WO2003041492A1 - Media compositions for faster growth of polygonatum cirrhifolium royle - Google Patents
Media compositions for faster growth of polygonatum cirrhifolium royle Download PDFInfo
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- WO2003041492A1 WO2003041492A1 PCT/IN2001/000200 IN0100200W WO03041492A1 WO 2003041492 A1 WO2003041492 A1 WO 2003041492A1 IN 0100200 W IN0100200 W IN 0100200W WO 03041492 A1 WO03041492 A1 WO 03041492A1
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- ranging
- medium
- compositions
- germination
- epicotyl
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- 239000000203 mixture Substances 0.000 title claims abstract description 99
- 241001448326 Polygonatum cirrhifolium Species 0.000 title claims abstract description 45
- 230000012010 growth Effects 0.000 title claims description 9
- 230000035784 germination Effects 0.000 claims abstract description 83
- 241000196324 Embryophyta Species 0.000 claims abstract description 82
- 239000001963 growth medium Substances 0.000 claims abstract description 74
- 238000000338 in vitro Methods 0.000 claims abstract description 63
- 238000000034 method Methods 0.000 claims abstract description 63
- 230000005059 dormancy Effects 0.000 claims abstract description 48
- 230000006698 induction Effects 0.000 claims abstract description 27
- 230000001360 synchronised effect Effects 0.000 claims abstract description 12
- 239000003375 plant hormone Substances 0.000 claims abstract description 10
- 239000000654 additive Substances 0.000 claims abstract description 6
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 claims description 67
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 66
- IXORZMNAPKEEDV-OBDJNFEBSA-N gibberellin A3 Chemical compound C([C@@]1(O)C(=C)C[C@@]2(C1)[C@H]1C(O)=O)C[C@H]2[C@]2(C=C[C@@H]3O)[C@H]1[C@]3(C)C(=O)O2 IXORZMNAPKEEDV-OBDJNFEBSA-N 0.000 claims description 59
- 239000002609 medium Substances 0.000 claims description 53
- 239000005980 Gibberellic acid Substances 0.000 claims description 42
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 32
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 claims description 32
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 30
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 28
- 230000000977 initiatory effect Effects 0.000 claims description 25
- 239000013028 medium composition Substances 0.000 claims description 24
- LXNHXLLTXMVWPM-UHFFFAOYSA-N pyridoxine Chemical compound CC1=NC=C(CO)C(CO)=C1O LXNHXLLTXMVWPM-UHFFFAOYSA-N 0.000 claims description 18
- 230000004069 differentiation Effects 0.000 claims description 17
- 239000004471 Glycine Substances 0.000 claims description 16
- 229930006000 Sucrose Natural products 0.000 claims description 16
- 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 16
- 235000001968 nicotinic acid Nutrition 0.000 claims description 16
- 229960003512 nicotinic acid Drugs 0.000 claims description 16
- 239000011664 nicotinic acid Substances 0.000 claims description 16
- 239000005720 sucrose Substances 0.000 claims description 16
- 229960003495 thiamine Drugs 0.000 claims description 16
- 229920001817 Agar Polymers 0.000 claims description 15
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 15
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 15
- 229910003887 H3 BO3 Inorganic materials 0.000 claims description 15
- 239000007836 KH2PO4 Substances 0.000 claims description 15
- 229910004729 Na2 MoO4 Inorganic materials 0.000 claims description 15
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 239000008272 agar Substances 0.000 claims description 15
- 239000001110 calcium chloride Substances 0.000 claims description 15
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 15
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 claims description 15
- 229910000366 copper(II) sulfate Inorganic materials 0.000 claims description 15
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 15
- 229910000402 monopotassium phosphate Inorganic materials 0.000 claims description 15
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 claims description 15
- 229940011671 vitamin b6 Drugs 0.000 claims description 15
- 229910000368 zinc sulfate Inorganic materials 0.000 claims description 15
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 claims description 15
- 239000011686 zinc sulphate Substances 0.000 claims description 15
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 14
- 229910000357 manganese(II) sulfate Inorganic materials 0.000 claims description 14
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 14
- 238000009472 formulation Methods 0.000 claims description 13
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 claims description 12
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 claims description 12
- 239000011764 pyridoxine hydrochloride Substances 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 12
- 239000002253 acid Substances 0.000 claims description 11
- 229960000367 inositol Drugs 0.000 claims description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 239000012153 distilled water Substances 0.000 claims description 7
