Title of Invention

MEDIA COMPOSITIONS FOR FASTER GROWTH OF POLYGONATUM CIRRHIFOLIUMN ROYLE

Abstract

The present invention relates to the novel culture medium compositions, said compositions comprising Murashige and Skoog (MS), a basal culture medium, varied concentrations of plant hormones, and other additives, leading to extraordinarily fast and synchronized in vitro induction of germination and release of epicotyl dormancy in Polygonatum Cirrhifolium Royle, an endangered medicinal plant species and a method for faster in vitro propagation of Polygonatum Cirrhifolium Royle.

Full Text

Field of invention 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. Background Art 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 Vaishya, 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). It is being over exploited from the wild habitats for its medicinal properties and this has led to its being placed among the threatened category of plants (Shah, N.C. 1983, In: An assessment of threatened plants of India, Botanical Survey of India, Calcutta, 40-49). According to seed dormancy classification of Mikolaeva (Mikolaeva, M.G. 1977, In : A. A.Khan (ed.) The physiology and biochemistry of seed dormancy and germination, 51- 74. North Holand Publ. Co., Amsterdam, New York and oxford) 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). Under in vitro conditions these requirements can be substituted by providing inorganic and organic nutrients, plant growth regulators and appropriating the thermo and photoperiodic conditions. Immature embryos can often be induced to germinate supplying nutrients (Dure,L.S. Ill, 1975 Ann. Rev. Plant Physiol. 26, 259-278). Stimulatory effect of GAs on germination of both dormant and non-dormant seeds has been widely reported (Lang,A. 1965, 848-893; Stokes,P. 1965, 746-803, Encydop. Plant Physiology. XV/2; Villiers, T.A. 1972, In: Seed biology (T.T.Kozlowski, ed.) Vol II, 220-281; Black, M. 1980/1981, Israil J. Bot. 29: 181- 192). Since 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. In epicotyl dormancy 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 GA3, BAP and NAA. (Chee, P.P. 1994. Hort Science, 29 (6), 695-97; Nhut,D.T. 1998. 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). Two of the applicants of the present invention (Dhar and Lattoo) studied the broader phenological pattern of germination in this particular species under laboratory and field conditions for three years of growth. Phenological observations indicated that the seeds of P. cirrhifolium exhibit epicotyl dormancy and have separate stratification requirements for hypocotyl, epicotyl and radicle emergence. Their's is the first report on record regarding the nature of dormancy in the referred species (under communication). They also found that epicotyl requires one, two or more startifications to release first foliage leaves. Objects of the present invention 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. Summary of the present invention 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. Detailed description of the present invention Accordingly 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. 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 ranging between 10 to 100 mg/1, b. 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 1 .Omg/1, and c. 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.Omg/1, Naphthalene acetic acid (NAA) about 1.Omg/1, and Gibberellic acid (GA3) ranging between 5 to 20 mg/1. Wherein, above-mentioned 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. In an embodiment of the present invention, plant hormones are selected a from group comprising Gibbrellic acid (GA3), alpha- naphthalene acetic acid (NAA), and 6-benzyl-aminopurine (BAP). In another embodiment of the present invention, Murashige and Skoog's (MS) basal culture medium is preferably consisting of 2.2g/l NH4NO3, 2.0g/l KNO3, O.44g/ 1 CaCl2. 2H2O, O.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/1 Na2 EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/lKI, 6.2 mg/1 H3 BO3, 22.3 mg/1 MnSO4. 4H20, 8.6 mg/1 ZnSO4. 7H20, 0.25mg/l Na2 MoO4. 2H20, 0.025 mg/1 CuSO4. 