WO2007107803B1 - Process and modified media for preparing callus- and cell suspension cultures of hypericum perforatum l. - Google Patents
Process and modified media for preparing callus- and cell suspension cultures of hypericum perforatum l.Info
- Publication number
- WO2007107803B1 WO2007107803B1 PCT/HU2007/000026 HU2007000026W WO2007107803B1 WO 2007107803 B1 WO2007107803 B1 WO 2007107803B1 HU 2007000026 W HU2007000026 W HU 2007000026W WO 2007107803 B1 WO2007107803 B1 WO 2007107803B1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- medium
- edta
- modified
- iii
- amount
- Prior art date
Links
- 206010020649 Hyperkeratosis Diseases 0.000 title claims abstract 17
- 238000004114 suspension culture Methods 0.000 title claims abstract 9
- 244000141009 Hypericum perforatum Species 0.000 title claims abstract 8
- 235000017309 Hypericum perforatum Nutrition 0.000 title claims abstract 6
- 238000000034 method Methods 0.000 title claims 8
- 229930195732 phytohormone Natural products 0.000 claims abstract 15
- 238000000338 in vitro Methods 0.000 claims abstract 13
- 241000196324 Embryophyta Species 0.000 claims abstract 3
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims 20
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 claims 17
- 239000004327 boric acid Substances 0.000 claims 15
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 claims 11
- 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 11
- 229930006000 Sucrose Natural products 0.000 claims 11
- 239000005720 sucrose Substances 0.000 claims 11
- 229960003512 nicotinic acid Drugs 0.000 claims 10
- 235000001968 nicotinic acid Nutrition 0.000 claims 10
- 239000011664 nicotinic acid Substances 0.000 claims 10
- 230000000408 embryogenic effect Effects 0.000 claims 8
- 230000006698 induction Effects 0.000 claims 4
- 230000000977 initiatory effect Effects 0.000 claims 4
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 claims 4
- 230000008929 regeneration Effects 0.000 claims 4
- 238000011069 regeneration method Methods 0.000 claims 4
- 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 3
- 230000003247 decreasing effect Effects 0.000 claims 3
- 229960000367 inositol Drugs 0.000 claims 3
- 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 3
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims 3
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 claims 3
- 229910000368 zinc sulfate Inorganic materials 0.000 claims 3
- 239000011686 zinc sulphate Substances 0.000 claims 3
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 claims 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 claims 2
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 claims 2
- 229960001230 asparagine Drugs 0.000 claims 2
- 235000009582 asparagine Nutrition 0.000 claims 2
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims 2
- QXYJCZRRLLQGCR-UHFFFAOYSA-N molybdenum(IV) oxide Inorganic materials O=[Mo]=O QXYJCZRRLLQGCR-UHFFFAOYSA-N 0.000 claims 2
- 235000008160 pyridoxine Nutrition 0.000 claims 2
- 239000011677 pyridoxine Substances 0.000 claims 2
- 229940011671 vitamin b6 Drugs 0.000 claims 2
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 claims 1
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 claims 1
- 239000005556 hormone Substances 0.000 claims 1
- 229940088597 hormone Drugs 0.000 claims 1
- 238000011081 inoculation Methods 0.000 claims 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims 1
- 239000013589 supplement Substances 0.000 claims 1
- 235000019157 thiamine Nutrition 0.000 claims 1
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 claims 1
- 229960003495 thiamine Drugs 0.000 claims 1
- 239000011721 thiamine Substances 0.000 claims 1
- 229940088594 vitamin Drugs 0.000 claims 1
- 235000013343 vitamin Nutrition 0.000 claims 1
- 239000011782 vitamin Substances 0.000 claims 1
- 229930003231 vitamin Natural products 0.000 claims 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims 1
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 abstract 3
- 239000002028 Biomass Substances 0.000 abstract 1
- 239000004480 active ingredient Substances 0.000 abstract 1
- 229910052742 iron Inorganic materials 0.000 abstract 1
- 238000004519 manufacturing process Methods 0.000 abstract 1
- 239000000463 material Substances 0.000 abstract 1
- 230000002195 synergetic effect Effects 0.000 abstract 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0018—Culture media for cell or tissue culture
- C12N5/0025—Culture media for plant cell or plant tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Developmental Biology & Embryology (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Botany (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Environmental Sciences (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Plant Substances (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention relates to a process for preparing callus- and suspension cultures of Hypericum perforatum L. in modified media in order to produce increased amount of biomass and active ingredients. According to the invention the ratio of the macro- and microelements is altered in the known MS, N6 and B5 media, further the media are completed with EDTA-Na-Fe selecton iron and optionally with phytohormones. In such manner, continuously producing in vitro systems are obtained which produce the synergetic, biologically active components of H. perforatum L. simultaneously with the plant material. The invention also relates to the modified media.