- 229920001213 Polysorbate 20 Polymers 0.000 claims description 6
- PYRZPBDTPRQYKG-UHFFFAOYSA-N cyclopentene-1-carboxylic acid Chemical compound OC(=O)C1=CCCC1 PYRZPBDTPRQYKG-UHFFFAOYSA-N 0.000 claims description 6
- 230000007721 medicinal effect Effects 0.000 claims description 6
- 229960002523 mercuric chloride Drugs 0.000 claims description 6
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 claims description 6
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 claims description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims description 6
- 239000008399 tap water Substances 0.000 claims description 6
- 235000020679 tap water Nutrition 0.000 claims description 6
- 239000011148 porous material Substances 0.000 claims description 5
- 239000007787 solid Substances 0.000 claims description 5
- 230000004044 response Effects 0.000 claims description 4
- 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 4
- KYMBYSLLVAOCFI-UHFFFAOYSA-N thiamine Chemical compound CC1=C(CCO)SCN1CC1=CN=C(C)N=C1N KYMBYSLLVAOCFI-UHFFFAOYSA-N 0.000 description 12
- 238000011282 treatment Methods 0.000 description 12
- 230000002195 synergetic effect Effects 0.000 description 11
- 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 description 10
- 241000894007 species Species 0.000 description 10
- 230000000694 effects Effects 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 7
- 235000021028 berry Nutrition 0.000 description 6
- 238000011534 incubation Methods 0.000 description 6
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 5
- JZRWCGZRTZMZEH-UHFFFAOYSA-N Thiamine Natural products CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N JZRWCGZRTZMZEH-UHFFFAOYSA-N 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 235000019157 thiamine Nutrition 0.000 description 4
- 239000011721 thiamine Substances 0.000 description 4
- 238000004891 communication Methods 0.000 description 3
- 230000018044 dehydration Effects 0.000 description 3
- 238000006297 dehydration reaction Methods 0.000 description 3
- IXORZMNAPKEEDV-UHFFFAOYSA-N gibberellic acid GA3 Natural products OC(=O)C1C2(C3)CC(=C)C3(O)CCC2C2(C=CC3O)C1C3(C)C(=O)O2 IXORZMNAPKEEDV-UHFFFAOYSA-N 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 235000008160 pyridoxine Nutrition 0.000 description 3
- 239000011677 pyridoxine Substances 0.000 description 3
- 238000013517 stratification Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 229930191978 Gibberellin Natural products 0.000 description 2
- 241000756042 Polygonatum Species 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 210000002257 embryonic structure Anatomy 0.000 description 2
- 238000011066 ex-situ storage Methods 0.000 description 2
- 239000003448 gibberellin Substances 0.000 description 2
- 238000011065 in-situ storage Methods 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 239000006870 ms-medium Substances 0.000 description 2
- 230000000737 periodic effect Effects 0.000 description 2
- 230000014284 seed dormancy process Effects 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 230000001256 tonic effect Effects 0.000 description 2
- 241000707825 Argyrosomus regius Species 0.000 description 1
- 241000660443 Encyclops Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 206010020649 Hyperkeratosis Diseases 0.000 description 1
- 241000234280 Liliaceae Species 0.000 description 1
- 241001448324 Polygonatum oppositifolium Species 0.000 description 1
- 241000037832 Polygonatum verticillatum Species 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 239000003048 aphrodisiac agent Substances 0.000 description 1
- 230000002509 aphrodisiac effect Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007640 basal medium Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 150000002500 ions Chemical group 0.000 description 1
- 235000021073 macronutrients Nutrition 0.000 description 1
- 239000011785 micronutrient Substances 0.000 description 1
- 235000013369 micronutrients Nutrition 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000005648 plant growth regulator Substances 0.000 description 1
- 230000037039 plant physiology Effects 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 210000003429 pore cell Anatomy 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000007226 seed germination Effects 0.000 description 1
- 229930002600 steroidal saponin Natural products 0.000 description 1
- 239000011885 synergistic combination Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 235000013311 vegetables Nutrition 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
- A01H4/002—Culture media for tissue culture
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
Definitions
- the present invention relates to novel culture media compositions for extraordinarily faster in vitro induction of germination and release of epicotyl dormancy in Polygonatum Cirrhifolium Royle, an endangered medicinal plant species.
- the Said compositions comprise Murashige and Skoog (MS), a basal culture medium, varied concentrations of plant hormones, and other additives.
