CN116349602B - Sweet potato detoxification tissue culture seedling carbon dioxide sugar-free domestication method - Google Patents
Sweet potato detoxification tissue culture seedling carbon dioxide sugar-free domestication method Download PDFInfo
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- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 title claims abstract description 260
- 239000001569 carbon dioxide Substances 0.000 title claims abstract description 130
- 229910002092 carbon dioxide Inorganic materials 0.000 title claims abstract description 130
- 244000017020 Ipomoea batatas Species 0.000 title claims abstract description 86
- 235000002678 Ipomoea batatas Nutrition 0.000 title claims abstract description 86
- 238000000034 method Methods 0.000 title claims abstract description 37
- 238000001784 detoxification Methods 0.000 title claims abstract description 10
- 238000012258 culturing Methods 0.000 claims abstract description 26
- 239000001963 growth medium Substances 0.000 claims abstract description 21
- 239000010451 perlite Substances 0.000 claims abstract description 15
- 235000019362 perlite Nutrition 0.000 claims abstract description 15
- 239000010455 vermiculite Substances 0.000 claims abstract description 15
- 235000019354 vermiculite Nutrition 0.000 claims abstract description 15
- 229910052902 vermiculite Inorganic materials 0.000 claims abstract description 15
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 14
- 239000010931 gold Substances 0.000 claims abstract description 14
- 229910052737 gold Inorganic materials 0.000 claims abstract description 14
- ZCCIPPOKBCJFDN-UHFFFAOYSA-N calcium nitrate Chemical compound [Ca+2].[O-][N+]([O-])=O.[O-][N+]([O-])=O ZCCIPPOKBCJFDN-UHFFFAOYSA-N 0.000 claims abstract description 12
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000004327 boric acid Substances 0.000 claims abstract description 7
- 239000012154 double-distilled water Substances 0.000 claims abstract description 6
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims abstract description 6
- 235000019796 monopotassium phosphate Nutrition 0.000 claims abstract description 6
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims abstract description 5
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims abstract description 5
- 238000005286 illumination Methods 0.000 claims description 25
- 241000196324 Embryophyta Species 0.000 claims description 21
- 230000003203 everyday effect Effects 0.000 claims description 12
- 230000001360 synchronised effect Effects 0.000 claims description 11
- 239000002689 soil Substances 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- BVTBRVFYZUCAKH-UHFFFAOYSA-L disodium selenite Chemical compound [Na+].[Na+].[O-][Se]([O-])=O BVTBRVFYZUCAKH-UHFFFAOYSA-L 0.000 claims description 4
- 235000003891 ferrous sulphate Nutrition 0.000 claims description 4
- 239000011790 ferrous sulphate Substances 0.000 claims description 4
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 claims description 4
- 229910000359 iron(II) sulfate Inorganic materials 0.000 claims description 4
- 229960001471 sodium selenite Drugs 0.000 claims description 4
- 235000015921 sodium selenite Nutrition 0.000 claims description 4
- 239000011781 sodium selenite Substances 0.000 claims description 4
- 150000003839 salts Chemical class 0.000 abstract description 6
- 229940082569 selenite Drugs 0.000 abstract description 6
- MCAHWIHFGHIESP-UHFFFAOYSA-L selenite(2-) Chemical compound [O-][Se]([O-])=O MCAHWIHFGHIESP-UHFFFAOYSA-L 0.000 abstract description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 abstract description 4
- 229910052799 carbon Inorganic materials 0.000 abstract description 4
- 230000001580 bacterial effect Effects 0.000 abstract description 2
- 230000000052 comparative effect Effects 0.000 description 7
- 239000011324 bead Substances 0.000 description 6
- 206010020649 Hyperkeratosis Diseases 0.000 description 4
- 239000012153 distilled water Substances 0.000 description 4
- 239000002245 particle Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 208000035143 Bacterial infection Diseases 0.000 description 3
- 208000022362 bacterial infectious disease Diseases 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 230000029553 photosynthesis Effects 0.000 description 3
- 238000010672 photosynthesis Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000700605 Viruses Species 0.000 description 2
- 230000001651 autotrophic effect Effects 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 238000011010 flushing procedure Methods 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 208000012868 Overgrowth Diseases 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 230000036579 abiotic stress Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229930002875 chlorophyll Natural products 0.000 description 1
- 235000019804 chlorophyll Nutrition 0.000 description 1
- ATNHDLDRLWWWCB-AENOIHSZSA-M chlorophyll a Chemical compound C1([C@@H](C(=O)OC)C(=O)C2=C3C)=C2N2C3=CC(C(CC)=C3C)=[N+]4C3=CC3=C(C=C)C(C)=C5N3[Mg-2]42[N+]2=C1[C@@H](CCC(=O)OC\C=C(/C)CCC[C@H](C)CCC[C@H](C)CCCC(C)C)[C@H](C)C2=C5 ATNHDLDRLWWWCB-AENOIHSZSA-M 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 159000000014 iron salts Chemical class 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 238000004161 plant tissue culture Methods 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000010008 shearing 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
- A01H4/008—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
- A01H4/002—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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention relates to a sweet potato detoxification tissue culture seedling carbon dioxide sugar-free domestication method, which comprises the steps of cutting off roots of 40-50 days full bottle seedlings, inoculating the roots stained with sugar-containing culture medium into a carbon dioxide sugar-free culture box, introducing carbon dioxide into the culture box, performing closed culture for 5 days, starting half-open cover culture on the 6 th day, starting full-open cover culture on the 8 th day, the 9 th day or the 10 th day, culturing until the 14 th-16 th day, cutting off redundant fibrous roots, and transplanting the seedlings into a greenhouse small arch shed. The carbon dioxide sugar-free culture box contains gold vermiculite, perlite and culture solution, wherein the culture solution contains 1.15g/L of calcium nitrate, 0.2g/L of monopotassium phosphate, 2.86mg/L of boric acid, 40mg/L of ferric salt, 20mg/L of selenite and the balance of double distilled water. The invention adopts carbon dioxide as a carbon source, the culture medium and the culture solution do not contain organic matters, the detoxified tissue culture seedling does not have the risk of bacterial and mould pollution, and simultaneously the invention effectively shortens the sugar-free culture time of the detoxified tissue culture seedling of the sweet potato, and improves the seedling supply efficiency and the live seedling rate.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a carbon dioxide sugar-free domestication method of sweet potato detoxified tissue culture seedlings.
