CN110896861A - Tissue culture rapid propagation and seedling raising method for agastache rugosus - Google Patents
Tissue culture rapid propagation and seedling raising method for agastache rugosus Download PDFInfo
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- 238000000034 method Methods 0.000 title claims abstract description 26
- 241001529821 Agastache Species 0.000 title claims description 27
- 241001162994 Rugosus Species 0.000 title claims description 26
- 239000001963 growth medium Substances 0.000 claims abstract description 76
- 230000004069 differentiation Effects 0.000 claims abstract description 43
- 238000005520 cutting process Methods 0.000 claims abstract description 18
- 238000012258 culturing Methods 0.000 claims abstract description 14
- 241000196324 Embryophyta Species 0.000 claims abstract description 11
- 238000004519 manufacturing process Methods 0.000 claims abstract description 9
- 238000005728 strengthening Methods 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 35
- 239000002609 medium Substances 0.000 claims description 23
- 238000005406 washing Methods 0.000 claims description 21
- 229930006000 Sucrose Natural products 0.000 claims description 18
- 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 18
- 239000000679 carrageenan Substances 0.000 claims description 18
- 229940113118 carrageenan Drugs 0.000 claims description 18
- 235000010418 carrageenan Nutrition 0.000 claims description 18
- 229920001525 carrageenan Polymers 0.000 claims description 18
- 229960004793 sucrose Drugs 0.000 claims description 18
- 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 18
- 239000012535 impurity Substances 0.000 claims description 17
- 238000005286 illumination Methods 0.000 claims description 15
- 229930192334 Auxin Natural products 0.000 claims description 14
- 239000002363 auxin Substances 0.000 claims description 14
- SEOVTRFCIGRIMH-UHFFFAOYSA-N indole-3-acetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CNC2=C1 SEOVTRFCIGRIMH-UHFFFAOYSA-N 0.000 claims description 14
- 239000008223 sterile water Substances 0.000 claims description 14
- 238000004140 cleaning Methods 0.000 claims description 12
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 9
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 8
- 230000000249 desinfective effect Effects 0.000 claims description 8
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 7
- JTEDVYBZBROSJT-UHFFFAOYSA-N indole-3-butyric acid Chemical compound C1=CC=C2C(CCCC(=O)O)=CNC2=C1 JTEDVYBZBROSJT-UHFFFAOYSA-N 0.000 claims description 7
- 238000011081 inoculation Methods 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 6
- 230000003020 moisturizing effect Effects 0.000 claims description 6
- 238000010008 shearing Methods 0.000 claims description 6
- 230000008569 process Effects 0.000 claims description 5
- 230000035755 proliferation Effects 0.000 claims description 5
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 claims description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 4
- 229960000367 inositol Drugs 0.000 claims description 4
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 claims description 4
- 229960003512 nicotinic acid Drugs 0.000 claims description 4
- 235000001968 nicotinic acid Nutrition 0.000 claims description 4
- 239000011664 nicotinic acid Substances 0.000 claims description 4
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 4
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 4
- 229960004172 pyridoxine hydrochloride Drugs 0.000 claims description 4
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 claims description 4
- 239000011764 pyridoxine hydrochloride Substances 0.000 claims description 4
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- 239000005720 sucrose Substances 0.000 claims description 4
- 239000003226 mitogen Substances 0.000 claims description 3
- 235000016709 nutrition Nutrition 0.000 claims description 3
- 230000035764 nutrition Effects 0.000 claims description 3
- 230000002062 proliferating effect Effects 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 2
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims description 2
- 229930003270 Vitamin B Natural products 0.000 claims description 2
- 241000607479 Yersinia pestis Species 0.000 claims description 2
- LFVGISIMTYGQHF-UHFFFAOYSA-N ammonium dihydrogen phosphate Chemical compound [NH4+].OP(O)([O-])=O LFVGISIMTYGQHF-UHFFFAOYSA-N 0.000 claims description 2
- 229910000387 ammonium dihydrogen phosphate Inorganic materials 0.000 claims description 2
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 2
- 238000001816 cooling Methods 0.000 claims description 2
- 229910052802 copper Inorganic materials 0.000 claims description 2
- 239000010949 copper Substances 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 2
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 2
- 229940099596 manganese sulfate Drugs 0.000 claims description 2
- 239000011702 manganese sulphate Substances 0.000 claims description 2
- 235000007079 manganese sulphate Nutrition 0.000 claims description 2
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 2
- 235000019837 monoammonium phosphate Nutrition 0.000 claims description 2
- 235000010333 potassium nitrate Nutrition 0.000 claims description 2
- 239000004323 potassium nitrate Substances 0.000 claims description 2