- the present invention also relates to a method for faster in vitro propagation of Polygonatum Cirrhifolium Royle.
- Polygonatum cirrhifolium Royle (Liliaceae) is an important medicinal plant of temperate Himalayas (Wealth of India Raw materials, 1976, 9, 365, CSIR, New Delhi). Its rhizomes constitute an important ingredient of Astavarga a group of eight drugs used extensively in Indian system of medicine mainly as a tonic and aphrodisiac (Misra, B. and Naishya, R. 1972, Bhavaprakasha Nighantu, pt.l, Chowkhamba Sanskrit Santhan, Sloka 120-122). These attributes are ascribed to the presence of steroidal saponins and polysaccharides in the rhizomatous root-stock of the plant. The plant is also useful in the preparation of cosmetics, skin tonic and as a vegetable (Singh, U. et al., 1983, In: Economic plants of India, IARI, new Delhi, 180).
- epicotyl dormancy is one of the seven types of morpho-physiological dormancy. Morphophysiologically dormant seeds have rudimentary embryos that require cold or some combination of warm or cold stratification to complete embryo growth (Baskin, J.M. and Baskin, C.C. 1985, Amer. J. Bot., 72(2), 185-290).
- gibberellins are known to activate hydrolytic enzymes especially in dark which results in the increase in osmotic content of the seed, increasing its water potential and giving the hypocotyl power to break through the seed coat.
- gibberellins have been found to be potent substitute for the cold stratifications, resulting in the maturity of embryo.
- Prior to present invention there is no particular embodiment of invention or report in which germination has been induced in P. cirrhifolium by in vitro or other means. The germination is protracted, meagre and asynchronous and takes 3 to 5 years for plants to grow to full size when raised from seeds (Dhar & Lattoo, under communication).
- Literature is replete with references where in vitro approaches have been successfully tried to release various forms of physiological dormancy employing various growth adjuvants especially GA 3 , BAP and NAA.
- Plant Cell Rep. 17(12), 913-16; Choi, Y.E. et al, 1999, Plant Cell Rep., 18(6), 493- 99; Chu,P.P. 1994, Hort Science, 29(6), 695-97; Buchheim J.A.T. 1994 Plant Cell Tissue Organan Culture, 36(1), 35-43; Gingas,V.M. and Lineberger,R.B. 1989. Plant Cell Tissue Organ Culture, 17(3), 191-203, Thomas,J.A. and Meyer,M.M. 1987. In vitro 23, 3pt 2, 70 A).
- the main object of the present invention is novel culture composition to induce faster germination in Polygonatum Cirrhifolium Royle, an endangered plant species.
- Another object of the present invention is novel culture composition to induce synchronized release of epicotyl, coleoptile, and radicle in said plant species.
- Yet another object of the present invention is novel culture composition to break the epicotyl dormancy in the said plant species.
- Still another object of the present invention is novel culture composition to achieve uniform and.consistent in vitro germination of the said plant species. Still another object of the present invention is a method to induce faster germination in Polygonatum Cirrhifolium Royle, an endangered plant species.
- Still another object of the present invention is a method to induce synchronized release of epicotyl, coleoptile, and radicle in said plant species.
- Still another object of the present invention is a method to break the epicotyl dormancy in the said plant species.
- Still another object of the present invention is a method to achieve uniform and consistent in vitro germination of the said plant species.
- the present invention relates to the development of culture media compositions, said compositions comprising Murashige and Skoog (MS), a basal culture medium, varied concentrations of plant hormones, and other additives.
- the said compositions lead to extraordinarily fast and synchronized in vitro induction of germination and release of epicotyl dormancy in Polygonatum Cirrhifolium Royle, an endangered medicinal plant species.
- the said germination is uniform and consistent. Synchronized release of epicotyl, coleoptile, and radicle is observed in said plant with above-mentioned compositions.
- the present invention relates to the Culture media compositions useful for initiating extraordinarily fast and synchronized in vitro induction of germination and release of epicotyl dormancy in Polygonatum Cirrhifolium Royle, an endangered medicinal plant species, said composition comprising Murashige and Skoog (MS) basal culture medium, varied concentrations of plant hormones, and other additives, said compositions are; a.