5H2O, 0.025 mg/1 CoCl2.6H20, supplemented with 250 mg/1 myo-inosital, 0.5mgl/l nicotinic acid, 0.5mg/l pyridoxine HC1, 3mg/l thiamine HC1, 2mg/l glycine, and 30g/l sucrose. In yet another embodiment of the present invention, 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 (GA3). In still another embodiment of the present invention, 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 (GA3) ranging between 50 to 100mg/l. In still another embodiment of the present invention, 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 (N A A) about l.Omg/1. In still another embodiment of the present invention, 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 (NAA) about l.Omg/1, and Gibberellic acid (GA3) ranging between 15 to 20 mg/1. In still another embodiment of the present invention, fast in vitro multiplication of the said medicinal plant species, with no dormancy period can make this plant commercially available in bulk. In still another embodiment of the present invention, said compositions are used for reliable and uniform germination. In still another embodiment of the present invention, said compositions are used for initiating faster germination, with only 81 days in vitro, to achieve the same. In still another embodiment of the present invention, said compositions is the first step towards release of the epicotyl dormancy. In still another embodiment of the present invention, said compositions can be used for all possible genotypes of said plant species to be grown in vitro. In still another embodiment of the present invention, said compositions can be used growing said plant species in vitro, in all geographical regions and all seasons. In still another embodiment of the present invention, said compositions are used for uniform and high germination rates. In still another embodiment of the present invention, said fast in vitro multiplication of the said medicinal plant species, is used for conservation of this endangered plant species. In still another embodiment of the present invention, said fast in vitro multiplication of the said plant species for wider utilization of its medicinal properties. In still another embodiment of the present invention, a method to for extraordinarily fast and synchronized in vitro induction of germination 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. In still another embodiment of the present invention, removing seeds from the berries after 15 days of dehydration at room temperature. 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. In still another embodiment of the present invention, rinsing the washed seeds thrice in distilled water. In still another embodiment of the present invention, sterilizing the rinsed seeds with 0.1% mercuric chloride (HgCb) for 3 minutes. In still another embodiment of the present invention, placing the sterilized seeds in sterile disposable plastic Petri plates (10x2 cm) containing semi-solid culture medium with 7.5% agar. In still another embodiment of the present invention, incubating the parafilm-sealed petri dishes at temperature ranging between 20 to 24 C and relative humidity (RH) ranging between 50 to 60%. In still another embodiment of the present invention, recording the emergence of hypocotyl in the said seeds at an interval of three days. In still another embodiment of the present invention, adjusting pH of the medium to 5.8 with IN NaOH or IN HC1. In still another embodiment of the present invention, sterilizing the medium for 20 minutes at 121°C andlS Ib. psi pressure. In still another embodiment of the present invention, dispensing the medium into petri dishes as 30 ml aliquots. In still another embodiment of the present invention, incorporating GAj into the medium after filter sterilization using 0.22 um pore size filter to cooled autoclaved medium. In still another embodiment of the present invention, transferring said seeds with emerged hypocotyl under aseptic conditions using Laminar Air Flow, to first medium culture consisting of MS basal culture medium, and Gibberellic acid (GA3) ranging between 10 to 100mg/l. In still another embodiment of the present invention, incubating the one set of said first medium culture at 30°C under continues dark conditions. In still another embodiment of the present invention, recording the germination percentage and duration of germination of the said seeds under the first medium culture. In still another embodiment of the present invention, incubating the second set of said first culture under diurnal temperature regime of 30/20°C, continues dark conditions. In still another embodiment of the present invention, recording the germination percentage and duration of germination of the said seeds under second set of first medium culture. In still another embodiment of the present invention, transferring the said germinating seeds to third set of first medium culture at 20°C under 16 hours photoperiod in a growth chamber. In still another embodiment of the present invention, recording differentiation of epicotyl with emergent coleoptile and radicle as germination synchronization in third set of first culture medium on daily basis. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, removing seeds from the berries after 15 days of dehydration at room temperature. 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. In still another embodiment of the present invention, rinsing the washed seeds thrice in distilled water. In still another embodiment of the present invention, sterilizing the rinsed seeds with 0.1% mercuric chloride (HgCli) for 3 minutes. In still another embodiment of the present invention, placing the sterilized seeds in sterile disposable plastic Petri plates (10x2 cm) containing semi-solid culture medium. In still another embodiment of the present invention, incubating the parafilm-sealed petri dishes at temperature ranging between 20 to 24 C and relative humidity (RH) ranging between 50 to 60%. In still another embodiment of the present invention, transferring secondary explants obtained from germinating seeds, consisting of epicotyl with emergent coleoptile and radicle with emerged hypocotyl under aseptic conditions, into the said dishes, using Laminar Air Flow. In still another embodiment of the present invention, adjusting pH of the medium to 5.8 withlNNaOHor 1NHC1. In still another embodiment of the present invention, sterilizing the medium for 20 minutes at 121°C and!5 Ib. psi pressure. In still another embodiment of the present invention, adding BAP and NAA to the above medium to form second medium culture, 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 1 .Omg/1. In still another embodiment of the present invention, dispensing the medium into petri dishes as 30 ml aliquots. In still another embodiment of the present invention, incubating the said culture at 20°C under 16 hr photoperiod with light intensity of 2000 lux provided by cool, white fluorescent tubes of 40 watts. In still another embodiment of the present invention, recording the optimal response in release of first foliage leaf from the explant. In still another embodiment of the present invention, 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.Omg/1, Naphthalene acetic acid (NAA) about 1.Omg/1, and Gibberellic acid (GA3) ranging between 5 to 20 mg/1. In still another embodiment of the present invention, incorporating GA3 into the fresh MS basal culture medium with NAA and BAP, after filter sterilization using 0.22 jam pore size filter to cooled autoclaved medium, to form third medium culture, said composition consisting of MS basal culture medium, 6- benzyl-aminopurine (BAP) about 2.Omg/1, Naphthalene acetic acid (NAA) about 1.Omg/1, and Gibberellic acid (GA3) ranging between 5 to 20 mg/1. In still another embodiment of the present invention, transferring fresh secondary explant consisting of epicotyl with emergent coleoptile and radicle, obtained from germinating seeds, under aseptic conditions using Laminar Air Flow, to third culture medium. In still another embodiment of the present invention, incubating the said culture at about 20°C under duration ranging between 10 to 6 hr photoperiod, with light intensity of about 2000 lux provided by cool. In still another embodiment of the present invention, maintaining the incubated cultures at 50-60% RH. In still another embodiment of the present invention, subculturing the said culture after every four weeks on the third medium formulations as mentioned above. In still another embodiment of the present invention, recording the de novo axillary bud differentiation and release of foliage leaves from them, In still another embodiment of the present invention, compiling the final data after 16 weeks of culture on the basis of periodical observations. In still another embodiment of the present invention, 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 NH4NO3, 2.0g/l KNO3, O.44g/1 CaCl2. 2H2O, 0.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/1 Na2 EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/lKI, 6.2 mg/1 H3 BO3, 22.3 mg/1 MnSO4. 4H20, 8.6 mg/1 ZnSO4. 7H20, 0.25mg/l Na2 MoO4. 2H20, 0.025 mg/1 CuSO4. 5H2O, 0.025 mg/1 CoCl2.6H20, 250mg/l myo- inositol, 0.5 mg/1 nicotinic acid, 0.5mg/l pyridoixine HC1, 3mg/l thiamine HC1, 2mg/l glycine, 30g/l sucrose, 7.5g/l agar and 2-50mg/l gibberellic acid (GA3) and on incubation at a temperature of 30°C (± 2°C) and diurnal temperature regime of 30/20°C (± 2°C) under continues darkness result in the induction of germination, indicated by the emergence of hypocotyl. In still another embodiment of the present invention, seeds with emerged hypocotyl on transfer to above basal culture medium containing altered levels of GA3 (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. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, invention constitutes the first and fundamental step to break the epicotyl dormancy in this particular species. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, invention results in breaking of epicotyl dormancy which under natural conditions takes 3-5 years to occur when raised from the seeds. In still another embodiment of the present invention, 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. Inventors have done years of research to come out with said novel culture media compositions. The said concentration ranges of said modified MS culture medium, said hormones and said additives is extremely critical. The criticality of the said media compositions, the sequence of use of said media compositions, and the parameters determined in the said media compositions is tested with much trial and error, and then only inventors were able to achieve the desired results. Any deviation from the same would not produce desired results. List of the accompanying drawings: 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. In still another embodiment of the present invention, Plants bearing ripe purplish berries were collected from the wild growing plants during October, 2000. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, it was followed by surface sterlization with 0.1% mercuric chloride (HgCl2) for 3 minutes. In still another embodiment of the present invention, after 4 washings in sterile distilled water, seeds were placed on semi-solid culture medium in sterile disposible plastic Petri plates (10x2 cm). In still another embodiment of the present invention, macro and micro nutrients in the basal medium were that of Murashige and Skoog's (Murashige,T. and Skoog,F. 1965. Physiol Plant 15: 433 -497) but modified to contain 2.2g/l NH4NO3, 2.0g/l KNO3, O.44g/1 CaCl2. 2H2O, O.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/1 Na2 EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/lKI, 6.2 mg/1 H3 BO3, 22.3 mg/1 MnSO4. 4H20, 8.6 mg/1 ZnSO4. 7H20, 0.25mg/l Na2 MoO4. 2H20, 0.025 mg/1 CuSO4. 5H2O, 0.025 mg/1 CoCl2.6H20. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, operations from surface sterilization to inoculation of seeds were carried under aseptic conditions in Laminar Air Flow. In still another embodiment of the present invention, 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). In still another embodiment of the present invention, observations regarding germination percentage as indicated by the emergence of hypocotyl were recorded at an interval of three days. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, observations regarding germination synchronization as indicated by differentiated epicotyl with emergent coleoptile and radicle were recorded on daily basis. In still another embodiment of the present invention, pH of medium was adjusted to 5.8 with IN NaOH or IN HC1 prior to adding agar. In still another embodiment of the present invention, medium was sterlized for 20 minutes at 121°C (15 Ib. psi pressure). In still another embodiment of the present invention, GA3 was incorporated after filter sterlization (0.22 um pore size millipore filter) to cooled (40-45°C) autoclaved medium. Medium was dispensed in petridishes as 30 ml aliquots. In still another embodiment of the present invention, 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- 60%). In still another embodiment of the present invention, 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 of 20°C (RH 50-60%). In still another embodiment of the present invention, invention is in vitro release of hypocotyl and synchronous release of epicotyl, coleoptile and radicle. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, invention is not limited to any particular variety or genotype but can be applied to genetically diverse and composite seeds of P.cirrhifolium. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, uniform and high germination rates for purpose of commercial cultivation of the reffered species. In still another embodiment of the present invention, for raising genetically variable progeny for selection of elite types and exploiting the variability to breed the varieties of commerce. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, 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 NH4NO3; 1.9g/l KNO3, O.44g/ 1 CaCl2. 2H2O, O.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/1 Na2 EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/lKI, 6.2 mg/1 H3 BO3, 22.3 mg/1 MnSO4. 4H20, 8.6 mg/1 ZnSO4. 7H20, 0.25mg/l Na2 MoO4. 2H20, 0.025 mg/1 CuSO4. 5H2O, 0.025 mg/1 CoCl2.6H20, 100mg/l myo-inositol, 0.5 mg/1 nicotinic acid, 0.5mg/l pyridoxine HC1, 0.1 mg/1 thiamine HC1, 2mg/l glycine, 30g/l sucrose, 7.0g/l agar, supplemented with l-6mg/l of 6-benzyl-aminopurine (BAP) and 0.1-l.Omg /I naphthalene acetic acid (NAA) in set 1 and 3mg/l of BAP, l.Omg/lNAA and 1-20 mg/1 of gibberellic acid (GA3) in set 2. In another embodiment of the present invention, in Polygonatum oppositifolium Royle and P. verticillatum L. BAP and NAA have stimulatory effect on the induction of shootlets in the axillary buds. In still another embodiment of the present invention, consequent to induction of germination there is no particular embodiment of invention or report in which epicotyl dormancy has been released in P. cirrhifolium by in vitro or other means. It takes 3-5 years for plants to grow to full size when raised from seeds (Dhar and Lattoo, under communication). In still another embodiment of the present invention, 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. In the diagram accompanying the specification 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. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, 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 NH4NO3; 1.9g/l KNO3, O.44g/ 1 CaCl2. 2H2O, O.37g/l MgSO4. 7H20, 0.17g/l, KH2PO4, 37.25 mg/1 Na2 EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/lKI, 6.2 mg/1 H3 BO3, 22.3 mg/1 MnSO4. 4H20, 8.6 mg/1 ZnSO4. 7H20, 0.25mg/l Na2 MoO4. 2H20, 0.025 mg/1 CuSO4. 5H2O, 0.025 mg/1 CoCl2.6H20, 100mg/l myo-inositol, 0.5 mg/1 nicotinic acid, 0.5mg/l pyridoxine HC1, 0.1 mg/1 thiamine HC1, 2mg/l glycine, 30g/l sucrose, 7.0g/l agar supplemented with l-6mg/l of BAP and 0.1- l.Omg /I of NAA. Set 2: MS basal culture medium containing 1.65g/l NH4NO3; 1.9g/l KNO3, O.44g/1 CaCl2. 2H2O, O.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/1 Na2 EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/lKI, 6.2 mg/1 H3 BO3, 22.3 mg/1 MnSO4. 4H20, 8.6 mg/1 ZnSO4. 7H20, 0.25mg/l Na2 MoO4. 2H20, 0.025 mg/1 CuSO4. 5H2O, 0.025 mg/1 CoCl2.6H20, 100mg/l myo-inositol, 0.5 mg/1 nicotinic acid, 0.5mg/l pyridoxine HC1, 0.1 mg/1 thiamine HC1, 2mg/l glycine, 30g/l sucrose, 7.0g/l agar and supplemented with 3.0mg/l of BAP, l.Omg/lNAA and l-20mg/l GA3 In still another embodiment of the present invention, cultures were incubated at 20-24°C under 10-16 hr photoperiod with light intensity of 2000-3500 lux provided by cool, white fluorescent tubes of 40 watts. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, pH of the medium was adjusted to 5.8 before adding agar. Medium was sterilized at 121°C (15 Ib. psi pressure) for 20 minutes. GAj, was incorporated after filter sterilization (0.22 um 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 25 and 35 ml aliquots respectively. In still another embodiment of the present invention, results in table 4 and 5 show the influence of various concentrations and combinations of BAP, NAA and GAa 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- 16 hr photoperiod (2000-3500 lux) at 50-60% of RH. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, 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. In still another embodiment of the present invention, specific embodiment of invention is not restricted to any particular geographic region or any season and can be utilized and practised anywhere in the world. In still another embodiment of the present invention, breaking of epicotyl dormancy in this particular species can be exploited to obtain continues multiplication of the species with no dormancy period for commercial cultivation. In still another embodiment of the present invention, raising genetically variable progeny for the selection of elite types and exploiting the variability to breed the varieties of commerce. In still another embodiment of the present invention, raising large populations of genetically hetergenous seedlings for in-situ and ex-situ conservation of the species as it is listed among the threatened category of plants. In still another embodiment of the present invention, 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 Murashige and Skoog's, (MS) basal nutrient culture medium and 