor, adding sterilized distilled water into the nano-copper mother liquor obtained in the step 1), and diluting to obtain a solution with the mass concentration of 10-2 % of the nano copper solution is sent into an oven, heated to 68 ℃ and then kept warm, and the activity of the nano copper is maintained;
3) preparing a disinfectant, namely adding Youjie 1003 disinfectant into sterile distilled water until the mass concentration is 0.2%, and performing high-pressure sterilization for 26min at 122 ℃ and 0.1Mpa to obtain the disinfectant;
4) mixed medium i was prepared by mixing the following media: MS + NAA 0.05ml/L + 6-BA 0.1ml/L + banana puree 50g/L + potato 50g/L + white sugar 30g/L + agar 7g/L, autoclaving at 122 deg.C and 0.1Mpa for 26min, cooling to 56 deg.C, and adding 20ml culture medium with mass concentration of 10 under aseptic condition-2 % of the nano copper solution, the mass concentration is 10-2% of the nano copper solution is added into the culture medium and stirred and mixed evenly to obtain a mixed culture medium I;
5) sterilizing Dendrobium officinale seeds, namely breaking the Dendrobium officinale fruit pods, taking out the seeds, putting the seeds into a gauze bag subjected to high-pressure sterilization treatment, and soaking the seeds in alcohol with the mass concentration of 70% for 50 seconds; then washing with sterile water for 3 times, sterilizing with 0.1% mercuric chloride for 7min, washing with sterile water for 3 times, soaking with 10% sodium hypochlorite for 7min, and washing with sterile water for 3 times; soaking for 2min by using the disinfectant in the step 3), taking out, putting on a sterile operating platform, and drying by using sterile absorbent paper to obtain sterilized seeds;
6) performing dendrobium officinale seed culture, uniformly placing the sterilized seeds in the step 5) in the mixed culture medium I in the step 4), performing dark culture until the seeds germinate, transferring the seeds to 1000lux of weak light, and culturing for 27 days to obtain young embryos;
7) performing differentiation culture on dendrobium officinale seeds, transferring the immature embryos obtained in the step 6) to a sterile environment with 24 ℃ and 1000lux, culturing for 6-8 days, transferring to a light condition, and continuously culturing until the immature embryos of the dendrobium officinale are differentiated;
8) mixed Medium II was prepared by mixing the following media: MS + NAA 0.1ml/L + 6-BA 1ml/L + banana puree 50g/L + potato 50g/L + white sugar 30g/L + agar 7g/L, autoclaving at 122 deg.C and 0.1Mpa for 26min, cooling to 56 deg.C, adding 20ml culture medium with mass concentration of 10 under aseptic condition-2 % of the nano copper solution, the mass concentration is 10-2% of the nano copper solution is added into the culture medium and stirred and mixed evenly to obtain a mixed culture medium II;
9) differentiating and culturing the young embryos, selecting the differentiated young embryos which are not polluted in the step 7), soaking the young embryos in sodium hypochlorite with the mass concentration of 10% for 7min, and washing the young embryos for 3 times by using sterile water; soaking for 2min by using the disinfectant in the step 3), taking out, putting on an aseptic operation table, sucking the solution by using aseptic absorbent paper, inoculating the solution into the mixed culture medium II in the step 8), and culturing for 7 days in an aseptic environment of 1000lux at the temperature of 25 ℃ to obtain a pollution-free bottle seedling;
10) bottle seedling culture and transplantation, namely culturing the pollution-free bottle seedlings obtained in the step 9) according to a conventional method, and after differentiation is completed, rooting, seedling hardening, transplanting and planting according to the conventional method.
The efficiency of blocking bacterial contamination of example 4 was 90%.
Claims (1)
1. A method for blocking dendrobium officinale tissue culture seedlings from being polluted by bacteria is characterized by comprising the following steps:
1) preparing nano-copper mother liquor, namely putting nano-copper into sterilized distilled water, carrying out ice bath for 20-40 minutes at the temperature of 40-60 ℃, and then putting the nano-copper into an ultrasonic cell disruption instrument for treatment for 20-40 minutes under the ice bath condition to obtain the nano-copper mother liquor with the mass concentration of 0.1%;
2) diluting the nano-copper mother liquor, adding sterilized distilled water into the nano-copper mother liquor obtained in the step 1), and diluting to obtain a solution with the mass concentration of 10-2 % ofFeeding the copper solution into an oven, heating to 60-70 ℃, and then preserving heat to maintain the activity of the nano copper;
3) preparing a disinfectant, namely adding the Youjie 1003 disinfectant into sterile distilled water until the mass concentration is 0.2%, and performing high-pressure sterilization for 20-30min at the temperature of 110-;
4) mixed medium i was prepared by mixing the following media: MS + NAA 0.05ml/L + 6-BA 0.1ml/L + banana puree 50g/L + potato 50g/L + white sugar 30g/L + agar 7g/L, sterilizing at 110--2 % of the nano copper solution, the mass concentration is 10-2% of the nano copper solution is added into the culture medium and stirred and mixed evenly to obtain a mixed culture medium I;
5) sterilizing Dendrobium officinale seeds, namely breaking the Dendrobium officinale fruit pods, taking out the seeds, putting the seeds into a gauze bag subjected to high-pressure sterilization treatment, and soaking the seeds in alcohol with the mass concentration of 70% for 40-60 seconds; then sequentially washing with sterile water for 2-4 times, sterilizing with 0.1% mercuric chloride for 6-10min, washing with sterile water for 2-4 times, soaking with 10% sodium hypochlorite for 6-10min, and washing with sterile water for 2-4 times; soaking for 1-2min by using the disinfectant in the step 3), taking out, putting on a sterile operating platform, and drying by using sterile absorbent paper to obtain sterilized seeds;
6) performing dendrobium officinale seed culture, uniformly placing the sterilized seeds in the step 5) in the mixed culture medium I in the step 4), performing dark culture until the seeds germinate, transferring the seeds to 1000lux of weak light, and culturing for 25-30 days to obtain young embryos;
7) performing differentiation culture on dendrobium officinale seeds, transferring the immature embryos obtained in the step 6) to a sterile environment with the temperature of 22-26 ℃ and the volume of 1000lux, culturing for 6-8 days, transferring to a light condition, and continuously culturing until the immature embryos of the dendrobium officinale are differentiated;
8) mixed Medium II was prepared by mixing the following media: MS + NAA 0.1ml/L + 6-BA 1ml/L + banana puree 50g/L + potato 50g/L + white sugar 30g/L + agar 7g/L, autoclaving at 110-The mass concentration of ml is 10-2 % of the nano copper solution, the mass concentration is 10-2% of the nano copper solution is added into the culture medium and stirred and mixed evenly to obtain a mixed culture medium II;
9) differentiating and culturing the young embryos, selecting the differentiated young embryos which are not polluted in the step 7), soaking the young embryos in sodium hypochlorite with the mass concentration of 10% for 6-10min, and washing the young embryos for 2-4 times by using sterile water; soaking for 1-2min by using the disinfectant in the step 3), taking out, putting on a sterile operating platform, sucking dry the solution by using sterile absorbent paper, inoculating the solution into the mixed culture medium II in the step 8), and culturing for 6-8 days at 23-26 ℃ in a sterile environment of 1000lux to obtain a pollution-free bottle seedling;
10) bottle seedling culture and transplantation, namely culturing and differentiating the pollution-free bottle seedling obtained in the step 9) according to a conventional method, and then rooting, hardening off, transplanting and planting according to the conventional method to achieve the purpose of blocking bacterial pollution.
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