- 229920006395 saturated elastomer Polymers 0.000 claims description 2
- 235000019156 vitamin B Nutrition 0.000 claims description 2
- 239000011720 vitamin B Substances 0.000 claims description 2
- 239000011691 vitamin B1 Substances 0.000 claims description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 2
- 229960001763 zinc sulfate Drugs 0.000 claims description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims 1
- 241000544602 Ageratum Species 0.000 abstract description 20
- 239000000306 component Substances 0.000 abstract description 13
- 241000700605 Viruses Species 0.000 abstract description 6
- 230000004083 survival effect Effects 0.000 abstract description 5
- 238000009395 breeding Methods 0.000 abstract description 4
- 230000001488 breeding effect Effects 0.000 abstract description 4
- 239000005026 oriented polypropylene Substances 0.000 abstract description 3
- 230000009467 reduction Effects 0.000 abstract description 3
- 230000015556 catabolic process Effects 0.000 abstract 1
- 238000006731 degradation reaction Methods 0.000 abstract 1
- 238000002054 transplantation Methods 0.000 abstract 1
- 235000015097 nutrients Nutrition 0.000 description 10
- 241001573881 Corolla Species 0.000 description 6
- 238000003306 harvesting Methods 0.000 description 6
- 239000002689 soil Substances 0.000 description 5
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 description 4
- 239000004062 cytokinin Substances 0.000 description 4
- 241001164374 Calyx Species 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 2
- 206010047700 Vomiting Diseases 0.000 description 2
- 235000015197 apple juice Nutrition 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 235000020415 coconut juice Nutrition 0.000 description 2
- 230000007850 degeneration Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 210000004907 gland Anatomy 0.000 description 2
- 230000000762 glandular Effects 0.000 description 2
- 229960002523 mercuric chloride Drugs 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 239000011573 trace mineral Substances 0.000 description 2
- 235000013619 trace mineral Nutrition 0.000 description 2
- 230000008673 vomiting Effects 0.000 description 2
- 206010000060 Abdominal distension Diseases 0.000 description 1
- 240000004510 Agastache rugosa Species 0.000 description 1
- 235000010686 Agastache rugosa Nutrition 0.000 description 1
- 235000009024 Ceanothus sanguineus Nutrition 0.000 description 1
- 241000732800 Cymbidium Species 0.000 description 1
- 241000320571 Cymbidium floribundum Species 0.000 description 1
- 206010012735 Diarrhoea Diseases 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 240000003553 Leptospermum scoparium Species 0.000 description 1
- 235000015459 Lycium barbarum Nutrition 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 240000007817 Olea europaea Species 0.000 description 1
- 240000002505 Pogostemon cablin Species 0.000 description 1
- 235000011751 Pogostemon cablin Nutrition 0.000 description 1
- 206010037660 Pyrexia Diseases 0.000 description 1
- 208000032140 Sleepiness Diseases 0.000 description 1
- 206010041349 Somnolence Diseases 0.000 description 1
- 241000130764 Tinea Species 0.000 description 1
- 208000002474 Tinea Diseases 0.000 description 1
- 208000019790 abdominal distention Diseases 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 206010061428 decreased appetite Diseases 0.000 description 1
- 239000006160 differential media Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000012533 medium component Substances 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 231100000989 no adverse effect Toxicity 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000037321 sleepiness Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 230000004206 stomach function Effects 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- 229960000344 thiamine hydrochloride Drugs 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Environmental Sciences (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a tissue culture rapid propagation and seedling raising method for ageratum, which comprises the steps of selecting ageratum branch bud tips or leaves as explants for propagation, culturing the explants by using a differentiation culture medium to culture protosphere cell clusters and form plantlets, cutting the induced plantlets, inoculating the cut plantlets into a new differentiation culture medium, carrying out multiple subcultures by using the differentiation culture medium, inoculating the plantlets obtained by aseptic differentiation into a special seedling strengthening culture medium for strong seedling culture, and finally carrying out bagged seedling transplantation. The invention adopts original culture medium components and adopts an oriented polypropylene film bag as a culture dish device. The method removes various plant viruses, avoids the loss caused by variety degradation and yield reduction caused by the viruses, keeps the original excellent properties, and has good seedling quality and high planting survival rate. Strong operability, high application value and originality, and opens up a new way for breeding seedlings of ageratum with fast breeding and high production speed.
Description
Technical Field
The invention relates to a method for breeding seedlings of medicinal materials, in particular to a method for quickly breeding and raising seedlings of agastache rugosus.