100mg/l myo-inositol, 0.5 mg/1 nicotinic acid, 0.5mg/l pyridoxine HC1, O.lmg/1 thiamine HC1, 2mg/l glycine, 30g/l sucrose, 7.0g/l agar, supplemented with l-6mg/l of 6- benzyl-aminopurine (BAP) and 0.1-l.Omg /I naphthalene acetic acid (NAA) or 3 mg/1 of BAP, l.Omg/lNAA and 1-20 mg/1 of gibberellic acid (GAS), In still another embodiment of the present invention, 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 and de novo axillary bud differentiation and their sprouting. In still another embodiment of the present invention, synergistic formulations of a culture media used for the in vitro release of epicotyl dormancy in P. cirrhifolium at temperature range of 20-24°C. In still another embodiment of the present invention, Synergistic formulations of a culture media above used for the in vitro release of epicotyl dormancy in P. cirrhifolium under 10- 16 hr photoperiod at light intensity of 2000-3500 lux. In still another embodiment of the present invention, synergistic formulations of a culture media used for the in vitro release of epicotyl dormancy in P. cirrhifolium at relative humidity of 50-60%. Brief description of the Accompanying drawings 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 Royle with emergent radicle (ra) and coleoptile (col). Fig 5 shows release of rosette of leaves from the coleoptile of plant species Polygonatum Cirrhifolium Royle . Fig 6. 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 Cirrhifolium Royle. In still another embodiment of the present invention, synergistic media formulation are described substantially herein with reference to the examples accompanying the specification. EXAMPLES The synergistic activity for the induction of germination and release of epicotyl, coleoptile and radicle in the dormant seeds of Polygonatum cirrhifolium of the medium is presented in the following examples which may not be construed to limit the scope of invention. Example 1. Experiment consisted of 4 treatments in the form of varying levels of GA3 (2, 10, 25 and 50 mg/1) supplemented to culture medium consisting of 2.2g/l NH4NO3, 2.0g/l KNO3, O.44g/ 1 CaCl2. 2H2O, O.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/1 Na2 EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/lKI, 6.2 mg/1 H3 BO3, 22.3 mg/1 MnSO4. 4H20, 8.6 mg/1 ZnSO4. 7H20, 0.25mg/l Na2 MoO4. 2H20, 0.025 mg/1 CuSO4. 5H2O, 0.025 mg/1 CoCl2.6H20, 250mg/l myo-inositol, 0.5 mg/1 nicotinic acid, 0.5mg/l pyridoxine HC1, 3mg/l thiamine HC1, 2mg/l glycine, 30g/l sucrose and 7.5g/l agar. Each treatment consisted of 15 replicates with 5 seeds in each. These were incubated at a constant temperature of 30°C under continuous darkness at an RH of 60%. With the increase in GA3 concentration, there is concomitant increase in germination (Table 1). Maximum germination of 98.53% was obtained at 25mg/l of GA3. Germination extended from 24-61 days (maximum duration) at 2mg/l of GA3 and 7-23 days (minimum duration) at 50mg/l of GA3 respectively. Table 1: Effect of different levels of GAs supplemented to modified MS culture medium on germination percentage and duration of germination at 30°C in dark (Table Removed) *Mean, ±S.E. Example 2. In this experiment also there were 4 treatments with varying levels of GA3 2- 50mg/l. Supplemented to the modified MS basal culture medium containing of2.2g/l NH4NO3, 2.0g/l KNO3, O.44g/ 1 CaCl2. 2H2O, O.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/1 Na2 EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/lKI, 6.2 mg/1 H3 BO3, 22.3 mg/1 MnSO4. 4H20, 8.6 mg/1 ZnSO4. 7H20, 0.25mg/l Na2 MoO4. 2H20, 0.025 mg/1 CuSO4. 5H2O, 0.025 mg/1 CoCl2.6H20, 250mg/l myo-inositol, 0.5 mg/1 nicotinic acid, 0.5mg/l pyridoxine HCI, 3mg/l thiamine HCI, 2mg/l glycine, 30g/l sucrose and 7.5g/l agar. Cultures were incubated under diurnal temperature regime of 30/20°C (dark). Each treatment consisted of 15 replicates with 5 seeds in each. Germination is induced in all the treatments (table 2). Maximum germination of 62.12% is obtained at 50 mg/1 Of GA3. Table 2: Effect of different levels of GAs supplemented to modified MS culture medium on germination percentage and duration of germination at 20/30°C in dark (Table Removed) 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 NH4NO3, 2.0g/l KNO3, O.44g/ 1 CaCl2. 2H2O, O.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/1 Na2 EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/lKI, 6.2 mg/1 H3 BO3, 22.3 mg/1 MnSO4. 4H20, 8.6 mg/1 ZnSO4. 7H20, 0.25mg/l Na2 MoO4. 2H20, 0.025 mg/1 CuSO4. 