Claims
1. In vitro cultivating system for suitable for preparing callus- and suspension cultures or in vitro regeneration of Hypericum perforatum L. characterized in that it contains
(1) modified MS-62 medium containing half amount of boric acid compared to the basic medium and completed with 35-45 mg/l EDTA-Na-Fe(III) and 0.1-10 mg/l phytohormones for induction of primary callus; (2) modified MS-62 medium containing half amount of boric acid and quarter or equal amount of macroelements compared to the basic medium and completed with 5-45 mg/l EDTA-Na-Fe(II!) and 0.1-10 mg/l phytohormones for induction of secondary callus;
(3) modified N6-78 medium containing half amount of macroelements compared to the basic medium and completed with Mo, Cu and Co in the form of B5- microeiements as well as with 35-45 mg/l EDTA-Na-Fe(III), 0.5-1.0 mg/l nicotinic acid and 20-30 g/l sucrose for initiation of embryogenic character;
(4) modified B5-68 medium containing an increased amount of CaCI2 compared to the basic medium and completed with 35-45 mg/l EDTA-Na-Fe(III), 0.5- 1.0 mg/l nicotinic acid, 1.0 mg/l 2,4-D and 12-25 g/l sucrose for increasing the differentiated level of embryogenic callus;
(5) modified MS-62 medium containing half amount of boric acid and quarter or equal amount of macroelements compared to the basic medium and completed with 5-45 mg/i EDTA-Na-Fe(III) and 0.1-10 mg/l phytohormones; or modified MS-62 medium containing quarter amount of macroelement, double amount of microelement, equal amount of boric acid compared to the basic medium and completed with 10 mg/l EDTA-Na-Fe(III) and 0.1-10 mg/l phytohormones; or modified MS-62 medium not containing Mo, Cu and Co but containing 0-1650 mg/l NH4NO3, boric acid decreased to quarter, MnSO4 decreased to the fifth, ZnSO4 decreased to the seventh, 1-5 fold amount of inositol and vitamins and it is completed with 250-500 mg/l asparagine or proline, 30-40 mg/l EDTA-Na-Fe(III) and 0-1.0 mg/l 2,4-D hormone, 30 g/l sucrose, 0-1.0 g/l caseine for suspension culture; and, if desired,
(6) modified hormon-free N6-78 medium completed with Mo, Cu and Co in the form of B5-microelements, 35-45 mg/i EDTA-Na-Fe(IH), 0.5-1.0 mg/l nicotinic acid and 20-30 g/l sucrose; or modified hormon-free B5-68 medium containing an increased amount of CaCI2 compared to the basic medium and completed with 35-45 mg/l EDTA-Na- Fe(III), 0.5-1.0 mg/l nicotinic acid and 15-25 g/l sucrose for in vitro regeneration of Hypericum perforatum L from an embryogenic callus or suspension culture.
2. In vitro cultivating system according to claim 1 wherein (1) modified MS-62 medium contains compared to the basic medium
(a) half amount of boric acid, 40 mg/l EDTA-Na-Fe(III), 1 mg/l IAA, 0.2 mg/l NAA and 0.1 mg/l KIN phytohormones (Medium 1); or
(b) half amount of boric acid, 40 mg/l EDTA-Na-Fe(III), 0.1-10 mg/l IAA, 0.1-0.4 mg/l NAA, 0.1-2.0 mg/l 2,4-D, 0.5 mg/l BAP or 0.2-0.5 mg/l KIN phytohormones (Medium 2); or
(c) half amount of boric acid, 40 mg/l EDTA-Na-Fe(III), 0.8 mg/l NAA and/or 1 mg/l 2,4-D and 0.5 mg/l KIN phytohormones (Medium 3).