Background
Agastache rugosus (academic name:Agastache rugosa(Fisch. et Mey.) O. Ktze.) perennial herbs. Erect stemThe height is 0.5-1.5 m, the thickness of the quadrangular prism is 7-8 mm, the upper part is extremely short and fine hair, the lower part is hairless, and the upper part is provided with fertile branches. Leaf heart-shaped oval to long round is coated with needle shape, the length is 4.5-11 cm, the width is 3-6.5 cm, the shape is gradually reduced upwards, the tip tail shape is gradually tapered, the base part is heart-shaped, the shape is thin and truncated, the edge is provided with thick teeth, the paper is made, the upper part is olive green, nearly has no hair, the lower part is slightly light, and the paper is slightly molted and dotted glands; the length of the petiole is 1.5-3.5 cm. The cymbidium floribundum floribunda is provided with multiple flowers, a top dense cylindrical panicle is formed on a main stem or a side branch, the panicle is 2.5-12 cm long, and the diameter is 1.8-2.5 cm; the bract at the base of inflorescence has length not more than 5mm, width of 1-2 mm, and is in the shape of needle, with long taper, and bract shape similar to it, smaller, and length of 2-3 mm; the cymbidium floridum has short stalk, the total stalk is about 3mm long, and the glandular minor hair is softened. The calyx has a tubular shape of inverted cone, a length of about 6 mm and a width of about 2 mm, and is slightly dyed into light purple or purple red by glandular hair and yellow gland, a slightly oblique throat, a triangular scaly scalded shape of the calyx teeth, a length of about 2.2 mm for the back 3 teeth and a little shorter for the front 2 teeth. The corolla is light purple blue, the length is about 8mm, the coat is slightly soft, the base of the corolla barrel is about 1.2 mm wide, slightly exceeds the calyx, the corolla barrel gradually widens upwards to the throat, the width of the throat is about 3mm, the two lips of the corolla brim are straight, the tip is slightly short, the lower lip is 3 cracks, the middle crack is wider, the length is about 2 mm, the width is about 3.5 mm, the corolla barrel is flat and flat, the edge is wavy, the base is wide, and the side cracks are semicircular. The stamen extends out of the corolla, and the filaments are thin, flat and hairless. The style and stamen are nearly equal in length, threadlike, and the tip is equal to 2 cracks. The flower disc is thick and annular. The top of the ovary lobe is provided with villi. The mature small nuts are oval and long, about 1.8 mm long, about 1.1 mm wide, ridged on the ventral surface, short bristles at the tip, and brown. The flowering period is 6-9 months, and the fruit period is 9-11 months. Geographic distribution: china is widely distributed in China, mainly in Sichuan, Jiangsu, Zhejiang, Hunan, Guangdong, etc., and Russia, Korea, Japan and North America. Reference 1, application No.: CN201410039484, name of the invention: a method for quickly rooting and seedling raising of cablin potchouli herb comprises six key links of selection of scion stock plants, scion harvesting, root forcing treatment, preparation of a cutting medium, cutting and management after cutting. The invention can make patchouli cuttage seedling root in advance and root quickly, the survival rate of cuttage is as high as 99%In the last step, the seedling is only required to be grown for 15 days, and the seedlings can be cut and raised all the year round. The comparative document 1 is different from the present invention.
The main function of ageratum is as follows: fragrant, resolving dampness, regulating stomach function, relieving vomiting, dispelling summerheat, and relieving exterior syndrome. Epigastric-abdominal distention and fullness, Min, which mainly obstructs the middle energizer due to dampness, inappetence, vomiting, diarrhea, headache due to summer-heat and dampness, fever with sleepiness at the beginning of damp-warm, chest-Min nausea, nasosinusitis, tinea of feet and hands.
Disclosure of Invention
The invention aims to provide a method for removing various plant viruses by using a tissue culture technology
The loss caused by variety degeneration and yield reduction caused by virus is avoided, so that the original excellent performance is maintained; the agastache rugosus seedling propagation method has the advantages of fast and large propagation speed, fast seedling formation, good seedling quality and high transplanting survival rate.
The invention takes the selected agastache bud and leaf as explants, removes impurities through disinfection, puts bud tip tissue and leaf on a differentiation medium for aseptic culture into plantlets, cuts the induced plantlets, inserts the cut plantlets into a new differentiation medium, adopts differentiation culture for multiple subcultures, and has the advantages of large quantity of clustered buds and fast growth. Then transferring the seedlings to a strong seedling culture medium for culture to form bagged seedlings, hardening the seedlings under natural illumination, then taking out the bagged seedlings for culture, and transplanting the bagged seedlings into a nutrition cup for seedling culture, wherein the survival rate is more than 98%. After three years of groping and screening, strong seedlings are cultured on a culture medium, the culture medium suitable for cell differentiation and strong seedlings is found as an invention point, and the invention is realized.