Background
The sweet potato has the characteristics of strong drought resistance, barren resistance, wide adaptability and the like, is widely distributed in China, and is planted in almost all provinces and cities. In the sweet potato planting process, virus diseases can cause serious influence on the yield and quality of sweet potatoes, no particularly effective chemical prevention and treatment agent exists at present, and the planting of detoxified healthy seedlings is the most effective method for preventing and treating the virus diseases and improving the yield of sweet potatoes.
The carbon source required by the detoxification tissue culture seedling of the sweet potato in the tissue culture bottle for photosynthesis mainly comes from sugar in the culture medium, the requirement on illumination is low, the photosynthesis of the detoxification tissue culture seedling is poor, and the detoxification tissue culture seedling belongs to heterotrophic life. The traditional sweet potato detoxification tissue culture seedling often adopts a transplanting method that a small arch shed is directly buried, so that the transplanted tissue culture Miao Lima is changed from heterotrophic to autotrophic and alive, so that some tissue culture seedlings with poor growth quality after transplanting can not adapt to dead seedlings, the survival rate is only 60-70%, the subsequent seedling supply number is seriously influenced, and the development of the sweet potato industry is restricted.
Plant sugarless culture techniques, also known as autotrophic micropropagation techniques, typically replace sugar in the medium with carbon dioxide as a carbon source. The existing carbon dioxide sugar-free domestication method for sweet potato virus-free tissue culture seedlings is less, and the existing technology for sugar-free culture of sweet potato virus-free tissue culture seedlings has long culture time for the tissue culture seedlings, usually more than 25 days and sometimes more than 30 days. In the existing method, the culture time of the sweet potato virus-free tissue culture seedlings is longer, the efficiency is lower, and the number of seedlings and the development of the sweet potato industry are greatly influenced.
Disclosure of Invention
The invention aims to provide a carbon dioxide sugar-free domestication method for sweet potato virus-free tissue culture seedlings, which can shorten the sugar-free culture time of the sweet potato virus-free tissue culture seedlings and improve the seedling supply efficiency and the live seedling rate.
The invention is realized by the following technical scheme, and the carbon dioxide sugar-free domestication method for the sweet potato detoxified tissue culture seedlings provided by the invention comprises the following steps:
(1) Culturing sweet potato virus-free tissue culture seedlings in a culture bottle;
(2) Selecting 40-50 days full bottle seedlings, cutting off roots stained with sugar-containing culture medium, inoculating the detoxified sweet potato tissue culture seedlings into a carbon dioxide sugarless culture box according to the amount of 26-30 seedlings per box, covering a culture box cover, and connecting a carbon dioxide supply pipe to supply carbon dioxide into the carbon dioxide sugarless culture box;
wherein, each box of the carbon dioxide sugar-free culture box is filled with gold vermiculite and perlite, and a certain amount of culture solution is added for uniform mixing; the culture solution contains 1.15g/L of calcium nitrate, 0.2g/L of monopotassium phosphate, 2.86mg/L of boric acid, 40mg/L of ferric salt, 20mg/L of selenite and the balance of double distilled water;
(3) The sweet potato detoxified tissue culture seedling is cultivated in a carbon dioxide sugarless cultivation box for 5 days in a closed way, two 18W red, blue and white light sources are selected as light sources during cultivation, the carbon dioxide sugarless cultivation box is 25cm away from the light sources, and the illumination intensity is 80 mu mol.m -2 ·s -1 Illuminating for 13 hours every day, wherein the carbon dioxide supply amount is 900-1100 ppM, the carbon dioxide supply time is synchronous with the illumination time, the culture temperature is 26-28 ℃, and the humidity in the culture box is 80-85%;
(4) Starting on the 6 th day of culture, the carbon dioxide sugar-free culture box is half-opened, four 18W red, blue and white light sources are selected during the culture period, the culture box is 25cm away from the light sources, and the illumination intensity is 160 mu mol.m -2 ·s -1 The light is irradiated for 13 hours every day, the carbon dioxide supply amount is 1000-1200 ppM, and the carbon dioxide is suppliedSynchronizing with illumination time, wherein the culture temperature is 26-28 ℃, and the humidity in the culture box is 60-70%; during which 100mL of culture medium was replenished every 2 days;
(5) The carbon dioxide sugar-free culture box is fully opened at the beginning of the culture at the 8 th day, the 9 th day or the 10 th day; the culture conditions are as in step (4), namely: four 18W red, blue and white light sources are selected during the culture period, the distance between a culture box and the light source is 25cm, and the illumination intensity is 160 mu mol.m -2 ·s -1 Illuminating for 13 hours every day, wherein the carbon dioxide supply amount is 1000-1200 ppM, the carbon dioxide supply time is synchronous with the illumination time, the culture temperature is 26-28 ℃, and the humidity in the culture box is 60-70%; during which 100mL of culture medium was replenished every 2 days;
(6) Culturing the sweet potato seedlings in a fully-opened cover until the sweet potato seedlings grow to 12-15 cm in 14-16 days, cutting off redundant fibrous roots, and transplanting the sweet potato seedlings into a small greenhouse arch shed.
