CN108077076B - Rapid propagation method of ammopiptanthus mongolicus - Google Patents
Rapid propagation method of ammopiptanthus mongolicus Download PDFInfo
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- 241000586291 Ammopiptanthus mongolicus Species 0.000 title claims abstract description 68
- 238000000034 method Methods 0.000 title claims abstract description 54
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 69
- 230000004069 differentiation Effects 0.000 claims abstract description 38
- 230000006698 induction Effects 0.000 claims abstract description 22
- 238000005520 cutting process Methods 0.000 claims abstract description 16
- 239000001963 growth medium Substances 0.000 claims description 95
- 238000005286 illumination Methods 0.000 claims description 78
- 238000002791 soaking Methods 0.000 claims description 40
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 32
- 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 31
- 229930006000 Sucrose Natural products 0.000 claims description 31
- 239000005720 sucrose Substances 0.000 claims description 31
- 229920001817 Agar Polymers 0.000 claims description 28
- 239000008272 agar Substances 0.000 claims description 28
- 239000005708 Sodium hypochlorite Substances 0.000 claims description 17
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 17
- 229960002523 mercuric chloride Drugs 0.000 claims description 15
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 15
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 15
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims description 13
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 12
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 12
- 239000012883 rooting culture medium Substances 0.000 claims description 11
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 10
- 239000001888 Peptone Substances 0.000 claims description 8
- 108010080698 Peptones Proteins 0.000 claims description 8
- 235000019319 peptone Nutrition 0.000 claims description 8
- 230000001954 sterilising effect Effects 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 6
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 6
- 229910000403 monosodium phosphate Inorganic materials 0.000 claims description 6
- 235000019799 monosodium phosphate Nutrition 0.000 claims description 6
- 239000004323 potassium nitrate Substances 0.000 claims description 6
- 235000010333 potassium nitrate Nutrition 0.000 claims description 6
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 claims description 6
- LMSDCGXQALIMLM-UHFFFAOYSA-N 2-[2-[bis(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetic acid;iron Chemical compound [Fe].OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O LMSDCGXQALIMLM-UHFFFAOYSA-N 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 5
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims description 5
- 239000004327 boric acid Substances 0.000 claims description 5
- GVPFVAHMJGGAJG-UHFFFAOYSA-L cobalt dichloride Chemical compound [Cl-].[Cl-].[Co+2] GVPFVAHMJGGAJG-UHFFFAOYSA-L 0.000 claims description 5
- 229910000365 copper sulfate Inorganic materials 0.000 claims description 5
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 5
- 229960000367 inositol Drugs 0.000 claims description 5
- 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 5
- 150000002505 iron Chemical class 0.000 claims description 5
- 229940099596 manganese sulfate Drugs 0.000 claims description 5
- 239000011702 manganese sulphate Substances 0.000 claims description 5
- 235000007079 manganese sulphate Nutrition 0.000 claims description 5
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 5
- 235000001968 nicotinic acid Nutrition 0.000 claims description 5
- 229960003512 nicotinic acid Drugs 0.000 claims description 5
- 239000011664 nicotinic acid Substances 0.000 claims description 5
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 claims description 5
- 229960004172 pyridoxine hydrochloride Drugs 0.000 claims description 5
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 claims description 5
- 239000011764 pyridoxine hydrochloride Substances 0.000 claims description 5
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims description 5
- 239000011684 sodium molybdate Substances 0.000 claims description 5
- 235000015393 sodium molybdate Nutrition 0.000 claims description 5
- TVXXNOYZHKPKGW-UHFFFAOYSA-N sodium molybdate (anhydrous) Chemical compound [Na+].[Na+].[O-][Mo]([O-])(=O)=O TVXXNOYZHKPKGW-UHFFFAOYSA-N 0.000 claims description 5
- 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 claims description 5
- 229960000344 thiamine hydrochloride Drugs 0.000 claims description 5
- 235000019190 thiamine hydrochloride Nutrition 0.000 claims description 5
- 239000011747 thiamine hydrochloride Substances 0.000 claims description 5
- 239000011573 trace mineral Substances 0.000 claims description 5
- 235000013619 trace mineral Nutrition 0.000 claims description 5
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 5
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 5
- 229960001763 zinc sulfate Drugs 0.000 claims description 5
- 230000001902 propagating effect Effects 0.000 claims description 4
- 230000000249 desinfective effect Effects 0.000 claims description 3
- 159000000014 iron salts Chemical class 0.000 claims description 3
- 241000196324 Embryophyta Species 0.000 abstract description 3
- 238000004519 manufacturing process Methods 0.000 abstract description 3
- 230000004083 survival effect Effects 0.000 abstract description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 29
- 239000002609 medium Substances 0.000 description 25
- 238000004659 sterilization and disinfection Methods 0.000 description 25
- 238000011081 inoculation Methods 0.000 description 20
- 239000000463 material Substances 0.000 description 18
- 230000000694 effects Effects 0.000 description 13
- 238000005406 washing Methods 0.000 description 13
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 230000001939 inductive effect Effects 0.000 description 12
- 239000008223 sterile water Substances 0.000 description 12
- 230000035784 germination Effects 0.000 description 11
- 239000003153 chemical reaction reagent Substances 0.000 description 10
- 239000011521 glass Substances 0.000 description 9
- 238000003756 stirring Methods 0.000 description 9
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 8
- 239000012153 distilled water Substances 0.000 description 7
- 238000012258 culturing Methods 0.000 description 4
- 239000003002 pH adjusting agent Substances 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 238000012136 culture method Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000011010 flushing procedure Methods 0.000 description 3
- 239000003365 glass fiber Substances 0.000 description 3
- 239000005556 hormone Substances 0.000 description 3
- 229940088597 hormone Drugs 0.000 description 3
- 229910017053 inorganic salt Inorganic materials 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 239000012882 rooting medium Substances 0.000 description 3
- 238000010008 shearing Methods 0.000 description 3
- 239000008399 tap water Substances 0.000 description 3
- 235000020679 tap water Nutrition 0.000 description 3
- 238000009395 breeding Methods 0.000 description 2
- 230000001488 breeding effect Effects 0.000 description 2
- RCTYPNKXASFOBE-UHFFFAOYSA-M chloromercury Chemical compound [Hg]Cl RCTYPNKXASFOBE-UHFFFAOYSA-M 0.000 description 2
- 230000000052 comparative effect Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 238000005034 decoration Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 238000003825 pressing Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000004576 sand Substances 0.000 description 2
- 241000157076 Ammopiptanthus Species 0.000 description 1
- 241000220485 Fabaceae Species 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
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- 230000007613 environmental effect Effects 0.000 description 1
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- 238000011177 media preparation Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
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- 230000007226 seed germination Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
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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)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a rapid propagation method of ammopiptanthus mongolicus, belonging to the technical field of asexual propagation. The rapid propagation method solves the problem that the ammopiptanthus mongolicus is difficult to root by cutting propagation on the basis of keeping the excellent properties of the ammopiptanthus mongolicus which is a valuable or endangered plant by setting the conditions of induction, differentiation culture and rooting culture of the callus of the aseptic seedlings of the ammopiptanthus mongolicus. The propagation period is short, the rooting rate is high, the survival rate is high, the culture cost is low, and a good early-stage foundation can be laid for the protection of ammopiptanthus mongolicus germplasm resources and the production and research of high-quality nursery stocks.