Embodiments of the invention are such that: a tissue culture and rapid propagation method of agastache rugosus is characterized in that agastache rugosus buds and leaves are used as explants, disinfection is carried out, a differentiation culture medium is prepared, a seedling strengthening culture medium is inoculated, and culture is carried out, wherein the technical process comprises the following steps:
step (1), explant collection: selecting and shearing tender branches of agastache rugosus, moisturizing and collecting continuous buds and leaves, selecting buds and tender leaves, washing with clear water, then washing with saturated washing powder water, and then rinsing with flowing clear water for 2-5 minutes;
step (2), sterile differentiation: rinsing the mopThe method comprises the steps of taking clean agastache rugosus buds or young leaves as explants, disinfecting the explants for 10-15 seconds by using 72-75% of alcohol on a workbench of an aseptic inoculation chamber, washing the explants for 2-5 times by using sterile water, disinfecting the explants in 0.1-0.2% of mercuric chloride for 3-15 minutes, and rinsing the explants for 4-6 times by using the sterile water to remove impurities on the surfaces. Placing the sterilized buds and leaves under an electron microscope with the weight more than 150 times, cutting impurities on the outer surface by using a scalpel, removing the tissues with 0.2-0.25 mm of the topmost bud tip after cleaning the impurities on the outer surface, inoculating the tissues onto a differentiation culture medium by using an inoculating needle, and culturing for 15-20 days to obtain plantlets, wherein each liter of the differentiation culture medium contains 15-35 mg of potassium nitrate, 1-6 mg of magnesium sulfate, 1-5 mg of ammonium dihydrogen phosphate, 0.5-3 mg of calcium chloride, 10-15 mg of manganese sulfate, 0.1-2 mg of zinc sulfate, 0.05-0.5 mg of copper blunderbuss, 0.001-0.2 mg of cobalt chloride, 0.001-0.2 mg of vitamin B, and 0.5-15 mg of vitamin B10.1-5 mg, 0.1-1 mg of pyridoxine hydrochloride, 0.5-5 mg of nicotinic acid, 10-100 mg of inositol, 15-45 g of sucrose, 6-10 g of carrageenan, 0.09-0.25 mg of auxin and 0.1-0.3 mg of 6BA of mitogen;
and (3) rapidly proliferating: after culturing for 15-20 days, cutting the induced plantlets into 0.3-0.5 mm, inoculating the plantlets into a new differentiation medium, carrying out subculture for multiple times by using the differentiation medium, continuously eliminating albino seedlings and pollution bag seedlings in each bag transferring process, cutting off withered yellow leaves, and carrying out bag transferring culture for 3-5 times to obtain plantlets for strong seedling culture;
step (4), strong seedling culture: inoculating the plantlets obtained through sterile differentiation and proliferation to a strong seedling culture medium for culturing for 40-60 days to obtain plantlets, wherein the strong seedling culture medium contains 10-35 g of cane sugar, 6-10 g of carrageenan, 0.2-5.0 mg of alpha-naphthylacetic acid and 0.1-3 mg of indolebutyric acid per liter, and the balance is the components in the culture medium in the step (2).
The differentiation culture medium in the step (2) contains 25-30 g of cane sugar, 8g of carrageenan and 0.09-0.25 mg of auxin per liter, and the balance is the components in the culture medium in the step (2).
The strong seedling culture medium in the step (4) contains 30g of cane sugar, 8g of carrageenan and 0.8mg of alpha-naphthylacetic acid per liter, and the balance is the components in the culture medium in the step (2).
The pH value of the culture medium used in the steps (2) to (4) is 5-6, the culture temperature is 20-28 ℃, the illumination is 8-12 hours per day, the illumination is 800-2500 Lx, the bud tip is a tissue with the growth point of the bud being 0.1-0.5 mm, the leaf is a tissue with the length of 0.1-0.8 mm at the position of an apex leaf 1/2, the height of a plantlet is preferably 1-3 cm, the height of a plantlet is preferably 3-8 cm, each plantlet has 2-8 roots, and the plantlet is albino variation.
Uniformly distributing the culture medium used in the steps (2) to (4) into film bags, wherein each bag is 30ml, sealing and sterilizing at high temperature for 15-25 minutes after being distributed, taking out and placing in a sterile room for cooling and storing, connecting corresponding explants or plantlets on an ultra-clean workbench of the sterile room, sealing the inoculated culture medium bags by using a sealing machine, placing in a culture room for culture, and distributing the culture medium bags into OPP film bags.
And when the plant formed by the plantlets grows to 3-8 cm, transferring the seedlings in the culture bag to natural light for 7-14 days, hardening the seedlings for 7-14 days, taking the seedlings out of the bag, cleaning a culture medium at the root, culturing in a light medium nutrition cup, and growing for 1-2 months to obtain medium seedlings which become seedlings for production and cultivation.
The medium seedlings are albino seedlings with the height of more than 15cm, each seedling has 2-5 branches and more than 3-8 roots, more fibrous roots, thick and green leaves, no diseases and pests and no variation.
Compared with the prior art, the invention has the advantages that: there are no patents and relevant reports on the production of ageratum seedlings by using a tissue culture technology at home and abroad, and only the application of seedlings and cutting seedling propagation is provided. The invention adopts the original culture medium and uses the oriented polypropylene film bag as the culture vessel, thereby reducing the cost, being capable of removing various plant viruses, avoiding the loss caused by variety degeneration and yield reduction caused by the viruses and keeping the original excellent properties; the propagation is fast and the quantity is large, the seedling formation is fast, the seedling quality is good, the transplanting survival rate is high, and the variety genetic material variation is small. The invention has the characteristics of strong operability, high application value, environmental protection, no adverse effect on the environment, originality and the like.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
Example 1
Step (1), explant collection: selecting and shearing tender branches of ageratum, moisturizing and refreshing buds and leaves, and harvesting, selecting buds, washing with clean water, washing with laundry powder water, and rinsing with flowing clean water.