- MS Murashige and Skoog
- first medium composition useful for initiating fast germination of said plant species with percentage germination ranging between 65 to 98%, and duration period to achieve the same ranging between 7 to.53 days, and also useful for initiating fast release of epicotyl, coleoptile and radicle in percentage seeds ranging between 78 to 100, with duration period for synchronization of the same ranging between 11 to 18 days, said composition consisting of MS basal culture medium, Gibberellic acid (GA 3 ) ranging between 10 to 100 mg/1, b.
- G 3 Gibberellic acid
- second medium composition useful for initiating fast release of first foliage leaf from epicotyl dormant explants, with percentage secondary explants producing leaves ranging between 65 to 86%, mean number of leaves per secondary explant ranging between 2.7 to 4, and mean leaf length ranging between 3 to 6 cms, said composition consisting of MS basal culture medium, 6- benzyl-aminopurine (BAP) ranging between 3 to 6 mg/1, and Naphthalene acetic acid (NAA) ranging between 0.5 to l.Omg/1, and c.
- BAP 6- benzyl-aminopurine
- NAA Naphthalene acetic acid
- third medium composition useful for initiating fast release of axillary buds from epicotyl dormant explant, with secondary explants producing axillary buds ranging between 70 to 100%, mean number of axillary buds per secondary explant ranging between 6 to 12, and axillary buds producing leaves ranging between 45 to 98%, said composition consisting of MS basal culture medium, 6- benzyl-aminopurine (BAP) about 2.0mg/l, Naphthalene acetic acid (NAA) about 1.0mg/l, and Gibberellic acid (GA3) ranging between 5 to 20 mg/1.
- BAP 6- benzyl-aminopurine
- NAA Naphthalene acetic acid
- GA3 Gibberellic acid
- first medium, second medium, and third medium are used in the same sequence for initiating extraordinarily fast and synchronized in vitro induction of germination and release of epicotyl dormancy in plant Polygonatum Cirrhifolium Royle.
- plant hormones are selected a from group comprising Gibbrellic acid (GA 3 ), alpha- naphthalene acetic acid (NAA), and 6-benzyl- aminopurine (BAP).
- Murashige and Skoog's (MS) basal culture medium is preferably consisting of 2.2g l NH 4 NO 3 , 2.0g/l KNO 3 , Q.44g/ 1 CaCl 2 . 2H 2 O, O.37g/l MgSO 4 . 7H 2 0, 0.17g/l KH 2 PO 4 , 37.25 mg/1 Na 2 EDTA, 27.8 mg/1 FeSO 4 .7H 2 0, 0.83 mg/lKI, 6.2 mg/1 H 3 BO 3 , 22.3 mg/1 MnSO 4 . 4H 2 0, 8.6 mg/1 ZnSO 4 . 7H 2 0, 0.25mg/l Na 2 MoO 4 .
- first medium composition is used for fast germination, with percentage induction of germination of about 98% is achieved in time duration ranging between 7 to 23 days, said composition consisting of MS basal culture medium and about 50mg/l Gibbrellic acid (GA 3 ).
- first medium composition is used for breaking epicotyl dormancy, with about 100% release of epicotyl, coleoptile and radicle, and to achieve the same in time duration ranging between 11 to 14 days, said composition consisting of MS basal culture medium and Gibbrelic acid (GA 3 ) ranging between 50 to
- second medium composition is used for initiating fast release of first foliage leaf from explants, with about 86% secondary explants producing leaves, about 3.4 mean number of leaves per secondary explant, and about 6 cms mean leaf length, said composition consisting of MS basal culture medium, 6- benzyl-aminopurine (BAP) ranging between 3 to 6 mg/1, and Naphthalene acetic acid
- NAA N-(NAA) about l.Omg/1.
- third medium composition used for initiating fast release of axillary buds from explants, with about 100%, secondary explants producing axillary buds, number of axillary buds per secondary explant ranging between 9 to 12, and axillary buds producing leaves ranging between 77 to 98%, said composition consisting of MS basal culture medium, 6- benzyl-aminopurine (BAP) about 2.0mg/l,
- Naphthalene acetic acid about 1.0mg/l
- GA3 Gibberellic acid
- compositions are used for reliable and uniform germination.
- compositions are used for initiating faster germination, with only 81 days in vitro, to achieve the same.
- said compositions is the first step towards release of the epicotyl dormancy.
- said compositions can be used for all possible genotypes of said plant species to be grown in vitro.
- compositions can be used growing said plant species in vitro, in all geographical regions and all seasons.
- compositions are used for uniform and high germination rates.
- said fast in vitro multiplication of the said medicinal plant species is used for conservation of this endangered plant species.