Further, the size of the carbon dioxide sugarless culture box is 22cm multiplied by 26cm multiplied by 16cm.
Preferably, the particle size of the gold vermiculite is 2-3 mm, and the particle size of the perlite is 1-3 mm.
Preferably, the ratio of the volume occupied by gold vermiculite to the volume occupied by perlite in the carbon dioxide sugarless culture box is 5.4:1.
Preferably, the ratio of the total volume occupied by gold vermiculite and perlite in the carbon dioxide sugar-free culture box to the added volume of the culture solution is 16:9.
Preferably, the ferric salt is ferrous sulfate, and the selenite is sodium selenite.
Preferably, the ratio of the number of red light beads to the number of blue light beads to the number of white light beads in the light source is 3:2:1.
Further, after transplanting the sweet potato seedlings in the step (6) into a small greenhouse, watering the soil thoroughly, ensuring that the air humidity in the small greenhouse is 75-85%, the temperature is 25-30 ℃, covering the small greenhouse with a shading net for the first two days, covering the shading net with a 10:00-16:00 for the 3 rd to 5 th days, removing the shading net after 5 days, removing the small greenhouse after 7-10 days, and enabling the sweet potato seedlings to grow normally.
The sweet potato detoxification tissue culture seedling carbon dioxide sugar-free domestication method can replace the step (6) with the following step (7):
(7) Culturing the sweet potato seedlings until the sweet potato seedlings grow to 12-15 cm in 14-16 days by fully opening the cover, cutting the sweet potato seedlings, and keeping 3-5 cm at the bottom as a mother plant for continuous culture in an original carbon dioxide sugar-free culture box;
transplanting the cut detoxified seedlings into a new carbon dioxide sugarless culture box for culture, and repeating the steps (3) to (6);
culturing the parent strain which is kept in the original carbon dioxide sugarless culture box and is continuously cultured until the sweet potato seedling grows to 12-15 cm according to the condition of the step (5), cutting the sweet potato seedling, and still keeping 3-5 cm at the bottom to serve as the parent strain and continuously culturing in the original carbon dioxide sugarless culture box; transplanting the cut detoxified seedlings into a new carbon dioxide sugarless culture box for culture, and repeating the steps (3) to (6);
the cycle is repeated according to the method.
Further, the parent plant which is kept in the original carbon dioxide sugarless culture box and is continuously cultured until the sweet potato seedling grows to 12-15 cm according to the condition of the step (5), then the parent plant is transplanted into a greenhouse small arch shed after redundant fibrous roots are cut off, then the parent plant is watered through soil slowly, the air humidity in the small arch shed is guaranteed to be 75-85%, the temperature is 25-30 ℃, the two days before the small arch shed is covered with a shading net in the whole day, the shading net is covered by 10:00-16:00 in the 3 th to 5 th days, the shading net is removed after 5 days, the small arch shed is removed after 7-10 days, and the sweet potato seedling grows normally.
Compared with the prior art, the invention has obvious advantages and beneficial effects. By means of the technical scheme, the invention can achieve quite technical progress and practicability, has wide utilization value, and has at least the following advantages:
(1) Compared with the prior art, the invention selects the mixture of 2-3 mm gold vermiculite and 1-3 mm perlite as the matrix, can effectively retain water while keeping the air permeability of the matrix, and the transplanted tissue culture seedlings can root in 3 days, and the survival rate is more than 99%. Carbon dioxide is used as a carbon source, and the matrix and the culture solution do not contain organic matters, so that the risk of bacterial and mould pollution is avoided.
(2) The invention fully considers the high potassium requirement of sweet potato growth when screening the culture solution, and is more beneficial to domestication culture of sweet potato detoxified seedlings. Meanwhile, selenite is added into the culture solution, so that the growth of sweet potato virus-free seedlings can be promoted, the abiotic stress resistance is enhanced, the photosynthesis is improved, and the sweet potato virus-free seedlings can be better helped to adapt to the change of the environment in the domestication process.
(3) Boric acid and ferric salt are added into the culture solution, so that the growth and division of cells can be promoted by the boric acid, the synthesis of hemicellulose and related cell wall substances is participated, the metabolism and lignification of enzymes are regulated, and the growth of shoot apex and root apex is vital. Iron salts are involved in plant respiration and are essential for chlorophyll synthesis.