Step (2), sterile differentiation: the rinsed ageratum buds are used as explants, sterilized by 72% -75% of alcohol for 10-15 seconds on a workbench of an aseptic inoculation room, rinsed by sterile water for 2-5 times, sterilized in 0.1% -0.2% of mercury bichloride for 3-15 minutes, rinsed by sterile water for 4-6 times, and impurities on the surfaces of the buds are removed. And putting the sterilized buds under an electron microscope with the weight of more than 150 times, cutting impurities on the outer surface by using a scalpel, cleaning the impurities on the outer surface, and inoculating the tissues with the bud tips of 0.2-0.25 mm to a differentiation culture medium by using an inoculating needle to culture to obtain plantlets. The differentiation culture medium contains 25-30 g of cane sugar, 8g of carrageenan, 0.09-0.25 mg of auxin, 6BA0.1mg of cytokinin and the balance of components in an SH culture medium per liter.
Step (3), strong seedling culture: inoculating the plantlets obtained by sterile differentiation to a strong seedling culture medium for culturing to obtain plantlets. The strong seedling culture medium contains 20-25 g of cane sugar, 8g of carrageenan, 0.5-3.0 mg of alpha-naphthylacetic acid, 0.3mg of indolebutyric acid, 0.09-0.25 mg of auxin and the balance of components in an SH culture medium per liter.
Step (4), transplanting bagged seedlings: and when the plant formed by the plantlets grows to 3-6 cm, transferring the seedlings in the culture bag to natural light for 7-14 days, taking the seedlings out of the bag, cleaning a culture medium at the root, transferring the seedlings to a nutrient cup filled with nutrient soil for cultivation to obtain medium seedlings, and forming the seedlings for production cultivation.
The culture medium used in the steps (2) to (3) is pH5.4-5.8, the culture temperature is 24-28 ℃, the illumination is 10-12 hours per day, and the illumination is 1500-2000 Lx.
The explant collected in the step (1) is buds of tender branches of the ageratum tree in the current year.
And (3) the bud tip in the step (2) is a bud growing point.
And (3) the height of the plantlet in the step (2) is 1-2 cm.
The height of the seedlings in the step (3) is 3-6 cm, and each seedling has 3-5 roots.
Example 2
Step (1), explant collection: selecting and shearing tender branches of ageratum, moisturizing and preserving buds and leaves, harvesting, selecting tender leaves, washing with clear water, washing with laundry powder water, and rinsing with flowing clear water.
Step (2), sterile differentiation: and taking the rinsed agastache rugosus tender leaves as explants, disinfecting the washed agastache rugosus tender leaves for 10 seconds by using 72 to 75 percent of alcohol on a workbench of an aseptic inoculation chamber, washing the agastache rugosus tender leaves for 2 to 5 times by using sterile water, disinfecting the agastache rugosus tender leaves for 5 to 15 minutes by placing the agastache rugosus tender leaves in 0.1 percent of mercury, and rinsing the agastache rugosus tender leaves for 4 to 6 times by using the sterile water. Placing the disinfected leaves under an electron microscope with the weight more than 150 times, cutting a tissue with the thickness of 0.3-0.5 mm at the position 1/2 below the apex of the tender leaves by using a scalpel, and inoculating the tissue to a differential medium for culturing to obtain plantlets. The differentiation culture medium contains 15ml of coconut juice, 22-30 g of cane sugar, 7-8 g of carrageenan, 100mg of inositol, 2mg of glycine, 0.5mg of nicotinic acid, 0.4-1.5 mg of pyridoxine hydrochloride, 0.09-0.25 mg of auxin, 6BA0.2mg of mitogen and the balance of 1/2 major elements and trace element components in an SH culture medium.
Step (3), strong seedling culture: inoculating the plantlets obtained by sterile differentiation to a strong seedling culture medium for culturing to obtain plantlets. The strong seedling culture medium contains 20-25 g of cane sugar, 8g of carrageenan, 0.5-3.0 mg of alpha-naphthylacetic acid, 40-100 mg of inositol, 4.5-12 mg of thiamine hydrochloride, 1.5-4.0 mg of glycine, 1.0-2.5 mg of nicotinic acid, 0.2mg of indolebutyric acid, 0.4-1.5 mg of pyridoxine hydrochloride and 0.09-0.25 mg of auxin per liter, and the balance of macroelements and 1/2 trace element components in an SH culture medium.