- said fast in vitro multiplication of the said plant species for wider utilization of its medicinal properties.
- washing the seeds thoroughly for 2 hours under tap water with 1-2 drops of Tween-20 In still another embodiment of the present invention, washing the seeds thoroughly for 2 hours under tap water with 1-2 drops of Tween-20.
- RH relative humidity
- a method to for extraordinarily faster release of epicotyl dormancy in Polygonatum Cirrhifolium Royle In still another embodiment of the present invention, collecting plants bearing ripe purplish berries from the wild growing plants of said species.
- RH relative humidity
- a method to for extraordinarily faster release of epicotyl dormancy from differentiated de novo axillary bud and release of foliage leaves in Polygonatum Cirrhifolium Royle using third media composition consisting of MS basal culture medium, 6- benzyl-aminopurine (BAP) about 2.0mg/l, Naphthalene acetic acid (NAA) about 1.0mg/l, and Gibberellic acid (GA3) ranging between 5 to 20 mg/1.
- BAP 6- benzyl-aminopurine
- NAA Naphthalene acetic acid
- GA3 Gibberellic acid
- Naphthalene acetic acid about 1.0mg/l
- GA3 Gibberellic acid
- invention in still another embodiment, compiling the final data after 16 weeks of culture on the basis of periodical observations.
- invention relates to a synergistic formulation of a culture medium for the induction of germination and release of epicotyl, coleoptile and radicle in the dormant seeds of Polygonatum cirrhifolium Royle by placing seeds on a culture medium containing 2.2g/l NH 4 NO 3 , 2.0g/l KNO 3 , O.44g/ 1 CaCl 2 . 2H 2 O, O.37g/l MgSO 4 .
- seeds with emerged hypocotyl on transfer to above basal culture medium containing altered levels of GA 3 (5-100 mg/1) and incubated at 20°C ( ⁇ 2°C) under 8-16 hr photoperiod (2000-3500 lux) result in the release of epicotyl, coleoptile and radicle within 81 days of incubation of seeds.
- invention results in more reliable and uniform germination and also hastens germination from upto 8-10 months in nature to only 81 days in vitro.
- the cultures on incubation at 20-24°C under 10-16hr photoperiod (2000-3500 lux) at 50-60% relative humidity (RH) results in the release of first foliage leaves from the emergent coleoptile in set I and set 2 and de novo axillary bud differentiation and their sprouting in set 2.
- invention constitutes the first and fundamental step to break the epicotyl dormancy in this particular species.
- present invention was undertaken to develop a method / process by which epicotyl dormancy in Polygonatum cirrhifolium could be released under in vitro conditions within shortest possible time with no intervening period of dormancy.
- invention results in breaking of epicotyl dormancy which under natural conditions takes 3-5 years to occur when raised from the seeds.
- present invention was undertaken to develop a method/process by which germination could be induced with emergent epicotyl, coleoptile and radicle under in vitro conditions with uniform and higher germination rates than what is manifest under natural conditions.
- Fig.l shows induction of germination marked by the emergence of hypocotyl.
- Fig.2 shows differentiated epicotyl with emerging radicle (ra) and coleoptile (col).
- Fig. 3 shows well differentiated coleoptile (col) with emergent radicle (ra) and coleoptile (col).
- Fig. 4 shows well differentiated coleoptile (col) with emergent radicle (ra) and coleoptile (col).
- Fig 5 shows release of rosette of leaves from the coleoptile
- Fig 6. shows release of first foliage leaves and de novo differentiation of axillary buds from the rhizomatous base.
- Fig 7 shows sprouting of axillary buds.
- Fig 8. shows excised axillary bud with emergent leaves.
- Plants bearing ripe purplish berries were collected from the wild growing plants during October, 2000.
- seeds were removed from the berries after 15 days of dehydration at room temperature (29°C ⁇ 2°C); throughly washed under tap water with 1-2 drops of Tween-20 for 2 hours; rinsed thrice in distilled water.
- it was followed by surface sterlization with 0.1% mercuric chloride (HgCl 2 ) for 3 minutes.
- this medium was supplemented with 250 mg/1 myo-inosital, 0.5mgl/l nicotinic acid, 0.5mg/l pyridoxine HC1, 3mg/l thiamine HC1, 2mg/l glycine, 30g/l sucrose, 7.5 g/1 agar and 2, 10, 25, and 50mg/l gibberellic acid.