(4) In the invention, the sweet potato virus-free tissue culture seedling can be transplanted into a shed after 14-16 days of domestication, so that the seedling period of the sweet potato virus-free tissue culture seedling is greatly shortened, and the propagation speed of the sweet potato virus-free tissue culture seedling is accelerated. When the light is irradiated, the red-blue-white light source is selected, the number ratio of the light beads of the red light source, the blue light source and the white light source is 3:2:1, and compared with a pure full white light source, the invention strengthens the irradiation of 660-665 nm red light and 460-470 nm blue light, not only can prevent overgrowth, but also can strengthen seedlings, and greatly increases the survival rate of transplanted seedlings.
Detailed Description
For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the present invention will be clearly and completely described below in conjunction with specific embodiments, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to fall within the scope of the invention.
The carbon dioxide sugar-free domestication method of the sweet potato detoxified tissue culture seedling mainly comprises the following steps:
(1) The sweet potato detoxification tissue culture seedling culture is carried out in a culture bottle, and the method comprises the following steps:
selecting a bud segment with the top end of about 2-3 cm, firstly flushing with distilled water for 2-3 times, then immersing in 75% alcohol for sterilization for 30s in an ultra-clean workbench, and then flushing with distilled water for 2-3 times. The water was blotted dry using sterilized filter paper for use.
In an ultra-clean workbench, under a continuous zoom stereo microscope, a dissecting needle is used for stripping stem tip growing points with the length of 0.2-0.3 mm, and 1-2 leaf primordia are arranged. The excised shoot tip growth points were cultured in a first medium. The first culture medium is MS culture medium +6-BA 0.5mg/L +IAA 0.1mg/L +GA 3 0.1mg/L. The culture temperature is 26-28 ℃, and the illumination intensity is 80 mu mol.m -2 ·s -1 Growing points turn green in 3-5 days, the basal part starts to form callus, and buds are generated in 18 days.
Cutting off the callus, transferring the buds into a second culture medium for rooting, wherein the second culture medium is MS culture medium+IBA 0.5mg/L+GA 3 1mg/L, the culture temperature is 26-28 ℃, and the illumination intensity is 80 mu mol.m -2 ·s -1 The light was irradiated for 13 hours/day. After 18-21 days of culture, transferring one leaf into MS without hormone, and continuously culturing at 26-28 deg.C under illumination intensity of 80 mu mol.m -2 ·s -1 And (5) illuminating for 13 hours/day, and forming seedlings after about 45 days.
(2) Selecting 40-50 days full bottle seedlings, cutting off roots stained with sugar-containing culture medium, inoculating the sweet potato detoxified seedlings into a carbon dioxide sugarless culture box according to the amount of 26-30 seedlings per box, covering a culture box cover, and connecting a carbon dioxide supply pipe to supply carbon dioxide into the carbon dioxide sugarless culture box.
Wherein, in one embodiment, the carbon dioxide sugarless culture box is about 22cm by 26cm by 16cm in size.
Preferably, based on the above embodiment, 1350mL of gold vermiculite (2-3 mm) and 250mL of perlite (1-3 mm) are contained in each carbon dioxide sugarless culture box, and 900mL of culture solution is added to mix uniformly, but this description should not be considered as limiting the invention, in other embodiments, the ratio of the volume occupied by gold vermiculite to the volume occupied by perlite in the carbon dioxide sugarless culture box is 5.4:1, and the ratio of the total volume occupied by gold vermiculite and perlite in the carbon dioxide sugarless culture box to the added volume of the culture solution is 16:9.
The culture solution contains 1.15g/L of calcium nitrate, 0.2g/L of monopotassium phosphate, 2.86mg/L of boric acid, 40mg/L of ferric salt, 20mg/L of selenite and the balance of double distilled water.
Preferably, the ferric salt is ferrous sulfate, and the selenite is sodium selenite.
(3) The sweet potato detoxified tissue culture seedling is cultivated in a carbon dioxide sugarless cultivation box for 5 days in a closed way, two 18W red, blue and white light sources are selected as light sources during cultivation, the carbon dioxide sugarless cultivation box is 25cm away from the light sources, and the illumination intensity is 80 mu mol.m -2 ·s -1 The light is irradiated for 13 hours every day, the carbon dioxide supply amount is 900-1100 ppM, the carbon dioxide supply time is synchronous with the light irradiation time, the culture temperature is 26-28 ℃, and the humidity in the culture box is 80-85%.
(4) Starting on the 6 th day of culture, the carbon dioxide sugar-free culture box is half-opened, four 18W red, blue and white light sources are selected during the culture period, the culture box is 25cm away from the light sources, and the illumination intensity is 160 mu mol.m -2 ·s -1 The light is irradiated for 13 hours every day, the carbon dioxide supply amount is 1000-1200 ppM, the carbon dioxide supply time is synchronous with the light irradiation time, the culture temperature is 26-28 ℃, and the humidity in the culture box is 60-70%. During which 100mL of culture broth was replenished every 2 days.