3. In vitro cultivating system according to claim 1 wherein (2) modified MS-62 medium contains compared to the basic medium
(a) half amount of boric acid, 40 mg/l EDTA-Na-Fe(III), 1 mg/l IAA1 0.2 mg/l NAA and 0.1 mg/l KIN phytohormones (Medium 1); or
(b) half amount of boric acid, 40 mg/l EDTA-Na-Fe(III), 0.1-10 mg/l IAA, 0.1-0.4 mg/l NAA, 0.1-2.0 mg/l 2,4-D, 0.5 mg/l BAP or 0.2-0.5 mg/l KIN phytohormones (Medium 2); or
(c) half amount of boric acid, 40 mg/l EDTA-Na-Fe(III)1 0.8 mg/l NAA and/or 1 mg/l 2,4-D and 0.5 mg/l KIN phytohormones (Medium 3); or
(d) half amount of boric acid, 40 mg/l EDTA-Na-Fe(IiI)1 1 mg/l NAA, 1 mg/l 2,4-D and 0.1 mg/l KIN phytohormones (Medium 4); or 29
(e) quarter amount of macroelement, half amount of boric acid, 10 mg/l EDTA-Na-FE(III), 0.8 mg/l IAA and 1.6 mg/l BAP phytohormones (Medium 5); or
(f) quarter amount macroelement, half amount of boric acid, 10 mg/l EDTA-Na-Fe(IfI)1 0.8 mg/l 2,4-D and 1.6 mg/l BAP phytohormones (Medium 6).
5
4. In vitro cultivating system according to claim 1 wherein (3) modified N6-78 medium contains half amount of macroelements, 0.25 mg/l Na2MoO2xH2θ, 0.025 mg/l CuSO4x5H2O, 0.025 mg/l CoCI2x6H20, 40 mg/l EDTA-Na-Fe(III)1 0.5 mg/l nicotinic acid and 30 g/l sucrose (Medium 10).
10
5. In vitro cultivating system according to claim 1 wherein (4) modified B5-68 medium contains 440 mg/l CaCI2x2H2O, 40 mg/l EDTA-Na-Fe(III), 1 mg/l nicotinic acid, 1 mg/l 2,4-D and 20g/l sucrose (Medium11 ).
"J 5 6. In vitro cultivating system according to claim 1 wherein (5) modified MS-62 medium is
(a) a medium according to claim 3 (Medium 1-6) or
(b) a medium containing quarter amount of macroelement, double amount of microelement, equal amount of boric acid and it is completed with 10 mg/l EDTA-
20 Na-Fe(III), 0.5-0.5 mg/l (AA and K!N phytohormones (Medium 7) or
(C) a medium containing 1.6 mg/l H3BO3, 4.4 mg/l MnSO4XH2O, 1.5 mg/l ZnSO4x7H2O, 40 mg/l EDTA-Na-Fe(III), 100 mg/l inositol, 1 mg/l nicotinic acid, 1 mg/l pyridoxine.HCI, 10 mg/l thiamine.HCI, 0.0-0.5-1.0 mg/l 2,4-D, 500 mg/l asparagine, 30 g/l sucrose and 0-1.0 g/l caseine (Medium 8) or
25 (d) a medium containing 1650 mg/l NH4NO3, 1.
6 mg/l H3BO3, 4.4 mg/l
MnSO4XH2O, 1.5 mg/l ZnSO4x7H2O, 40 mg/l EDTA-Na-Fe(III), 500 mg/l inositol, 1 mg/l nicotinic acid, 1 mg/l pyridoxine.HCI, 10 mg/l thyamine.HCI, 1.0 mg/l 2,4-D, 250 mg/l proline and 30 g/l sucrose (Medium 9).
30 7. In vitro cultivating system according to claim 1 wherein (6) modified hormon- free N6-78 medium contains 0.25 mg/l Na2MoO2XH2O, 0.025 mg/l CuSO4x5H2O and 0.025 mg/l CoCI2x6H2O, 40 mg/l EDTA-Na-Fe(III), 0.5 mg/l nicotinic acid and 30 g/l sucrose as supplements (Medium 10). 30
8. In vitro cultivating system according to claim 1 wherein (6) modified horrnon- free B5-68 medium contains 440 mg/l CaCI2x2H2O, 40 mg/l EDTA-Na-Fe(III), 1 mg/l nicotinic acid and 20 g/l sucrose (Medium11).