- operations from surface sterilization to inoculation of seeds were carried under aseptic conditions in Laminar Air Flow.
- parafilm-sealed petri dishes were incubated in two separate growth chambers at a temperature of 30°C (+ 2°C; dark) and diurnal temperature of 30/20°C ( ⁇ 2°C; dark) at 50-60% relaltive humidity (RH).
- RH relaltive humidity
- seeds with emerged hypocotyl were transferred under aseptic conditions to the above defined basal culture meidium containing altered levels of gibberellic acid 5, 25, 50 and 100 mg/1.
- cultures were incubated at 20°C ( ⁇ 2°C) under 8-16 hr photoperiod with light intensity of 2000 lux provided by cool , white fluorescent tubes of 40 watts. Cultures were maintained at 50-60% RH.
- observations regarding germination synchronization as indicated by differentiated epicotyl with emergent coleoptile and radicle were recorded on daily basis.
- pH of medium was adjusted to 5.8 with IN NaOH or IN HC1 prior to adding agar.
- medium was sterlized for 20 minutes at 121°C (15 lb. psi pressure).
- GA 3 was incorporated after filter sterlization (0.22 ⁇ m pore size millipore filter) to cooled (40-45°C) autoclaved medium.
- results in table 1 and . 2 show the induction of germination as indicated by the emergence of hypocotyl at temperature of 30°C ( ⁇ 2°C ; dark; RH 50-60%) and diurnal temperature of 30/20°C (+ 2°C ; dark; RH 50-
- the consequent step of synchronization of germination is presented in table 3 in which epicotyl with emergent coleoptile and radicle is manifest under 8-16 hr photoperiod (light intensity 2000-3500 lux) at an incubation temperature of20°C (RH 50-60%).
- invention is in vitro release of hypocotyl and synchronous release of epicotyl, coleoptile and radicle.
- invention not only results in more uniform, reliable germination, but also hastens germination from upto 8-10 months in nature to only 81 days in vitro.
- invention is not limited to any particular variety or genotype but can be applied to genetically diverse and composite seeds of P. cirrhifolium.
- this specific embodiment of invention is also not restricted to any particular geogrpahic region or any season and can be utilized and practised anywhere in the World.
- invention can be used as a reliable and effective technique for in-situ and ex-situ conservation of this species as it is listed among the threatened category of plant.
- a synergistic formulation used for induction of germination and release of epicotyl, coleoptile and radicle in the dormant seeds of Polygonatum cirrhifolium at temperature range of 28-32°C and diurnal temperature range of 28-32/18-22°C under dark for induction of hypocotyl.
- a synergistic formulation used for induction of germination and release of epicotyl, coleoptile and radicle in the dormant seeds of Polygonatum cirrhifolium at temperature range of 18-22°C under 8-16 hr photoperiod (light intensity 2000-3500 lux) for induction of epicotyl, coleoptile and radicle.
- a synergistic formulation used for induction of germination and release of epicotyl, coleoptile and radicle in the dormant seeds of Polygonatum cirrhifolium at 50- 60% relative humidity.
- invention relates to synergistic formulations of culture media for the in vitro release of epicotyl dormancy in Polygonatum cirrhifolium Royle indicated by the release of rosette of leaves and differentiation of de novo axillary buds and their sprouting by placing in vitro germinated seeds with epicotyl, coleoptile and radicle on two sets of Murashige and Skoog's, (MS) basal culture medium containing 1.65g/l NH 4 NO 3; 1.9g/l KNO 3 , O.44g/ 1 CaCl 2 . 2H 2 O, O.37g/l MgSO 4 .
- MS Murashige and Skoog's,
- present invention was undertaken to develop a method / process by which epicotyl dormancy in Polygonatum cirrhifolium could be released under in vitro conditions within shortest possible time with no intervening period of dormancy.
- figure 1 explains the release of rosette of leaves from the coleoptile.
- Figure 2 shows the release of first foliage leaves and de novo differentiation of axillary buds from the rhizomatous base.
- Figure 3 shows the sprouting of axillary buds and figure 4 depicts the excised axillary bud with emergent leaves.
- the cultures on incubation at 20-24°C under 10-16hr photoperiod (2000-3500 lux) at 50-60% relative humidity (RH) results in the release of first foliage leaves from the emergent coleoptile in set I and set 2 and de novo axillary bud differentiation and their sprouting in set 2.