(5) The carbon dioxide sugarless culture box is fully opened at the beginning of the culture at the 8 th day, the 9 th day or the 10 th day. The culture conditions are as in step (4), namely: four 18W red, blue and white light sources are selected during the culture period, the distance between a culture box and the light source is 25cm, and the illumination intensity is 160 mu mol.m -2 ·s -1 The light is irradiated for 13 hours every day, the carbon dioxide supply amount is 1000-1200 ppM, the carbon dioxide supply time is synchronous with the light irradiation time, the culture temperature is 26-28 ℃, and the humidity in the culture box is 60-70%. During which 100mL of culture broth was replenished every 2 days.
(6) Culturing the sweet potato seedlings in a fully opened cover until the sweet potato seedlings grow to 12-15 cm, cutting off redundant fibrous roots, and transplanting the sweet potato seedlings into a small greenhouse. Slowly watering the soil after transplanting, ensuring that the air humidity in the small arch shed is 75-85%, the temperature is 25-30 ℃, covering the small arch shed with a shading net for the first two days, covering the shading net for the first two days, and removing the shading net after 5 days, wherein the temperature is 7-10 days, the sweet potato seedlings grow normally, and the live seedling rate is more than 99%.
By adopting the technical scheme, the sweet potato detoxified tissue culture seedlings can be transplanted into a greenhouse small arch shed after being cultured in the carbon dioxide sugar-free culture box for 14-16 days, so that the domestication time is greatly shortened, and the seedling supply efficiency and the live seedling rate are improved.
The sugar-free acclimation method of the present invention may have the following further scheme. Replacing the step (6) with the following step (7):
(7) Culturing in a full-open cover until the seedling grows to 12-15 cm, shearing the seedling, and reserving 3-5 cm at the bottom as a mother plant for continuous culture in an original carbon dioxide sugar-free culture box.
Transplanting the cut detoxified tissue culture seedlings into a new carbon dioxide sugar-free culture box for culture, repeating the steps (3) to (6), and finally transplanting the detoxified tissue culture seedlings of the sweet potatoes subjected to sugar-free domestication into a greenhouse and growing normally.
Culturing the parent strain which is kept in the original carbon dioxide sugarless culture box and is continuously cultured until the sweet potato seedling grows to 12-15 cm according to the condition of the step (5), cutting the sweet potato seedling, and still keeping 3-5 cm at the bottom to serve as the parent strain and continuously culturing in the original carbon dioxide sugarless culture box; transplanting the cut detoxified seedlings into a new carbon dioxide sugarless culture box for culture, repeating the steps (3) to (6), and finally transplanting the detoxified sweet potato tissue culture seedlings subjected to sugarless domestication into a greenhouse and growing normally. The cycle is repeated according to the method.
Or, the parent plant which is kept in the original carbon dioxide sugarless culture box and is continuously cultured until the sweet potato seedling grows to 12-15 cm according to the condition of the step (5), then redundant fibrous roots are cut off, the sweet potato seedling is directly transplanted into a greenhouse small arch shed, then, the soil is watered slowly, the air humidity in the small arch shed is ensured to be 75-85%, the temperature is 25-30 ℃, the two days before the small arch shed is covered by a shading net for all days, the shading net is covered by 10:00-16:00 for 3-5 days, the shading net is removed after 5 days, the small arch shed is removed after 7-10 days, and the sweet potato seedling grows normally.
By adopting the scheme, a batch of mother plants can be cultivated for multiple times, and the mother plants are directly transplanted into a greenhouse small arch shed after being cultivated until the transplanting conditions are met, or the seedlings are sheared, the sheared seedlings are transplanted into a new carbon dioxide sugar-free cultivation box for cultivation until the transplanting conditions are met, and then redundant fibrous roots are sheared and transplanted into the greenhouse small arch shed; and the bottom after seedling cutting is used as a mother plant to be continuously cultivated in an original carbon dioxide sugarless cultivation box, and the steps are repeated. The scheme can further enlarge the seedling feeding quantity and improve the seedling feeding efficiency.
The red, blue and white light source can be implemented by adopting lamp tubes containing red, blue and white light beads, and the number ratio of the light beads of the red, blue and white light sources is 3:2:1.
The invention is illustrated in more detail by the following examples:
example 1
(1) The sweet potato virus-free seedling culture is carried out in a culture bottle, and the specific method comprises the following steps:
selecting a bud segment with the top of about 2-3 cm of the sweet potato seedling, washing with distilled water for 2-3 times, immersing in 75% alcohol in an ultra-clean workbench for sterilization for 30s, and washing with distilled water for 2-3 times. The water was blotted dry using sterilized filter paper for use.
In an ultra-clean workbench, under a continuous zoom stereo microscope, a dissecting needle is used for stripping stem tip growing points with the length of 0.2-0.3 mm, and 1-2 leaf primordia are arranged. The excised shoot tip growth points were cultured in a first medium. The first culture medium is MS culture medium +6-BA 0.5mg/L +IAA 0.1mg/L +GA 3 0.1mg/L. The culture temperature is 26-28 ℃, and the illumination intensity is 80 mu mol.m -2 ·s -1 Growing points turn green in 3-5 days, the basal part starts to form callus, and buds are generated in 18 days.