Description
Technical Field
The invention relates to the technical field of asexual propagation, in particular to a rapid propagation method of ammopiptanthus mongolicus.
Background
Ammopiptanthus mongolicus (Ammopiptanthus mongolicus) belongs to the genus Ammopiptanthus in the family Leguminosae, is a small number of evergreen sandy shrubs in desert regions, is widely distributed in inner Mongolia, Ningxia and Gansu, and is mainly distributed in Hexi corridors and black river drainage basins in Gansu. The ammopiptanthus mongolicus has strong adaptability to environmental conditions, is drought resistant, cold resistant, barren resistant, salt and alkali resistant, has developed root system, and has very important functions of preventing sand and fixing sand, preventing water and soil loss and improving environment. Therefore, the plant has good ecological benefit in the aspect of improving the environment and is a potential ecological tree species for recovering and reconstructing vegetation in arid and semiarid regions to be developed and utilized.
However, most of the existing ammopiptanthus mongolicus adopts seed sowing and propagation, and genetic character separation and variation often occur after seedling formation; when the conventional asexual propagation cutting method is adopted, the ammopiptanthus mongolicus is difficult to root, and the cuttings are easy to rot in the cutting process, so that the propagation of the ammopiptanthus mongolicus is influenced. The prior art has no research report on a system in the aspect of tissue culture asexual rapid propagation of ammopiptanthus mongolicus with high rooting rate.
Disclosure of Invention
The invention aims to provide a method for quickly propagating ammopiptanthus mongolicus. The rapid propagation method provided by the invention has the advantages of short propagation period, high rooting rate, high survival rate and low culture cost, and can lay a good early foundation for ammopiptanthus mongolicus germplasm resource protection and production and research of high-quality nursery stocks.
The invention provides a method for quickly propagating ammopiptanthus mongolicus, which comprises the following steps of:
1) cutting an aseptic ammopiptanthus mongolicus seedling to obtain a stem section, and performing induced callus culture on an induced callus culture medium for 25-30 days to obtain a callus; the induction callus culture medium takes an improved B5 culture medium as a reference, and comprises 0.5-0.8 mg/L of KT, 0.15-0.2 mg/L of 2,4-D, 25-30 g/L of sucrose and 4.5g/L of agar, wherein the pH value is 5.8-6.0;
2) carrying out differentiation culture on the callus in the step 1) on an adventitious bud differentiation culture medium for 35-42 d to obtain adventitious buds with stem nodes; the adventitious bud differentiation medium is based on an improved B5 medium, and comprises 0.5-1.0 mg/L NAA, 0.2-0.5 mg/L6-BA, 25-30 g/L sucrose and 4.5g/L agar, wherein the pH value is 5.8-6.0;
3) carrying out rooting culture on the adventitious buds with the stem nodes in the step 2) on a rooting culture medium for 20-25 d to obtain ammopiptanthus mongolicus plants; the rooting medium takes an improved B5 medium as a reference, and comprises 1.0-1.5 mg/L of 2,4-D, 1000-1300 mg/L of peptone, 10-15 g/L of sucrose and 4.5g/L of agar, and the pH value is 5.8-6.0;
the improved B5 culture medium comprises macroelements, microelements, iron salt and organic components;
the macroelements include: 2000.00mg/L potassium nitrate, 100.00mg/L calcium chloride, 333.33mg/L magnesium sulfate, 100.00mg/L sodium dihydrogen phosphate and 89.30mg/L ammonium sulfate;
the trace elements include: 3.00mg/L boric acid, 2.00mg/L zinc sulfate, 0.75mg/L potassium iodide, 0.025mg/L copper sulfate, 0.25mg/L sodium molybdate, 10.00mg/L manganese sulfate and 0.025mg/L cobalt chloride;
the iron salts include: 28.00mg/L of iron ethylenediaminetetraacetic acid;
the organic component comprises: 100.00mg/L inositol, 1.00mg/L nicotinic acid, 1.00mg/L pyridoxine hydrochloride and 10.00mg/L thiamine hydrochloride.
Preferably, the preparation method of the aseptic seedlings in the step 1) comprises the following steps:
a) soaking and disinfecting the ammopiptanthus mongolicus seeds to obtain disinfected ammopiptanthus mongolicus seeds;
b) inoculating the disinfected ammopiptanthus mongolicus seeds to an aseptic seedling culture medium to carry out aseptic seedling culture for 10-15 days to obtain aseptic seedlings; the aseptic seedling culture medium takes the improved B5 culture medium as a reference, and comprises 25g/L of sucrose and 4.5g/L of agar, and the pH value is 5.8-6.0.
Preferably, the soaking time in the step a) is 8-12 h.
Preferably, the sterilization method in step a) is as follows: and soaking the soaked ammopiptanthus mongolicus seeds for 5-10 min by adopting a sodium hypochlorite solution with the mass concentration of 2%, soaking the ammopiptanthus mongolicus seeds for 30-40 s by adopting an ethanol solution with the mass concentration of 70% and soaking the ammopiptanthus mongolicus seeds for 3-5 min by adopting a mercuric chloride solution with the mass concentration of 0.08%.
Preferably, the aseptic seedling culture in the step b) comprises dark culture and illumination culture which are sequentially carried out, the dark culture time is 1-2 d, the illumination culture is carried out after the dark culture, and the illumination culture conditions are as follows: the temperature is 24 +/-2 ℃, the illumination intensity is 1500-1800 lx, and the illumination time is 12-15 h/d.
Preferably, the induced callus culture in the step 1) comprises dark culture and illumination culture which are sequentially performed, the dark culture time is 3-5 days, the illumination culture is performed after the dark culture, and the illumination culture conditions are as follows: the temperature is 24 +/-2 ℃, the illumination intensity is 1800-2000 lx, and the illumination time is 12-15 h/d.