Step (4), transplanting bagged seedlings: and when the plant formed by the plantlets grows to 3-6 cm, transferring the seedlings in the culture bag to natural light for 7-14 days, taking the seedlings out of the bag, cleaning a culture medium at the root, transferring the seedlings to a nutrient cup filled with nutrient soil for cultivation to obtain medium seedlings, and forming the seedlings for production cultivation.
The culture medium used in the steps (2) to (3) is pH5.4-6.1, the culture temperature is 24-30 ℃, the illumination is 10 hours per day, and the illumination is 1500-2000 Lx.
The explant collected in the step (1) is the leaf of the tender branch of the ageratum in the current year.
The blade in the step (2) is a 0.3-0.5 mm tissue at the position of the tip blade 1/2.
And (3) the height of the plantlet in the step (2) is 1-2 cm.
The height of the seedlings in the step (3) is 3-6 cm, and each seedling has 3-5 roots.
Example 3
Step (1), explant collection: the method comprises the steps of selecting locally-planted ageratum tender branches, shearing the ageratum tender branches, moisturizing and refreshing the connected buds and leaves, harvesting the buds, selecting buds, washing the buds with clean water, washing the buds with laundry powder water, and rinsing the buds with flowing clean water.
Step (2), sterile differentiation: the rinsed ageratum buds are used as explants, sterilized by 71% -76% alcohol on a workbench of an aseptic inoculation room for 15 seconds, rinsed by sterile water for 2-5 times, sterilized in 0.2% mercuric chloride for 7-15 minutes, rinsed by sterile water for 4-6 times, and impurities on the surfaces of the ageratum buds are removed. Placing the sterilized bud under an electron microscope with the weight of more than 150 times, cutting foreign matters on the outer surface by using a scalpel, cleaning the foreign matters on the outer surface, and inoculating a tissue with the bud tip of 0.3mm onto a differentiation culture medium by using an inoculating needle to culture to obtain a plantlet. The differentiation culture medium contains 10ml of coconut juice, 25-30 g of cane sugar, 8g of carrageenan, 0.09-0.25 mg of auxin, 6BA0.1mg of cytokinin and the balance of SH culture medium components per liter.
Step (3), strong seedling culture: inoculating the plantlets obtained by sterile differentiation to a strong seedling culture medium for culturing to obtain plantlets. The strong seedling culture medium contains 20-25 g of cane sugar, 8g of carrageenan, 0.5-3.0 mg of alpha-naphthylacetic acid, 0.6mg of indolebutyric acid, 0.09-0.25 mg of auxin and the balance of components in an SH culture medium per liter.
Step (4), transplanting bagged seedlings: and when the plant formed by the plantlets grows to 3-6 cm, transferring the seedlings in the culture bag to natural light for 7-14 days, taking the seedlings out of the bag, cleaning a culture medium at the root, transferring the seedlings to a nutrient cup filled with nutrient soil for cultivation to obtain medium seedlings, and forming the seedlings for production cultivation.
The culture medium used in the steps (2) to (3) is pH5.4-6.0, the culture temperature is 23-29 ℃, the illumination is 12 hours per day, and the illumination is 1500-2000 Lx.
The explant collected in the step (1) is buds of tender branches of ageratum in the current year.
And (3) the bud tip in the step (2) is a bud growing point.
And (3) the height of the plantlet in the step (2) is 1-3 cm.
The height of the seedlings in the step (3) is 3-6 cm, and each seedling has 3-5 roots.
Example 4
Step (1), explant collection: the method comprises the steps of selecting and cutting tender ageratum branches planted in Luchuan county of Yulin city of Guangxi, harvesting buds and leaves for moisture preservation and freshness preservation, selecting buds, washing with clean water, washing with washing powder water, and rinsing with flowing clean water.
Step (2), sterile differentiation: the rinsed ageratum buds are used as explants, sterilized by 72% -75% of alcohol for 10-15 seconds on a workbench of an aseptic inoculation room, rinsed by sterile water for 2-5 times, sterilized in 0.1% -0.2% of mercury bichloride for 3-15 minutes, rinsed by sterile water for 4-6 times, and impurities on the surfaces of the buds are removed. And (3) putting the sterilized buds and leaves under an electron microscope with the weight of more than 150 times, cutting impurities on the outer surface by using a scalpel, cleaning the impurities on the outer surface, and inoculating a tissue with the bud tip of 0.2-0.25 mm to a differentiation culture medium by using an inoculating needle to culture to obtain a plantlet. The differentiation culture medium contains 25-30 g of cane sugar, 8g of carrageenan, 0.09-0.25 mg of auxin, 6BA0.1mg of cytokinin and the balance of SH culture medium components per liter.
Step (3), strong seedling culture: inoculating the plantlets obtained by sterile differentiation to a strong seedling culture medium for culturing to obtain plantlets. The strong seedling culture medium contains 20-25 g of cane sugar, 8g of carrageenan, 0.5-3.0 mg of alpha-naphthylacetic acid, 0.55mg of indolebutyric acid, 0.1-0.35 mg of auxin and 35g of apple juice per liter, and the balance is components in an SH culture medium.