- secondary explants consisting of epicotyl with emergent coleoptile and radicle obtained from the in vitro germinated seeds (co-pending application) were transferred aseptically in a parallel set of experiments on semi-solid culture medium viz. set 1: Ms basal culture medium containing 1.65g/l NH 4 NO 3; 1.9g/l KNO 3 , O.44g/ 1 CaCl 2 . 2H 2 O, O.37g/l MgSO 4 .
- cultures were maintained at 50-60% RH. Subculturing was done after every four weeks in both sets of experiments on the same media formulations. Observations were made periodically but final data was compiled after 16 weeks of culture.
- pH of the medium was adjusted to 5.8 before adding agar.
- Medium was sterilized at 121°C (15 lb. psi pressure) for 20 minutes.
- GA 3 was incorporated after filter sterilization (0.22 ⁇ m pore cell, milipore filter to cooled (40-45°C) autoclaved medium. BAP and NAA were added prior to autoclaving. Medium was dispensed in culture tubes (25x150 mm, Borosil) and conical vials (100 ml, Borosil) as
- results in table 4 and 5 show the influence of various concentrations and combinations of BAP, NAA and GA 3 on the in vitro release of epicotyl dormancy indicated by the release of rosette of leaves and induction of de novo axillary bud differentiation and their sprouting at 20-24°C under 10-
- result of the present invention is in vitro release of epicotyl dormancy in P. cirrhifolium, indicated by the release of rosette of leaves and differentiation of de novo axillary buds and their sprouting.
- present invention not only results in breaking of dormancy which under natural conditions takes 3-5 years to occur but also results in differentiation of axillary buds with no dormancy period.
- breaking of epicotyl dormancy in this particular species can be exploited to obtain continuos multiplication of the species with no dormancy period for commercial cultivation.
- synergistic formulations of culture media for the in vitro release of epicotyl dormancy in Polygonatum cirrhifolium Royle indicated by the release of rosette of leaves and differentiation ofde novo axillary buds and their sprouting by placing in vitro germinated seeds with epicotyl, coleoptile and radicle on Murashige and Skoog's, (MS) basal nutrient culture medium and lOOmg/1 myo-inositol, 0.5 mg/1 nicotinic acid, 0.5mg/l pyridoxine HCl, O.lmg/1 thiamine HCl, 2mg/l glycine, 30g/l sucrose, 7.0g/l agar, supplemented with l-6mg/l of 6- benzyl-aminopurine (BAP) and
- NAA 0.1-l.Omg /l naphthalene acetic acid
- BAP 3mg/l of BAP
- l.Omg/lNAA 1-20 mg/1 of gibberellic acid (GA3)
- the cultures on incubation at 20-24°C under 10-16hr photoperiod (2000-3500 lux) at 50-60% relative humidity (fa) results in the release of first foliage leaves from the emergent coleoptile and de novo axillary bud differentiation and their sprouting.
- synergistic formulations of a culture media used for the in vitro release of epicotyl dormancy in P. cirrhifolium at relative humidity of 50-60% are used for the in vitro release of epicotyl dormancy in P. cirrhifolium at relative humidity of 50-60%.
- Fig.l shows induction of germination in plant species Polygonatum Cirrhifolium Royle marked by the emergence of hypocotyl.
- Fig.2 shows differentiated epicotyl in plant species Polygonatum Cirrhifolium Royle with emerging radicle (ra) and coleoptile (col).
- Fig. 3 shows well differentiated coleoptile (col) of plant species Polygonatum Cirrhifolium Royle with emergent radicle (ra) and coleoptile (col).
- Fig. 4 shows well differentiated coleoptile (col) of plant species Polygonatum Cirrhifolium
- Fig 5 shows release of rosette of leaves from the coleoptile of plant species Polygonatum
- Cirrhifolium Royle shows release of first foliage leaves and de novo differentiation of axillary buds from the rhizomatous base of plant species Polygonatum Cirrhifolium Royle .
- Fig 7 shows sprouting of axillary buds in plant species Polygonatum Cirrhifolium Royle .
- Fig 8. shows excised axillary bud with emergent leaves in plant species Polygonatum
- Example 1 Experiment consisted of 4 treatments in the form of varying levels of GA 3 (2, 10, 25 and 50 mg/1) supplemented to culture medium consisting of 2.2g/l NH 4 NO 3 , 2.0g/l KNO 3 , O.44g/ 1 CaCl 2 . 2H 2 O, O.37g/l MgSO 4 . 7H 2 0, 0.17g/l KH 2 PO 4 , 37.25 mg/1 Na 2 EDTA, 27.8 mg/1 FeSO 4 .7H 2 0, 0.83 mg/lKI, 6.2 mg/1 H 3 BO 3 , 22.3 mg/1 MnSQ 4 . 4H 2 0, 8.6 mg/1 ZnSO 4 . 7H 2 0, 0.25mg/l Na 2 MoO 4 .