Preferably, the length of the stem section in the step 1) is 0.4-0.6 cm.
Preferably, the differentiation culture of step 2) is: the illumination intensity is 1800-2000 lx at 24 +/-2 ℃, and the illumination time is 12-15 h/d.
Preferably, the rooting culture in the step 3) comprises dark culture and illumination culture which are sequentially performed, the dark culture time is 3-5 days, the illumination culture is performed after the dark culture, and the illumination culture conditions are as follows: the temperature is 25 ℃, the illumination intensity is 2000-2500 lx, and the illumination time is 14 h/d.
The invention provides a rapid propagation method of ammopiptanthus mongolicus. According to the method, the problem that the ammopiptanthus mongolicus is difficult to root by cutting propagation is solved on the basis of keeping the excellent properties of the ammopiptanthus mongolicus which is a treasure or endangered plant by setting callus induction, differentiation culture and rooting culture conditions; the method has the advantages of short breeding period, fast breeding and low seedling raising cost; the setting of each culture medium and the method of the invention increases the rooting rate of the ammopiptanthus mongolicus regenerated seedlings, and lays an early foundation for ammopiptanthus mongolicus germplasm resource protection and production and research of high-quality nursery stocks. The result shows that the callus induction rate of the ammopiptanthus mongolicus is 85-90%, the adventitious bud differentiation rate is 90-92%, and the rooting rate is as high as 90-95%.
Detailed Description
The invention provides a method for quickly propagating ammopiptanthus mongolicus, which comprises the following steps of:
1) cutting an aseptic ammopiptanthus mongolicus seedling to obtain a stem section, and performing induced callus culture on an induced callus culture medium for 25-30 days to obtain a callus; the induction callus culture medium takes an improved B5 culture medium as a reference, and comprises 0.5-0.8 mg/L of KT, 0.15-0.2 mg/L of 2,4-D, 25-30 g/L of sucrose and 4.5g/L of agar, wherein the pH value is 5.8-6.0;
2) carrying out differentiation culture on the callus in the step 1) on an adventitious bud differentiation culture medium for 35-42 d to obtain adventitious buds with stem nodes; the adventitious bud differentiation medium is based on an improved B5 medium, and comprises 0.5-1.0 mg/L NAA, 0.2-0.5 mg/L6-BA, 25-30 g/L sucrose and 4.5g/L agar, wherein the pH value is 5.8-6.0;
3) carrying out rooting culture on the adventitious buds with 1-2 stem nodes in the step 2) on a rooting culture medium for 20-25 d to obtain ammopiptanthus mongolicus plants; the rooting medium takes an improved B5 medium as a reference, and comprises 1.0-1.5 mg/L of 2,4-D, 1000-1300 mg/L of peptone, 10-15 g/L of sucrose and 4.5g/L of agar, and the pH value is 5.8-6.0;
the improved B5 culture medium comprises macroelements, microelements, iron salt and organic components;
the macroelements include: 2000.00mg/L potassium nitrate, 100.00mg/L calcium chloride, 333.33mg/L magnesium sulfate, 100.00mg/L sodium dihydrogen phosphate and 89.30mg/L ammonium sulfate;
the trace elements include: 3.00mg/L boric acid, 2.00mg/L zinc sulfate, 0.75mg/L potassium iodide, 0.025mg/L copper sulfate, 0.25mg/L sodium molybdate, 10.00mg/L manganese sulfate and 0.025mg/L cobalt chloride;
the iron salts include: 28.00mg/L of iron ethylenediaminetetraacetic acid;
the organic component comprises: 100.00mg/L inositol, 1.00mg/L nicotinic acid, 1.00mg/L pyridoxine hydrochloride and 10.00mg/L thiamine hydrochloride.
In the invention, the inorganic salt (i.e. macroelements) in the improved B5 culture medium is reduced by 1/3 on the original basis, and 2/3 of the inorganic salt is kept, and other components are not changed. The reduction of the concentration of the inorganic salt is beneficial to the growth and development of the root system when the ammopiptanthus mongolicus roots are cultured. The macroelements of unmodified B5 medium included: potassium nitrate 3000.00mg/L, calcium chloride 150.00mg/L, magnesium sulfate 500.00mg/L, sodium dihydrogen phosphate 150.00mg/L, ammonium sulfate 134.00 mg/L;
in the invention, the preparation method of the aseptic seedling comprises the following steps:
a) soaking and disinfecting the ammopiptanthus mongolicus seeds to obtain disinfected ammopiptanthus mongolicus seeds;
b) inoculating the disinfected ammopiptanthus mongolicus seeds to an aseptic seedling culture medium to carry out aseptic seedling culture for 10-15 days to obtain aseptic seedlings; the aseptic seedling culture medium takes the improved B5 culture medium as a reference, and comprises 25g/L of sucrose and 4.5g/L of agar, and the pH value is 5.8-6.0.
According to the invention, ammopiptanthus mongolicus seeds are soaked and disinfected to obtain disinfected ammopiptanthus mongolicus seeds. In the invention, the ammopiptanthus mongolicus seeds are preferably full and consistent ammopiptanthus mongolicus seeds harvested in the same year, the ammopiptanthus mongolicus seeds are preferably washed with water before being soaked, and the washing time is preferably 4 hours. In the present invention, the washing water is preferably running water. The water for soaking is not specially specified, and the water for soaking seeds commonly used by the technicians in the field can be adopted, and particularly, the water for soaking is preferably distilled water. In the invention, the soaking time is 8-12 h, and more preferably 10 h.
In the invention, the disinfection method comprises the following steps: soaking the soaked ammopiptanthus mongolicus seeds in a sodium hypochlorite solution with the mass concentration of 2% for 5-10 min, soaking in an ethanol solution with the mass concentration of 70% for 30-40 s, and soaking in mercuric chloride with the mass concentration of 0.08% for 3-5 min. In the invention, in the disinfection process, the sodium hypochlorite, the ethanol and the mercuric chloride solution are preferably stirred in the soaking process, so that the disinfection is more thorough. In the invention, the soaking time of the sodium hypochlorite solution is preferably 8min, the soaking time of the ethanol solution is preferably 35s, and the soaking time of the mercuric chloride solution is preferably 4 min.