Step (4), transplanting bagged seedlings: and when the plant formed by the plantlets grows to 3-6 cm, transferring the seedlings in the culture bag to natural light for 7-14 days, taking the seedlings out of the bag, cleaning a culture medium at the root, transferring the seedlings to a nutrient cup filled with nutrient soil for cultivation to obtain medium seedlings, and forming the seedlings for production cultivation.
The culture medium used in the steps (2) to (3) is pH5.5-6.0, the culture temperature is 24-30 deg.C, the illumination is 12 hours per day, and the illumination is 1500-2200 Lx.
The explant collected in the step (1) is buds of tender branches of ageratum in the current year.
And (3) the bud tip in the step (2) is a bud growing point.
And (3) the height of the plantlet in the step (2) is 1-2 cm.
The height of the seedlings in the step (3) is 3-6 cm, and each seedling has 3-5 roots.
Example 5
Step (1), explant collection: selecting and shearing tender ageratum branches, moisturizing and refreshing buds and leaves, and harvesting the buds, selecting buds, washing the buds with clean water, then washing the buds with laundry powder water, and then rinsing the buds with flowing clean water.
Step (2), sterile differentiation: the method comprises the steps of taking a rinsed oil tea tree bud as an explant, disinfecting the explant by using 72-75% of alcohol on a workbench of an aseptic inoculation room for 10-15 seconds, washing the explant by using sterile water for 2-5 times, disinfecting the explant in 0.1-0.2% of mercury bichloride for 3-15 minutes, rinsing the explant by using the sterile water for 4-6 times, and removing impurities on the surface. And (3) putting the sterilized buds and leaves under an electron microscope with the weight of more than 150 times, cutting impurities on the outer surface by using a scalpel, cleaning the impurities on the outer surface, and inoculating a tissue with the bud tip of 0.2-0.25 mm to a differentiation culture medium by using an inoculating needle to culture to obtain a plantlet. The differentiation medium contains 25-30 g of cane sugar, 8g of carrageenan, 0.09-0.25 mg of auxin, 6BA0.1mg of cytokinin and the balance of SH medium components per liter.
And (3) rapidly proliferating: after culturing for 15-20 days, cutting the induced plantlets and inoculating the plantlets into a new differentiation culture medium, so that the number of cluster buds is large and the growth is fast. And adopting a differentiation culture medium for multiple subcultures. During each bag rotation process, apical dominance should be cut off, and clumped buds cannot be cut too small to affect proliferation rate. Meanwhile, albino seedlings and pollution bag seedlings are continuously eliminated, withered yellow leaves are cut off, and after the bags are changed for more than 3 times, the proliferation speed can reach 2.5-3 times.
Step (4), strong seedling culture: inoculating the plantlets obtained by culture and proliferation to a strong seedling culture medium for culture to obtain plantlets. The strong seedling culture medium contains 20-25 g of cane sugar, 8g of carrageenan, 0.5-3.0 mg of alpha-naphthylacetic acid, 0.3mg of indolebutyric acid, 0.09-0.25 mg of auxin, 10-15 g of apple juice and the balance of SH culture medium components per liter.
Step (5), transplanting bagged seedlings: and when the plant formed by the plantlets grows to 3-6 cm, transferring the seedlings in the culture bag to natural light for 7-14 days, then taking the seedlings out of the bag, cleaning the culture medium at the root, transferring the seedlings to a nutrient cup filled with nutrient soil for culture, and obtaining medium seedlings after 2-4 months to obtain the seedlings for production and culture.
The culture medium used in the steps (2) to (3) is pH5.4-5.8, the culture temperature is 24-28 ℃, the illumination is 10-12 hours per day, and the illumination is 1500-2000 Lx.
The explant collected in the step (1) is buds of tender branches of ageratum in the current year.
And (3) the bud tip in the step (2) is a bud growing point.
And (3) the height of the plantlet in the step (2) is 1-2 cm.
The height of each seedling in the steps (3) to (4) is 3-6 cm, and each seedling has 3-5 roots.
The height of the medium seedling in the step (5) is more than 15cm, each seedling has more than 3-5 roots, and the fibrous roots are more.