Cutting off the callus, transferring the buds into a second culture medium for rooting, wherein the second culture medium is MS culture medium+IBA 0.5mg/L+GA 3 1mg/L, the culture temperature is 26-28 ℃, and the illumination intensity is 80 mu mol.m -2 ·s -1 The light was irradiated for 13 hours/day. After 18-21 days of culture, transferring one leaf into MS without hormone, and continuously culturing at 26-28 deg.C under illumination intensity of 80 mu mol.m -2 ·s -1 And (5) illuminating for 13 hours/day, and forming seedlings after about 45 days.
(2) Selecting 45-day full bottle seedlings, cutting off roots stained with sugar-containing culture medium, inoculating the sweet potato detoxified seedlings into a carbon dioxide sugarless culture box according to the amount of 26-30 seedlings per box, covering a culture box cover, and connecting a carbon dioxide supply pipe.
Wherein the carbon dioxide sugarless culture box has the size of about 22cm×26cm×16cm. Each box of the carbon dioxide sugar-free culture box is filled with 1350mL of gold vermiculite (2-3 mm) and 250mL of perlite (1-3 mm), and 900mL of culture solution is added for uniform mixing. The culture solution contains 1.15g/L of calcium nitrate, 0.2g/L of monopotassium phosphate, 2.86mg/L of boric acid, 40mg/L of ferrous sulfate, 20mg/L of sodium selenite and the balance of double distilled water.
(3) The sweet potato detoxified tissue culture seedling is cultivated in a carbon dioxide sugarless cultivation box for 5 days in a closed way, two 18W red, blue and white light sources are selected as light sources during cultivation, the carbon dioxide sugarless cultivation box is 25cm away from the light sources, and the illumination intensity is 80 mu mol.m -2 ·s -1 The light is irradiated for 13 hours every day, the carbon dioxide supply amount is 1000ppM, the carbon dioxide supply time is synchronous with the light irradiation time, the culture temperature is 26-28 ℃, and the humidity in the culture box is 80-85%.
(4) Starting the culture until the 6 th day, half-opening a carbon dioxide sugar-free culture box, wherein four 18W red, blue and white light sources are selected during the culture, the culture box is 25cm away from the light sources, and the illumination intensity is 160 mu mol m -2 ·s -1 The light is irradiated for 13 hours every day, the carbon dioxide supply amount is 1000ppM, the carbon dioxide supply time is synchronous with the light irradiation time, the culture temperature is 26-28 ℃, and the humidity in the culture room is 60-70%. During which 100mL of culture broth was replenished every 2 days.
(5) The carbon dioxide sugarless culture box is fully opened from the beginning of the culture until the 8 th day. The culture conditions are as in step (4), namely: four 18W red, blue and white light sources are selected during the culture period, the distance between a culture box and the light source is 25cm, and the illumination intensity is 160 mu mol.m -2 ·s -1 The light is irradiated for 13 hours every day, the carbon dioxide supply amount is 1000ppM, the carbon dioxide supply time is synchronous with the light irradiation time, the culture temperature is 26-28 ℃, and the humidity in the culture room is 60-70%. During which 100mL of culture broth was replenished every 2 days.
(6) Culturing in a fully opened cover until the seedling grows to 12-15 cm on day 14, cutting off redundant fibrous roots, and transplanting into a greenhouse small arch shed. Slowly watering the soil after transplanting, ensuring that the air humidity in the small arch shed is 75-85%, the temperature is 25-30 ℃, covering the small arch shed with a shading net all the day in the first two days, covering the shading net at a ratio of 10:00-16:00 in 3-5 days, removing the shading net after 5 days, and removing the small arch shed for normal growth after 7 days.
Example 2
And (2) selecting full seedlings for 40 days.
The carbon dioxide supply in step (3) was 1100ppM.
And (3) starting the culture in the step (4) until the 6 th day, and half-opening the carbon dioxide sugarless culture box, wherein the carbon dioxide supply amount is 1200ppM.
And (3) starting the culture in the step (5) until the 9 th day, and fully opening the carbon dioxide sugarless culture box. The carbon dioxide supply amount was 1200ppM.
And (6) culturing the seedlings in a fully opened mode until the seedlings grow to 12-15 cm on the 15 th day, cutting off redundant fibrous roots, and transplanting the seedlings into a greenhouse small arch shed. Slowly watering the soil after transplanting, ensuring that the air humidity in the small arch shed is 75-85%, the temperature is 25-30 ℃, covering the small arch shed with a shading net all the day in the first two days, covering the shading net at a ratio of 10:00-16:00 in 3-5 days, removing the shading net after 5 days, and removing the small arch shed for normal growth after 10 days.
Other steps and culture conditions were the same as in example 1.
Example 3
And (2) selecting 50-day full seedlings.
The carbon dioxide supply in the step (3) was 900ppM.
Starting the culture in the step (4) until the 6 th day, half-opening the cover of the carbon dioxide sugarless culture box, and supplying the carbon dioxide with the quantity of 1100ppM.
And (5) starting the culture until the 10 th day, and fully opening the carbon dioxide sugarless culture box. The carbon dioxide supply was 1100ppM.