In the invention, after the sodium hypochlorite is soaked and before the sodium hypochlorite is soaked in the ethanol solution, the sodium hypochlorite is preferably washed by sterile water, and the washing frequency is preferably 4-6 times, and more preferably 5 times; after the ethanol solution is soaked and before the mercuric chloride solution is soaked, preferably washing with sterile water for 4-6 times, and more preferably 5 times; after the mercuric chloride solution is soaked, the mercuric chloride solution is preferably washed by sterile water for 4-6 times, and more preferably 5 times. According to the invention, sodium hypochlorite, ethanol and mercuric chloride with low concentrations are adopted, and three sterilization reagents are combined for use, so that no damage is caused to seeds, the seeds are sterilized more thoroughly, and the pollution rate of the sterilized seeds is greatly reduced.
After the disinfected ammopiptanthus mongolicus seeds are obtained, inoculating the disinfected ammopiptanthus mongolicus seeds to an aseptic seedling culture medium to perform aseptic seedling culture for 10-15 days to obtain aseptic seedlings; the aseptic seedling culture medium takes an improved B5 culture medium as a reference, and also comprises 25g/L of sucrose and 4.5g/L of agar, and the pH value is 5.8-6.0. The method of adjusting the pH is not particularly limited, and the pH can be adjusted by using a pH adjusting agent known to those skilled in the art, for example, a sodium hydroxide solution with a mass concentration of 4% or a hydrochloric acid solution with a mass concentration of 8.3%.
In the invention, the aseptic seedling culture comprises dark culture and illumination culture which are sequentially carried out, the dark culture time is 1-2 d, the illumination culture is carried out after the dark culture, and the illumination culture conditions are as follows: the temperature is 24 +/-2 ℃, the illumination intensity is 1500-1800 lx, and the illumination time is 12-15 h/d. In the invention, the setting of the culture condition of the aseptic seedling ensures that the germination rate of the seeds is high, and the seeds can germinate after being inoculated for 3 days. In the present invention, the illumination intensity is preferably 1600 lx. In the present invention, the illumination time is preferably 14 h/d.
After obtaining the sterile seedlings, cutting the sterile seedlings of ammopiptanthus mongolicus to obtain stem sections, and performing induced callus culture on an induced callus culture medium for 25-30 days to obtain callus; the induction callus culture medium takes an improved B5 culture medium as a reference, 0.5-0.8 mg/L of KT, 0.15-0.2 mg/L of 2,4-D, 25-30 g/L of sucrose and 4.5g/L of agar, and the pH value is 5.8-6.0. In the invention, the concentration of KT is preferably 0.6mg/L, the concentration of 2,4-D is preferably 0.18mg/L, and the concentration of sucrose is preferably 28 g/L. The method of adjusting the pH is not particularly limited, and the pH can be adjusted by using a pH adjusting agent known to those skilled in the art, for example, a sodium hydroxide solution with a mass concentration of 4% or a hydrochloric acid solution with a mass concentration of 8.3%.
In the invention, the length of the aseptic seedling cultured for 10-15 days is 5-8 cm, and the stem section of the aseptic seedling after cutting is used as an explant for callus induction. In the invention, the induced callus culture comprises dark culture and illumination culture which are sequentially carried out, the dark culture time is 3-5 days, the illumination culture is carried out after the dark culture, and the illumination culture conditions are as follows: the temperature is 24 +/-2 ℃, the illumination intensity is 1800-2000 lx, and the illumination time is 12-15 h/d. In the present invention, the illumination intensity is preferably 1900lx, and the illumination time is preferably 14 h/d. In the invention, the length of the stem section is 0.4-0.6 cm, and preferably 0.5 cm. In the present invention, the stem section is preferably inoculated by plating, and the stem section is sufficiently contacted with the culture medium by lightly pressing. The callus tissue grown under the callus induction culture condition has loose structure and yellow-green color, and the callus tissue induction rate is 85-90%.
After obtaining the callus, carrying out differentiation culture on the callus on an adventitious bud differentiation culture medium for 35-42 days to obtain adventitious buds with stem nodes; the adventitious bud differentiation medium is based on an improved B5 medium, 0.5-1.0 mg/L NAA, 0.2-0.5 mg/L6-BA, 25-30 g/L sucrose and 4.5g/L agar, and the pH value is 5.8-6.0. In the invention, the concentration of the NAA is preferably 0.75mg/L, the concentration of the 6-BA is preferably 0.3mg/L, and the concentration of the sucrose is preferably 27 g/L. The method of adjusting the pH is not particularly limited, and the pH can be adjusted by using a pH adjusting agent known to those skilled in the art, for example, a sodium hydroxide solution with a mass concentration of 4% or a hydrochloric acid solution with a mass concentration of 8.3%.
In the present invention, the differentiation culture is: culturing at 24 +/-2 ℃, the illumination intensity of 1800-2000 lx and the illumination time of 12-15 h/d. In the present invention, the illumination intensity is preferably 1900lx, and the illumination time is preferably 14 h/d. In the present invention, the callus is preferably inoculated by plating, and the callus is sufficiently contacted with the medium by lightly pressing. The adventitious bud differentiation rate in the culture medium under the differentiation culture condition is 90-92%, and the differentiated adventitious buds are light green.
After obtaining adventitious buds with stem nodes, the invention cuts the adventitious buds into stem segments with 1-2 stem nodes, and performs rooting culture on a rooting culture medium for 20-25 days to obtain ammopiptanthus mongolicus plants; the rooting medium is based on an improved B5 medium, and comprises 1.0-1.5 mg/L of 2,4-D, 1000-1300 mg/L of peptone, 10-15 g/L of sucrose and 4.5g/L of agar, and the pH value is 5.8-6.0. In the present invention, the concentration of 2,4-D is preferably 1.2mg/L, the concentration of peptone is preferably 1200mg/L, and the concentration of sucrose is preferably 12 g/L. The method of adjusting the pH is not particularly limited, and the pH can be adjusted by using a pH adjusting agent known to those skilled in the art, for example, a sodium hydroxide solution with a mass concentration of 4% or a hydrochloric acid solution with a mass concentration of 8.3%.
In the present invention, the rooting culture is preferably: culturing in dark for 3-5 days, and culturing in a culture room with illumination intensity of 2000-2500 lx and illumination time of 14h/d at 25 ℃. In the present invention, the illumination intensity is preferably 2200 lx. In the invention, the adventitious bud with 1-2 stem nodes is preferably selected for rooting culture. Under the rooting culture condition, the adventitious bud rooting rate is 90-95%. In the invention, dark culture is carried out in the early inoculation stage of the rooting culture stage, adventitious buds start to root in the earliest 7 days, and the main roots are thick and strong, the fibrous roots are more, the color of root seedlings is light green, the growth is vigorous, and the bottle-out seedling hardening transplantation is facilitated.