- Table 1 Effect of different levels of GA 3 supplemented to modified MS culture medium on germination percentage and duration of germination at 30°C in dark
- Example 2 In this experiment also there were 4 treatments with varying levels of GA 3 2-
- Table 2 Effect of different levels of GA 3 supplemented to modified MS culture medium on germination percentage and duration of germination at 20/30°C in dark
- Example 3 In this experiment seeds with emerged hypocotyl were transferred to the culture to modified MS basal culture medium containing of 2.2g/l NH NO 3 , 2.0g/l KNO 3 , O.44g/ 1 CaCl 2 . 2H 2 O, O.37g/l MgSO 4 . 7H 2 0, 0.17g/l KH 2 PO 4 , 37.25 mg/1 Na 2 EDTA, 27.8 mg/1 FeSO 4 .7H 2 0, 0.83 mg/lKI, 6.2 mg/1 H 3 BO 3 , 22.3 mg/1 MnSO 4 . 4H 2 0, 8.6 mg/1 ZnSO 4 . 7H 2 0, 0.25mg/l Na 2 MoO 4 . 2H 2 0, 0.025 mg/1 CuSO 4 .
- Example 4 Experiment consisted of 9 treatments in the form of various combinations of BAP (1-6 mg/1) and NAA (0.1-1.0 mg/1) supplemented to MS basal culture medium containing 1.65g/l NH 4 NO 3, 1.9g/l KNO 3 , O.44g/ 1 CaCl 2 . 2H 2 O, O.37g/l MgSO 4 . 7H 2 0, 0.17g/l KH 2 PO 4 , 37.25 mg/1 Na 2 EDTA, 27.8 mg/1 FeSO 4 .7H 2 0, 0.83 mg/lKI, 6.2 mg/1 H 3 BO 3 , 22.3 mg/1 MnSO 4 . 4H 2 0, 8.6 mg/1 ZnSO 4 .
- BAP to NAA ratio of 3:1 yields the optimum response resulting in release of first foliage leaves in 85.15% of secondary explants.
- 6mg/l of BAP in combination with lmg/1 of NAA gave maximum number of leaves (3.40) and leaf length (5.56 cm) respectively.
- Example 5 In this experiment there were 5 treatments with varying levels of GA 3 (1-20 mg/1) in combination with 3mg/l of BAP and 1.0 mg/1 of NAA supplemented to MS basal culture medium containing 1.65g/l NH 4 NO 3; 1.9g/l KNO 3 , O.44g/ 1 CaCl 2 . 2H 2 O, O.37g/l MgSO 4 . 7H 2 0, 0.17g/l KH 2 PO 4 , 37.25 mg/1 Na 2 EDTA, 27.8 mg/1 FeSO 4 .7H 2 0, 0.83 mg/lKI, 6.2 mg/1 H 3 BO 3 , 22.3 mg/1 MnSO 4 . 4H 2 0, 8.6 mg/1 ZnSO 4 .
- Table 5 Effect of various levels of GA 3 in combination with 2mg/l of BAP and l.Omg/1 of NAA on de novo axillary bud differentiation and the release of foliage leaves from them on MS medium (data from periodic observations compiled after 16 weeks of culture).
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CA2466653A CA2466653C (en) | 2001-11-15 | 2001-11-15 | Media compositions for faster growth of polygonatum cirrhifolium royle |
DE10197281T DE10197281T5 (en) | 2001-11-15 | 2001-11-15 | Media compositions for faster growth of Polygonatum Cirrhifolium Royle |
PCT/IN2001/000200 WO2003041492A1 (en) | 2001-11-15 | 2001-11-15 | Media compositions for faster growth of polygonatum cirrhifolium royle |
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2001
- 2001-11-15 DE DE10197281T patent/DE10197281T5/en not_active Withdrawn
- 2001-11-15 CA CA2466653A patent/CA2466653C/en not_active Expired - Fee Related
- 2001-11-15 WO PCT/IN2001/000200 patent/WO2003041492A1/en not_active Application Discontinuation
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