9. A method for preparing callus- and cell suspension cultures or in vitro regeneration of Hypericum perforatum L characterized in that primary callus is induced from the expiants of cotyledon, young foliage leaf, root or leafy shoots of the in vitro regenerated plant of H. perforatum L. or from leaves developed in the upper third of the flowery shooting of the intact plant by initiation in a medium according to claim 1 (1) at pH 5.8 and 24.5-25.5°C in dark; then secondary callus is prepared by inoculation a primary callus into a medium according to claim 1 (2) (pH 5.0-5.8), at 23-27°C in light and dark; the secunder callus is selected and inoculated further into an other medium according to claim 1 (1) or a medium according to claim 1 (3) for initiation of embryogenic character or into a medium according to claim 1 (4) for increasing the differentiated level of embryogenic callus; after the selection a suspension culture is prepared in a medium according to claim 1 (5), which is cultivated at pH 5.0-5.8 and 23.5-250C in dark and under a photoperiod of 16 h light/8 h dark; and, if desired, shoots or rooty shoots of Hypericum perforatum L. are regenerated form thus- obtained embryogenic callus or suspension culture in light in modified hormon-free N6-78 or modified hormon-free B5-68 medium according to claim 1 (6).
10, The method according to claim 9 characterized in that a modified MS-62 medium according to claim 2 is used for induction of primary callus.
11. The method according to claim 9 or 10 characterized in that a modified MS-62 medium according to claim 3 is used for induction of secondary callus.
12. The method according to any claims of 9-11 characterized in that a modified N6-78 medium according to claim 4 is used for initiation of embryogenic character. 31
13. The method according to any claims of 9-12 characterized in that a modified B5-68 medium according to claim 5 is used for increasing the differentiated level of embryogenic callus.
5 14. The method according to any claims of 9-13 characterized in that a modified MS-62 medium according to claim 6 is used for preparing suspension culture.
15. The method according to any claims of 9-14 characterized in that a "• Q modified N6-78 medium according to claim 7 or a modified B5-68 medium according to claim 8 is used for the regeneration.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| HUP0600245 | 2006-03-23 | ||
| HU0600245A HU227572B1 (en) | 2006-03-23 | 2006-03-23 | Process and modified media for preparing callus- and cell suspension cultures of hypericum perforatum l. |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| WO2007107803A2 WO2007107803A2 (en) | 2007-09-27 |
| WO2007107803A3 WO2007107803A3 (en) | 2007-11-22 |
| WO2007107803B1 true WO2007107803B1 (en) | 2008-01-24 |
Family
ID=89986663
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/HU2007/000026 WO2007107803A2 (en) | 2006-03-23 | 2007-03-21 | Process and modified media for preparing callus- and cell suspension cultures of hypericum perforatum l. |
Country Status (2)
| Country | Link |
|---|---|
| HU (1) | HU227572B1 (en) |
| WO (1) | WO2007107803A2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101180950B (en) * | 2007-12-26 | 2010-12-08 | 浙江森禾种业股份有限公司 | Tissue cultivation rapid breeding method of spring dendrobium stem |
| CN109496841B (en) * | 2018-11-13 | 2022-03-22 | 中国林业科学研究院林业研究所 | Tissue culture and rapid propagation method for golden silk plum |
| CN114303944B (en) * | 2020-09-30 | 2023-08-11 | 伽蓝（集团）股份有限公司 | Callus culture medium and extract of rosa tenuifolia, preparation method and application |
| CN114667933A (en) * | 2022-04-28 | 2022-06-28 | 蒙草生态环境(集团)股份有限公司 | Culture medium for tissue culture of sillima-type chenopodium camellinum |
| CN116034876A (en) * | 2023-01-10 | 2023-05-02 | 重庆市铜梁区果之王园艺研究院 | GF677 peach stock culture medium and cultivation method thereof |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS603826B2 (en) * | 1980-10-06 | 1985-01-30 | クロレラ工業株式会社 | Plant tissue or cell culture methods |
| WO2000022138A1 (en) * | 1998-10-14 | 2000-04-20 | Agriculture And Agri-Food Canada | Genetic factor of s. meliloti usda 1170 containing nodulation efficiency factor |
| DE60006367T2 (en) * | 1999-03-25 | 2004-08-12 | University Of Guelph, Guelph | MICROPROPAGATION AND PRODUCTION OF PHYTOPHARMACEUTICAL PLANTS |
-
2006
- 2006-03-23 HU HU0600245A patent/HU227572B1/en unknown
-
2007
- 2007-03-21 WO PCT/HU2007/000026 patent/WO2007107803A2/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| HU0600245D0 (en) | 2006-05-29 |
| WO2007107803A2 (en) | 2007-09-27 |
| HUP0600245A2 (en) | 2008-01-28 |
| WO2007107803A3 (en) | 2007-11-22 |
| HU227572B1 (en) | 2011-08-29 |
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