The method for rapid propagation of ammopiptanthus mongolicus according to the present invention is further described in detail with reference to the following embodiments, and the technical solutions of the present invention include, but are not limited to, the following embodiments.
Example 1
Preparation of a modified B5 culture medium:
1) macroelements: 2000.00mg/L potassium nitrate, 100.00mg/L calcium chloride, 333.33mg/L magnesium sulfate, 100.00mg/L sodium dihydrogen phosphate and 89.30mg/L ammonium sulfate;
2) trace elements: 3.00mg/L boric acid, 2.00mg/L zinc sulfate, 0.75mg/L potassium iodide, 0.025mg/L copper sulfate, 0.25mg/L sodium molybdate, 10.00mg/L manganese sulfate and 0.025mg/L cobalt chloride;
3) iron salt: 28.00mg/L of iron ethylenediaminetetraacetic acid;
4) organic components: 100.00mg/L inositol, 1.00mg/L nicotinic acid, 1.00mg/L pyridoxine hydrochloride and 10.00mg/L thiamine hydrochloride. The four groups of reagents are respectively dissolved and mixed according to groups, and the volume is respectively determined to 1000 mL.
II, the main steps are as follows:
1. seed disinfection treatment:
the method comprises the steps of selecting full ammopiptanthus mongolicus seeds with the same size harvested in the current year, flushing the seeds for 4 hours under flowing tap water, soaking the seeds in distilled water for 12 hours, pouring the distilled water, and placing the seeds which are soaked fully on a super-clean workbench for disinfection treatment.
Firstly, soaking seeds for 5min by using 2% sodium hypochlorite, continuously stirring a glass rod for sterilization during the soaking period so as to ensure that the seeds are sterilized more thoroughly, and then washing the seeds for 4 times by using sterile water; soaking the glass fiber in 70% ethanol for 40s, and washing with sterilized glass rod while stirring and sterile water for 4 times; and thirdly, soaking for 3min by using 0.1 percent mercuric chloride, continuously stirring by using a sterilized glass rod during soaking, then washing for 5 times by using sterile water, and finally placing the treated seeds on sterilized filter paper for inoculation after the surfaces of the seeds are soaked in water and dried in air.
2. And (3) sterile seedling culture:
1) the method, the formula and the effect are as follows: and inoculating the treated seeds to a sterile seedling culture medium, wherein the sterile seedling culture medium is based on the improved B5 culture medium, and further comprises 25g/L of sucrose and 4.5g/L of agar, and the pH value is 5.8. 5 seeds are inoculated in each bottle of culture medium, the seeds are soaked before disinfection treatment, the seeds can germinate after being inoculated for 3 days, the aseptic seedlings after germination grow to 5-8 cm, the seedlings can be cut and induced by callus, and the germination rate of the seeds is 100% (the highest germination rate of the seeds is about 90% after the conventional technology adopts hormone treatment).
2) The culture conditions are as follows: after inoculation, the seeds are placed in an artificial incubator for dark culture for 2d, and then placed in a culture room with the temperature of 24 ℃, the illumination intensity of 1800lx and the illumination time of 12h/d for culture.
3. Inducing callus culture
1) The method, the formula and the effect are as follows: 0.5cm of stem segments of aseptic seedlings growing for 15 days are cut as a material for inducing callus, the material is spread and inoculated in an inducing callus culture medium, then the material is lightly pressed by a pair of tweezers so as to ensure that the explants are fully contacted with the inducing callus culture medium, and 10 explant stem segments are inoculated in each bottle of culture medium. The induction callus culture medium takes the improved B5 culture medium as a reference, and also comprises 0.5mg/L KT, 0.2 mg/L2, 4-D, 25g/L sucrose and 4.5g/L agar, wherein the pH value is 6.0, the grown callus has a loose structure and is yellow-green, and the callus induction rate is 88.36%.
2) The culture conditions are as follows: after inoculation, the seeds are placed in an artificial incubator for dark culture for 4d, and then are placed in a culture room with the temperature of 24 +/-2 ℃, the illumination intensity of 2000lx and the illumination time of 12h/d for culture.
4. Differential culture of adventitious bud
1) The method, the formula and the effect are as follows: the callus which has grown for 28 days is cut by a sterilized scalpel blade to be used as an explant material for adventitious bud induction, the explant material is spread and inoculated on an adventitious bud differentiation culture medium, then the explant material is lightly pressed so as to ensure that the inoculated callus is fully contacted with the culture medium, and 5 callus pieces are inoculated in each bottle of the culture medium. The adventitious bud differentiation medium is based on an improved B5 medium, and further comprises 0.5mg/L NAA, 0.2 mg/L6-BA, 25g/L sucrose and 4.5g/L agar, the pH value is 6.0, the adventitious bud differentiation rate in the adventitious bud differentiation medium is 90.37%, and the differentiated adventitious buds are light green.
2) The culture conditions are as follows: after inoculation, the seeds are placed in a culture room with the temperature of 24 +/-2 ℃, the illumination intensity of 2000lx and the illumination time of 12h/d for culture.
5. Rooting culture
1) The method, the formula and the effect are as follows: the method comprises the following steps of shearing an adventitious bud in a differentiation culture medium to grow for about 35 days, transferring the adventitious bud with 1-2 stem nodes into a rooting culture medium to perform rooting culture, wherein the rooting culture medium takes an improved B5 culture medium as a reference, and comprises 1.0mg/L of 2,4-D, 1000mg/L of peptone, 10g/L of sucrose and 4.5g/L of agar, the pH value is 6.0, the rooting rate of the adventitious bud in the culture medium is 91.76%, and the ammopiptanthus mongolicus cutting shoot is difficult to root by adopting a conventional cutting method. Because the dark culture is carried out in the initial stage of inoculation, adventitious buds start to root in the earliest 7 days, and the main roots are thick and strong, the fibrous roots are more, the color of the root seedlings is light green, the growth is vigorous, and the bottle-out seedling hardening transplantation is facilitated.
2) The culture conditions are as follows: after inoculation, the seeds are placed in an artificial incubator for dark culture for 3d, and then are placed in a culture room with the temperature of 25 ℃, the illumination intensity of 2200lx and the illumination time of 14h/d for culture.