And (6) culturing the seedlings in a fully opened mode until the seedlings grow to 12-15 cm on the 16 th day, cutting off redundant fibrous roots, and transplanting the seedlings into a greenhouse small arch shed. Slowly watering the soil after transplanting, ensuring that the air humidity in the small arch shed is 75-85%, the temperature is 25-30 ℃, covering the small arch shed with a shading net all the day in the first two days, covering the shading net at a ratio of 10:00-16:00 in 3-5 days, removing the shading net after 5 days, and removing the small arch shed for normal growth after 9 days.
Other steps and culture conditions were the same as in example 1.
Example 4
Following the procedure of example 1, step (6) is replaced with: and (3) culturing the seedlings by fully opening the cover until the 14 th day, cutting the seedlings after the seedlings grow to 12-15 cm, and keeping 3-5 cm at the bottom as a mother plant for continuous culture in an original carbon dioxide sugarless culture box. Transplanting the cut detoxified seedlings into a new carbon dioxide sugarless culture box for culture, and repeating the steps (3) to (5) of the example 1 to start a new culture period. Culturing the seedlings until 15 days, growing the seedlings to 12-15 cm, cutting off redundant fibrous roots, transplanting the seedlings into a greenhouse small arch shed, watering the seedlings thoroughly by slow water after transplanting, ensuring that the air humidity in the small arch shed is 75-85%, the temperature is 25-30 ℃, covering the seedlings with a shading net for the first two days, covering the shading net for the 3-5 days by using a shading net for 10:00-16:00, removing the shading net after 5 days, and removing the small arch shed for normal growth after 7 days.
And the mother plant remained in the original carbon dioxide sugarless culture box for continuous culture is cultured until the seedling grows to 12-15 cm according to the condition of the step (5) of the embodiment 1, and the seedling is cut, and the mother plant still remains at the bottom of 3-5 cm as the mother plant for continuous culture in the original carbon dioxide sugarless culture box. Transplanting the cut detoxified seedlings into a new carbon dioxide sugarless culture box for culture, and repeating the steps (3) to (6) of the example 1. The remaining stock strain was continued to be cultured in a raw carbon dioxide sugarless culture box under the conditions of step (5) of example 1. The above process is repeated.
Example 5
The difference from example 4 is that: culturing parent plants which are kept in an original carbon dioxide sugar-free culture box until sweet potato seedlings grow to 12-15 cm according to the condition of the step (5) of the embodiment 1, cutting off redundant fibrous roots, transplanting the parent plants into a greenhouse small arch shed, watering the parent plants slowly after transplanting to thoroughly soak the soil, ensuring that the air humidity in the small arch shed is 75-85%, the temperature is 25-30 ℃, covering the parent plants with a shading net for the first two days, covering the shading net for the first two days, and removing the shading net after 5 days, and removing the small arch shed for normal growth after 7 days.
Comparative example 1
Referring to the method of example 1, the light sources in step (3) to step (5) were all replaced with full white light sources, and the other conditions were unchanged.
Comparative example 2
With reference to the method of example 1, the culture broth was replaced with a solution containing 1.51g/L calcium nitrate, 170mg/L potassium dihydrogen phosphate and 0.15g/L ammonium nitrate, the remainder being double distilled water, with the other conditions unchanged.
Comparative example 3
With reference to the method of example 1, 1600mL of gold vermiculite (vermiculite particle size 2-3 mm) is packed in each carbon dioxide sugarless culture box, and other conditions are unchanged.
Comparative example 4
With reference to the method of example 1, 1600mL of perlite (perlite particle size of 1 to 3 mm) was contained in each of the carbon dioxide sugarless culture cassettes, with the other conditions unchanged.
The live seedling rate and bacterial infection rate statistics of the above example 1 and comparative examples 1 to 4 are as follows:
TABLE 1 live seedling Rate and bacterial infection Rate statistics for example 1 and four comparative example culture methods
As can be seen from the comparison, compared with comparative examples 1-4, the sweet potato virus-free seedling obtained by the carbon dioxide sugarless domestication method has good growth vigor, high survival rate and low bacterial infection rate.
The carbon dioxide sugar-free domestication method has short domestication time for sweet potato virus-free tissue culture seedlings, meets the transplanting conditions after 14-16 days, can be directly transplanted into a greenhouse small arch shed, can remove the small arch shed for normal growth after 7-10 days after transplanting, and has high live seedling rate.
The foregoing is merely an embodiment of the present invention, and the present invention is not limited in any way, and may have other embodiments according to the above structures and functions, which are not listed. Therefore, any simple modification, equivalent variation and modification of the above embodiments according to the technical substance of the present invention without departing from the scope of the technical solution of the present invention will still fall within the scope of the technical solution of the present invention.