Claims (7)
1. A tissue culture and rapid propagation method of agastache rugosus is characterized in that agastache rugosus buds and leaves are used as explants, disinfection is carried out, a differentiation culture medium is prepared, a seedling strengthening culture medium is inoculated, and culture is carried out, wherein the technical process comprises the following steps:
step (1), explant collection: selecting and shearing tender branches of agastache rugosus, moisturizing and collecting continuous buds and leaves, selecting buds and tender leaves, washing with clear water, then washing with saturated washing powder water, and then rinsing with flowing clear water for 2-5 minutes;
step (2), sterile differentiation: taking rinsed agastache rugosus buds or young leaves as explants, disinfecting the agastache rugosus buds or young leaves for 10 to 15 seconds on a workbench of an aseptic inoculation room by using 72 to 75 percent of alcohol, washing the agastache rugosus buds or young leaves for 2 to 5 times by using sterile water, disinfecting the agastache rugosus buds or young leaves in 0.1 to 0.2 percent of mercury bichloride for 3 to 15 minutes, rinsing the agastache rugosus buds or young leaves for 4 to 6 times by using the sterile water, removing impurities on the surfaces of the aga,
placing the sterilized buds and leaves under an electron microscope with the weight more than 150 times, cutting impurities on the outer surface by using a scalpel, removing the tissues with 0.2-0.25 mm of the topmost bud tip after cleaning the impurities on the outer surface, inoculating the tissues onto a differentiation culture medium by using an inoculating needle, and culturing for 15-20 days to obtain plantlets, wherein each liter of the differentiation culture medium contains 15-35 mg of potassium nitrate, 1-6 mg of magnesium sulfate, 1-5 mg of ammonium dihydrogen phosphate, 0.5-3 mg of calcium chloride, 10-15 mg of manganese sulfate, 0.1-2 mg of zinc sulfate, 0.05-0.5 mg of copper blunderbuss, 0.001-0.2 mg of cobalt chloride, 0.001-0.2 mg of vitamin B, and 0.5-15 mg of vitamin B10.1-5 mg, 0.1-1 mg of pyridoxine hydrochloride, 0.5-5 mg of nicotinic acid, 10-100 mg of inositol, 15-45 g of sucrose, 6-10 g of carrageenan, 0.09-0.25 mg of auxin and 0.1-0.3 mg of 6BA of mitogen;
and (3) rapidly proliferating: after culturing for 15-20 days, cutting the induced plantlets into 0.3-0.5 mm, inoculating the plantlets into a new differentiation medium, carrying out subculture for multiple times by using the differentiation medium, continuously eliminating albino seedlings and pollution bag seedlings in each bag transferring process, cutting off withered yellow leaves, and carrying out bag transferring culture for 3-5 times to obtain plantlets for strong seedling culture;
step (4), strong seedling culture: inoculating the plantlets obtained through sterile differentiation and proliferation to a strong seedling culture medium for culturing for 40-60 days to obtain plantlets, wherein the strong seedling culture medium contains 10-35 g of cane sugar, 6-10 g of carrageenan, 0.2-5.0 mg of alpha-naphthylacetic acid and 0.1-3 mg of indolebutyric acid per liter, and the balance is the components in the culture medium in the step (2).
2. The method according to claim 1, wherein the differentiation medium of step (2) contains 25-30 g of sucrose, 8g of carrageenan, 0.09-0.25 mg of auxin and the balance of the components in the medium of step (2) per liter.
3. The method according to claim 1, wherein the medium for strong seedlings in step (4) contains 30g of sucrose, 8g of carrageenan and 0.8mg of alpha-naphthylacetic acid per liter, and the balance is the components of the medium in step (2).
4. The method for tissue culture and rapid propagation of agastache rugosus according to claim 1, wherein the pH of the culture medium used in the steps (2) to (4) is 5 to 6, the culture temperature is 20 to 28 ℃, the illumination is 8 to 12 hours per day, the illumination is 800 to 2500Lx, the bud tip is a tissue with the growth point of bud 0.1 to 0.5mm, the leaf is a tissue with the height of 0.1 to 0.8mm at the position of the tip leaf 1/2, the height of the plantlet is preferably 1 to 3cm, the height of the plantlet is preferably 3 to 8cm, each plantlet has 2 to 8 roots and is not albino variation.
5. The method for tissue culture and rapid propagation of agastache rugosus as claimed in claim 1, wherein the culture medium used in steps (2) to (4) is divided into equal portions and filled into film bags, each bag is 30ml, the bags are sealed after being divided and sterilized at high temperature for 15-25 minutes, the bags are taken out and placed in a sterile room for cooling and storage, the bag with the inoculated culture medium is sealed by a sealing machine after the corresponding explant or plantlet is inoculated on a clean bench in the sterile room, the bag is placed in a culture room for culture, and the film bags with the culture medium subpackaged are all OPP film bags.
6. A method for raising the seedlings of agastache rugosus is characterized in that when the plant formed by the young seedlings grows to 3-8 cm, the seedlings in a culture bag are transferred to natural light to be hardened for 7-14 days, then the seedlings are taken out of the bag, a culture medium at the root is cleaned, the seedlings are cultured in a light medium nutrition cup, and the seedlings are grown for 1-2 months to obtain medium seedlings which become seedlings for production and cultivation.
7. The method of claim 6, wherein the medium-sized seedlings are seedlings with a height of more than 15cm, each seedling has 2-5 branches, 3-8 roots, more fibrous roots, thick and green leaves, no diseases and pests, no variation and albino seedlings.
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