Example 2
Modified B5 medium was prepared as in example 1:
the method mainly comprises the following steps:
1. seed disinfection treatment:
the method comprises the steps of selecting full ammopiptanthus mongolicus seeds with the same size harvested in the current year, flushing the seeds for 4 hours under flowing tap water, soaking the seeds in distilled water for 12 hours, pouring the distilled water, and placing the seeds which are soaked fully on a super-clean workbench for disinfection treatment.
The seeds are disinfected by firstly soaking for 2min by using 20 percent sodium hypochlorite, continuously stirring a glass rod for disinfection during the soaking so as to ensure that the disinfection is more thorough, and then washing for 3 times by using sterile water; soaking the glass fiber in 70% ethanol for 35s, and washing with sterile water for 5 times while stirring the sterilized glass rod; and thirdly, soaking for 5min by using 0.1 percent mercuric chloride, continuously stirring by using a sterilized glass rod during soaking, then washing for 6 times by using sterile water, and finally placing the treated seeds on sterilized filter paper for inoculation after the surfaces of the seeds are soaked in water and dried in air.
2. And (3) sterile seedling culture:
1) the method, the formula and the effect are as follows: and inoculating the treated seeds to a sterile seedling culture medium, wherein the sterile seedling culture medium is based on the improved B5 culture medium, and further comprises 25g/L of sucrose and 4.5g/L of agar, and the pH value is 5.8. 5 seeds are inoculated in each bottle of culture medium, the seeds are soaked before disinfection treatment, the seeds can germinate after being inoculated for 3 days, the aseptic seedlings after germination grow to 5-8 cm, the seedlings can be cut and induced by callus, and the germination rate of the seeds is 100% (the highest germination rate of the seeds is about 90% after the conventional technology adopts hormone treatment).
2) The culture conditions are as follows: after inoculation, the seeds are placed in an artificial incubator for dark culture for 1d, and then are placed in a culture room with the temperature of 24 ℃, the illumination intensity of 1600lx and the illumination time of 12h/d for culture.
3. Inducing callus culture
1) The method, the formula and the effect are as follows: 0.4cm of stem segments of aseptic seedlings growing for 13 days are cut as a material for inducing callus, the material is spread and inoculated in an inducing callus culture medium, then the material is lightly pressed by a pair of tweezers so as to ensure that the explants are fully contacted with the inducing callus culture medium, and 10 explant stem segments are inoculated in each bottle of culture medium. The induction callus culture medium takes the improved B5 culture medium as a reference, and also comprises 0.8mg/L KT, 0.15 mg/L2, 4-D, 30g/L sucrose and 4.5g/L agar, wherein the pH value is 6.0, the grown callus has a loose structure and is yellow-green, and the callus induction rate is 88.09%.
2) The culture conditions are as follows: after inoculation, the seeds are placed in an artificial incubator for dark culture for 5d, and then are placed in a culture room with the temperature of 24 +/-2 ℃, the illumination intensity of 2000lx and the illumination time of 12h/d for culture.
4. Stem bud differentiation culture
1) The method, the formula and the effect are as follows: the callus which grows for 30 days is cut by a sterilized scalpel blade to be used as an explant material for adventitious bud induction, the explant material is spread and inoculated on an adventitious bud differentiation culture medium, then the explant material is lightly pressed so as to ensure that the inoculated callus is fully contacted with the culture medium, and 5 callus pieces are inoculated in each bottle of the culture medium. The adventitious bud differentiation medium is based on an improved B5 medium, and further comprises 1.0mg/L NAA, 0.3 mg/L6-BA, 30g/L sucrose and 4.5g/L agar, the pH value is 6.0, the adventitious bud differentiation rate in the adventitious bud differentiation medium is 91.14%, and the differentiated adventitious buds are light green.
2) The culture conditions are as follows: after inoculation, the seeds are placed in a culture room with the temperature of 24 +/-2 ℃, the illumination intensity of 2000lx and the illumination time of 12h/d for culture.
5. Rooting culture
1) The method, the formula and the effect are as follows: the method comprises the steps of shearing an adventitious bud differentiation culture medium to grow for about 40 days, transferring the adventitious bud with 1-2 stem nodes into a rooting culture medium to perform rooting culture, wherein the rooting culture medium takes an improved B5 culture medium as a reference, and comprises 1.2mg/L of 2,4-D, 1200mg/L of peptone, 12g/L of sucrose and 4.5g/L of agar, the pH value is 5.8, the rooting rate of the adventitious bud in the culture medium is 93.55%, and the ammopiptanthus mongolicus cutting shoot is difficult to root by adopting a conventional cutting method. Because the dark culture is carried out in the initial stage of inoculation, adventitious buds start to root in the earliest 7 days, and the main roots are thick and strong, the fibrous roots are more, the color of the root seedlings is light green, the growth is vigorous, and the bottle-out seedling hardening transplantation is facilitated.
2) The culture conditions are as follows: after inoculation, the seeds are placed in an artificial incubator for dark culture for 4d, and then are placed in a culture room with the temperature of 25 ℃, the illumination intensity of 2500lx and the illumination time of 14h/d for culture.
Example 3
Modified B5 Medium preparation As shown in example 1
1) Macroelements: 2000.00mg/L potassium nitrate, 100.00mg/L calcium chloride, 333.33mg/L magnesium sulfate, 100.00mg/L sodium dihydrogen phosphate and 89.30mg/L ammonium sulfate;
2) trace elements: 3.00mg/L boric acid, 2.00mg/L zinc sulfate, 0.75mg/L potassium iodide, 0.025mg/L copper sulfate, 0.25mg/L sodium molybdate, 10.00mg/L manganese sulfate and 0.025mg/L cobalt chloride;
3) iron salt: 28.00mg/L of iron ethylenediaminetetraacetic acid;
4) organic components: 100.00mg/L inositol, 1.00mg/L nicotinic acid, 1.00mg/L pyridoxine hydrochloride and 10.00mg/L thiamine hydrochloride. The four groups of reagents are respectively dissolved and mixed according to groups, and the volume is respectively determined to 1000 mL.
The method mainly comprises the following steps:
1. seed disinfection treatment:
the method comprises the steps of selecting full ammopiptanthus mongolicus seeds with the same size harvested in the current year, flushing the seeds for 4 hours under flowing tap water, soaking the seeds in distilled water for 12 hours, pouring the distilled water, and placing the seeds which are soaked fully on a super-clean workbench for disinfection treatment.