Claims (6)
1. The carbon dioxide sugar-free domestication method for the sweet potato detoxified tissue culture seedling is characterized by comprising the following steps of:
(1) Culturing sweet potato virus-free tissue culture seedlings in a culture bottle;
(2) Selecting 40-50 days full bottle seedlings, cutting off roots stained with sugar-containing culture medium, inoculating the sweet potato virus-free tissue culture seedlings subjected to root removal into a carbon dioxide sugarless culture box according to the amount of 26-30 seedlings per box, covering a culture box cover, and connecting a carbon dioxide supply pipe to supply carbon dioxide into the carbon dioxide sugarless culture box;
wherein, each box of the carbon dioxide sugar-free culture box is filled with gold vermiculite with the grain diameter of 2-3 mm and perlite with the grain diameter of 1-3 mm, and a certain amount of culture solution is added for uniform mixing; the culture solution contains 1.15g/L of calcium nitrate, 0.2g/L of monopotassium phosphate, 2.86mg/L of boric acid, 40mg/L of ferrous sulfate, 20mg/L of sodium selenite and the balance of double distilled water; the ratio of the volume occupied by gold vermiculite to the volume occupied by perlite in the carbon dioxide sugar-free culture box is 5.4:1, and the ratio of the total volume occupied by gold vermiculite to perlite to the added volume of culture solution is 16:9;
(3) The sweet potato detoxified tissue culture seedling is cultivated in a carbon dioxide sugarless cultivation box for 5 days in a closed way, two 18W red, blue and white light sources are selected as light sources during cultivation, the carbon dioxide sugarless cultivation box is 25cm away from the light sources, and the illumination intensity is 80 mu mol.m -2 ·s -1 Illuminating for 13 hours every day, wherein the carbon dioxide supply amount is 900-1100 ppM, the carbon dioxide supply time is synchronous with the illumination time, the culture temperature is 26-28 ℃, and the humidity in the culture box is 80-85%;
(4) Starting on the 6 th day of culture, the carbon dioxide sugar-free culture box is half-opened, four 18W red, blue and white light sources are selected during the culture period, the culture box is 25cm away from the light sources, and the illumination intensity is 160 mu mol.m -2 ·s -1 Illuminating for 13 hours every day, wherein the carbon dioxide supply amount is 1000-1200 ppM, the carbon dioxide supply time is synchronous with the illumination time, the culture temperature is 26-28 ℃, and the humidity in the culture box is 60-70%; 100mL of culture medium is supplemented every 2 days;
(5) Starting on day 8 or day 9 or day 10 of culture, carbon dioxide is sugar-freeThe culture box is fully opened; the culture conditions are as in step (4), namely: four 18W red, blue and white light sources are selected during the culture period, the distance between a culture box and the light source is 25cm, and the illumination intensity is 160 mu mol.m -2 ·s -1 Illuminating for 13 hours every day, wherein the carbon dioxide supply amount is 1000-1200 ppM, the carbon dioxide supply time is synchronous with the illumination time, the culture temperature is 26-28 ℃, and the humidity in the culture box is 60-70%; 100mL of culture medium is supplemented every 2 days;
(6) Culturing the sweet potato seedlings in a fully opened mode until the sweet potato seedlings grow to 12-15 cm in 14-16 days, cutting off redundant fibrous roots, and transplanting the sweet potato seedlings into a small greenhouse arch shed.
2. The method for carbon dioxide sugarless domestication of sweet potato virus-free tissue culture seedlings according to claim 1, wherein the size of the carbon dioxide sugarless culture box is 22cm multiplied by 26cm multiplied by 16cm.
3. The sweet potato detoxification tissue culture seedling carbon dioxide sugarless domestication method according to claim 1, wherein the ratio of the number of red light bulbs to the number of blue light bulbs to the number of white light bulbs in the light source is 3:2:1.
4. The carbon dioxide sugarless domestication method of sweet potato virus-free tissue culture seedlings, which is characterized in that after the sweet potato seedlings are transplanted into a greenhouse small arch shed in the step (6), slow water is used for watering soil, the humidity of the air in the small arch shed is guaranteed to be 75-85%, the temperature is 25-30 ℃, the sweet potato seedlings are covered with a shading net in the first two days, the shading net is covered with 10:00-16:00 in the 3 rd day to the 5 th day, the shading net is removed after 5 days, the small arch shed is removed after 7 days to 10 days, and the sweet potato seedlings grow normally.
5. The sweet potato virus-free tissue culture seedling carbon dioxide sugar-free domestication method according to claim 1 or 4, wherein the step (6) is replaced by the following step (7):
(7) Culturing the sweet potato seedlings in a full-open cover manner until the sweet potato seedlings grow to 12-15 cm, cutting the sweet potato seedlings, and reserving 3-5 cm at the bottom to serve as a mother plant to continue culturing in an original carbon dioxide sugarless culture box;
transplanting the cut detoxified seedlings into a new carbon dioxide sugarless culture box for culturing, and repeating the steps (3) to (6) of the claim 1;
culturing the parent strain which is kept in the original carbon dioxide sugarless culture box and is continuously cultured until the sweet potato seedling grows to 12-15 cm according to the condition of the step (5) of claim 1, cutting the sweet potato seedling, and still keeping 3-5 cm at the bottom to serve as the parent strain to be continuously cultured in the original carbon dioxide sugarless culture box; transplanting the cut detoxified seedlings into a new carbon dioxide sugarless culture box for culturing, and repeating the steps (3) to (6) of the claim 1;
the cycle is repeated according to the method.
6. The method for carbon dioxide sugarless domestication of sweet potato virus-free tissue culture seedlings according to claim 5, wherein the method for treating the mother plant which is left in the original carbon dioxide sugarless culture box and is continuously cultured in the step (7) is replaced by: culturing under the condition of the step (5) according to the claim 1 until the sweet potato seedlings grow to 12-15 cm, cutting off redundant fibrous roots, and transplanting into a greenhouse.
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