The seeds are disinfected by firstly soaking for 8min by adopting 2 percent sodium hypochlorite, continuously stirring a glass rod for disinfection during the soaking period so as to ensure that the disinfection is more thorough, and then washing for 5 times by using sterile water; soaking the glass fiber in 70% ethanol for 30s, and washing with sterilized glass rod while stirring and sterile water for 4 times; and thirdly, soaking for 4min by using 0.1 percent mercuric chloride, continuously stirring by using a sterilized glass rod during soaking, then washing for 5 times by using sterile water, and finally placing the treated seeds on sterilized filter paper for inoculation after the surfaces of the seeds are soaked in water and dried in air.
2. And (3) sterile seedling culture:
1) the method, the formula and the effect are as follows: and inoculating the treated seeds to a sterile seedling culture medium, wherein the sterile seedling culture medium is based on the improved B5 culture medium, and further comprises 25g/L of sucrose and 4.5g/L of agar, and the pH value is 5.8. 5 seeds are inoculated in each bottle of culture medium, the seeds are soaked before disinfection treatment, the seeds can germinate after being inoculated for 3 days, the aseptic seedlings after germination grow to 5-8 cm, the seedlings can be cut and induced by callus, and the germination rate of the seeds is 100% (the highest germination rate of the seeds is about 90% after the conventional technology adopts hormone treatment).
2) The culture conditions are as follows: after inoculation, the seeds are placed in an artificial incubator for dark culture for 2d, and then placed in a culture room with the temperature of 24 ℃, the illumination intensity of 1800lx and the illumination time of 12h/d for culture.
3. Inducing callus culture
1) The method, the formula and the effect are as follows: 0.4cm of stem segments of 14d aseptic seedlings are cut as a material for inducing callus, the material is spread and inoculated in an inducing callus culture medium, then the material is lightly pressed by a pair of tweezers so as to ensure that the explants are fully contacted with the inducing callus culture medium, and 10 explant stem segments are inoculated in each bottle of culture medium. The induction callus culture medium takes the improved B5 culture medium as a reference, and also comprises 0.6mg/L KT, 0.18 mg/L2, 4-D, 25g/L sucrose and 4.5g/L agar, wherein the pH value is 6.0, the grown callus has a loose structure and is yellow-green, and the callus induction rate is 86.83%.
2) The culture conditions are as follows: after inoculation, the seeds are placed in an artificial incubator for dark culture for 3d, and then are placed in a culture room with the temperature of 24 +/-2 ℃, the illumination intensity of 2000lx and the illumination time of 12h/d for culture.
4. Differential culture of adventitious bud
1) The method, the formula and the effect are as follows: the callus which grows for 30 days is cut by a sterilized scalpel blade to be used as an explant material for adventitious bud induction, the explant material is spread and inoculated on an adventitious bud differentiation culture medium, then the explant material is lightly pressed so as to ensure that the inoculated callus is fully contacted with the culture medium, and 5 callus pieces are inoculated in each bottle of the culture medium. The adventitious bud differentiation medium is based on the improved B5 medium, and further comprises 0.5mg/L NAA, 0.4 mg/L6-BA, 25g/L sucrose and 4.5g/L agar, the pH value is 6.0, the adventitious bud differentiation rate in the adventitious bud differentiation medium is 91.67%, and the differentiated adventitious buds are light green.
2) The culture conditions are as follows: after inoculation, the seeds are placed in a culture room with the temperature of 24 +/-2 ℃, the illumination intensity of 2000lx and the illumination time of 12h/d for culture.
5. Rooting culture
1) The method, the formula and the effect are as follows: shearing an adventitious bud differentiation culture medium to grow for about 42 days, transferring the adventitious bud with 1-2 stem nodes into a rooting culture medium for rooting culture, wherein the rooting culture medium takes an improved B5 culture medium as a reference, and comprises 1.5mg/L of 2,4-D, 1300mg/L of peptone, 15g/L of sucrose and 4.5g/L of agar, the pH value is 6.0, the rooting rate of the adventitious bud in the culture medium is 92.95%, and the ammopiptanthus mongolicus cutting shoot is difficult to root by adopting a conventional cutting method. Because the dark culture is carried out in the initial stage of inoculation, adventitious buds start to root in the earliest 7 days, and the main roots are thick and strong, the fibrous roots are more, the color of the root seedlings is light green, the growth is vigorous, and the bottle-out seedling hardening transplantation is facilitated.
2) The culture conditions are as follows: after inoculation, the seeds are placed in an artificial incubator for dark culture for 5d, and then are placed in a culture room with the temperature of 25 ℃, the illumination intensity of 2000lx and the illumination time of 14h/d for culture.
Comparison example 1
The results of different agent disinfection were compared (see table 1).
TABLE 1 Disinfection results with different reagents
Description of the drawings: in the above table, sodium hypochlorite solution is soaked for 5min, ethanol solution is soaked for 30s, and mercuric chloride solution is soaked for 4 min.
The results of seed disinfection are shown in table 1, when 2% sodium hypochlorite, 5% sodium hypochlorite, 70% ethanol, 75% ethanol, 0.05% mercuric chloride, 0.08% mercuric chloride were used alone or in combination. The difference of the contamination rates is small when the same reagent is sterilized at different concentrations, and the difference of the contamination rates is large when different reagents are combined at different concentrations. Wherein, when three reagent combinations of 2% sodium hypochlorite, 70% ethanol and 0.08% mercury chloride and three reagent combinations of 2% sodium hypochlorite, 75% ethanol and 1% mercury chloride are adopted for sterilization, the pollution rates are minimum, namely 7.18% and 8.33% respectively, and when one or two reagent combinations are adopted for sterilization independently, the pollution rates are minimum and reach 44.51% and 36.12% respectively; therefore, the combination of the three reagents can achieve good sterilization effect.
Comparative example two
The minimal medium in the present invention was MS, B5 medium, and the culture results were obtained under the same conditions as in example 1 (see Table 2).
TABLE 2 Ammopiptanthus mongolicus growth in different media
The results of culturing in MS, B5 and modified B5 media under the same conditions as those of the present invention are shown in Table 2. The germination rate of the aseptic seedlings in the three culture media is over 90 percent, wherein the seeds completely germinate when the improved B5 culture medium is used for culture; when callus is induced and adventitious bud is differentiated and cultured, the culture results of the three basic culture media have larger difference, and the size difference is within 10 percent; however, when ammopiptanthus mongolicus rooting culture is carried out, the results of the three culture media are greatly different, the rooting rate in the improved B5 culture medium is as high as 95.16%, the rooting rate in the B5 culture medium is 73.56%, and the rooting rate is only 68.91% when MS is used as a basic culture medium.
Comparative example three
The minimal medium in the present invention was modified B5 medium, and the culture results were obtained when the culture mode was different (see Table 3).
TABLE 3 Rapid propagation of Ammopiptanthus mongolicus under different cultivation methods
As shown in Table 3, when two different culture methods are adopted, the seeds can germinate after 3d by combining dark culture after inoculation, while the seeds germinate at the earliest time of 8d by conventional culture methods with larger difference, and the seed germination difference is smaller in the two culture methods; when the callus is induced and adventitious bud is differentiated and cultured, the difference between the callus induction rate and the adventitious bud differentiation rate between the two culture modes is small, the size difference is kept about 5%, and the induction and differentiation time is also different; however, in the rooting culture, the two culture modes are greatly different, when the dark culture and the illumination culture are combined, the root can be rooted within 7 days, the rooting rate is as high as 95.85%, and when the dark culture and the illumination culture are combined, the root is rooted within 11 days at the earliest time, and the rooting rate is only 77.19%.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (9)
1. A method for quickly propagating ammopiptanthus mongolicus comprises the following steps:
1) cutting an aseptic ammopiptanthus mongolicus seedling to obtain a stem section, and performing induced callus culture on an induced callus culture medium for 25-30 days to obtain a callus; the induction callus culture medium takes an improved B5 culture medium as a basic culture medium, and comprises 0.5-0.8 mg/L of KT, 0.15-0.2 mg/L of 2,4-D, 25-30 g/L of sucrose and 4.5g/L of agar, wherein the pH value is 5.8-6.0;
2) carrying out differentiation culture on the callus in the step 1) on an adventitious bud differentiation culture medium for 35-42 d to obtain adventitious buds with stem nodes; the adventitious bud differentiation culture medium takes an improved B5 culture medium as a basic culture medium, and comprises 0.5-1.0 mg/L of NAA, 0.2-0.5 mg/L of 6-BA, 25-30 g/L of sucrose and 4.5g/L of agar, wherein the pH value is 5.8-6.0;
3) carrying out rooting culture on the adventitious buds with the stem nodes in the step 2) on a rooting culture medium for 20-25 d to obtain ammopiptanthus mongolicus plants; the rooting culture medium takes an improved B5 culture medium as a basic culture medium, and comprises 1.0-1.5 mg/L of 2,4-D, 1000-1300 mg/L of peptone, 10-15 g/L of sucrose and 4.5g/L of agar, and the pH value is 5.8-6.0;
the improved B5 culture medium comprises macroelements, microelements, iron salt and organic components;
the macroelements include: 2000.00mg/L potassium nitrate, 100.00mg/L calcium chloride, 333.33mg/L magnesium sulfate, 100.00mg/L sodium dihydrogen phosphate and 89.30mg/L ammonium sulfate;
the trace elements include: 3.00mg/L boric acid, 2.00mg/L zinc sulfate, 0.75mg/L potassium iodide, 0.025mg/L copper sulfate, 0.25mg/L sodium molybdate, 10.00mg/L manganese sulfate and 0.025mg/L cobalt chloride;
the iron salts include: 28.00mg/L of iron ethylenediaminetetraacetic acid;
the organic component comprises: 100.00mg/L inositol, 1.00mg/L nicotinic acid, 1.00mg/L pyridoxine hydrochloride and 10.00mg/L thiamine hydrochloride.
2. The method as claimed in claim 1, wherein the step 1) of preparing the aseptic seedlings comprises the steps of:
a) soaking and disinfecting the ammopiptanthus mongolicus seeds to obtain disinfected ammopiptanthus mongolicus seeds;
b) inoculating the disinfected ammopiptanthus mongolicus seeds to an aseptic seedling culture medium to carry out aseptic seedling culture for 10-15 days to obtain aseptic seedlings; the aseptic seedling culture medium takes the improved B5 culture medium as a basic culture medium, and comprises 25g/L of sucrose and 4.5g/L of agar, and the pH value is 5.8-6.0.
3. The method according to claim 2, wherein the soaking time in the step a) is 8-12 h.
4. The method of claim 2, wherein the sterilizing of step a) is by: and soaking the soaked ammopiptanthus mongolicus seeds for 5-10 min by adopting a sodium hypochlorite solution with the mass concentration of 2%, soaking the ammopiptanthus mongolicus seeds for 30-40 s by adopting an ethanol solution with the mass concentration of 70% and soaking the ammopiptanthus mongolicus seeds for 3-5 min by adopting a mercuric chloride solution with the mass concentration of 0.08%.
5. The method as claimed in claim 2, wherein the sterile seedling culture of step b) comprises dark culture and illumination culture which are sequentially carried out, the dark culture time is 1-2 days, the illumination culture is carried out after the dark culture, and the conditions of the illumination culture are as follows: the temperature is 24 +/-2 ℃, the illumination intensity is 1500-1800 lx, and the illumination time is 12-15 h/d.
6. The method according to claim 1, wherein the callus induction culture in the step 1) comprises dark culture and light culture which are sequentially carried out, wherein the dark culture time is 3-5 days, and the light culture is carried out after the dark culture, and the conditions of the light culture are as follows: the temperature is 24 +/-2 ℃, the illumination intensity is 1800-2000 lx, and the illumination time is 12-15 h/d.
7. The method according to claim 1, wherein the length of the stem segment of step 1) is 0.4-0.6 cm.
8. The method according to claim 1, wherein the differentiation culture conditions of step 2) are: the temperature is 24 +/-2 ℃, the illumination intensity is 1800-2000 lx, and the illumination time is 12-15 h/d.
9. The method according to claim 1, wherein the rooting culture in the step 3) comprises dark culture and illumination culture which are sequentially performed, the dark culture time is 3-5 days, the illumination culture is performed after the dark culture, and the conditions of the illumination culture are as follows: the temperature is 25 ℃, the illumination intensity is 2000-2500 lx, and the illumination time is 14 h/d.
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| CN102612968B (en) * | 2012-04-09 | 2013-04-10 | 天津师范大学 | Ammopiptanthus introducing and cultivating method |
| CN107173231A (en) * | 2017-07-03 | 2017-09-19 | 甘肃省科学院生物研究所 | A kind of method of the Mongolian Ammopiptanthus mongolicus rapid propagation in vitro of rare or endangered species |
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| CN102612968B (en) * | 2012-04-09 | 2013-04-10 | 天津师范大学 | Ammopiptanthus introducing and cultivating method |
| CN107173231A (en) * | 2017-07-03 | 2017-09-19 | 甘肃省科学院生物研究所 | A kind of method of the Mongolian Ammopiptanthus mongolicus rapid propagation in vitro of rare or endangered species |
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