5H2O, 0.025 mg/1 CoCl2.6H20, 250mg/l myo-inositol, 0.5 mg/1 nicotinic acid, 0.5mg/l pyridoxine HCI, 3mg/l thiamine HCI, 2mg/l glycine, 30g/l sucrose and 7.5g/l agar and supplemented with 4 concentrations of GA3 viz. 10,25,50 and 100 mg/1. Cultures were incubated at 20°C under 16 hr photoperiod in a growth chamber. There were four treatments in total and each treatment consisted of 15 replicates with 4 seeds with emerged hypocotyl in each replicate. The effect of various treatments on the release of epicotyl, coleoptile radicle and days to release/synchronization is described in table 3. 100 mg/1 of GA3 results in 100% synchronization after 11 days of culture. Table 3. Effect of different levels of GAs supplemented to modified MS culture medium for The release of epicotyl, coleoptile radicle and days to synchronization. (Table Removed) The synegistic activity for the in vitro release of epicotyl dormancy in P.cirrhifolium indicated by the release of rosette of leaves and differentiation of de nova axillary buds and their sprouting of the medium is presented in the following examples which may not be construed to limit the scope of invention. 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 NH4NO3; 1.9g/l KNO3, O.44g/ 1 CaCl2. 2H2O, O.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/1 Na2 EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/lKI, 6.2 mg/1 H3 BO3, 22.3 mg/1 MnSO4. 4H20, 8.6 mg/1 ZnSO4. 7H20, 0.25mg/l Na2 MoO4. 2H20, 0.025 mg/1 CuSO4. 5H2O, 0.025 mg/1 CoCl2.6H20, 100mg/l myo-inositol, 0.5 mg/1 nicotinic acid, 0.5mg/l pyridoxine HC1, 0.1 mg/1 thiamine HC1, 2mg/l glycine, 30g/l sucrose, 7.0g/l agar. Each treatment consisted of 24 cultures. These were incubated at 20°C under 16 hr photoperiod (2000 lux) at 60% RH. Synergistic combination of BAP and NAA is effective at all levels (table 1). However 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. Table 4: Influence of 3 levels of BAP in combination with 3 levels of NAA on the release of first foliage leaves cultured on MS medium (data from periodic observations compiled after 16 weeks of culture). (Table Removed) Example 5: In this experiment there were 5 treatments with varying levels of GA3 (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 NH4NO3; 1.9g/l KNO3, O.44g/1 CaCl2. 2H2O, O.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/1 Na2 EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/lKI, 6.2 mg/1 H3 BO3, 22.3 mg/1 MnSO4. 4H20, 8.6 mg/1 ZnSO4. 7H20, 0.25mg/l Na2 MoO4. 2H20, 0.025 mg/1 CuSO4. 5H2O, 0.025 mg/1 CoCl2.6H20, 100mg/l myo-inositol, 0.5 mg/1 nicotinic acid, 0.5mg/l pyridoxine HC1, 0.1 mg/1 thiamine HC1, 2mg/l glycine, 30g/l sucrose, 7.0g/l agar. Each treatment consisted of 24 cultures. These were incubated at 20°C under 16 hr photoperiod (2000 lux) at 60% RH. Effect of various levels of GA3 in combination with BAP (3mg/l) and NAA (1.0 mg/1) on percentage of secondary explants producing axillary buds per explant and percentage of axillary buds producing leaves is described in table 5. There is a concomitant increase in the elicitation of response with the increasing concentrations of GA3. Table 5: Effect of various levels of GAs 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). (Table Removed) We claim: 1. Media compositions for faster growth of Polygonatum Cirrhifolium Royle, wherein the said composition comprising: a. first medium composition consisting of MS basal culture medium and Gibberellic acid (GA3) ranging between 10 to 100 mg/1, b. second medium 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 1 .Omg/1, and c. third medium composition consisting of MS basal culture medium, 6- benzyl- aminopurine (BAP) at a concentration of 2.Omg/1, Naphthalene acetic acid (NAA) at a concentration of 1 .Omg/1, and Gibberellic acid (GA3) ranging between 5 to 20 mg/1, wherein, the above-mentioned 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. 2. Composition as claimed in claim 1, wherein the Murashige and Skoog's (MS) basal culture medium preferably consists of 2.2g/l NH4NO3, 2.0g/l KNO3, O.44g/ 1 CaCl2. 2H2O, O.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/1 Na2 EDTA, 27.8 mg/1 FeS04.7H20, 0.83 mg/lKI, 6.2 mg/1 H3 BO3, 22.3 mg/1 MnSO4. 4H20, 8.6 mg/1 ZnSO4. 7H20, 0.25mg/l Na2 MoO4. 2H20, 0.025 mg/1 CuSO4. 5H2O, 0.025 mg/1 CoCl2.6H20, supplemented with 250 mg/1 myo-inosital, 0.5mgl/l nicotinic acid, 0.5mg/l pyridoxine HC1, 3mg/l thiamine HC1, 2mg/l glycine, and 30g/l sucrose. 3. Composition as claimed in claim 1, wherein the said compositions are used for initiating faster germination in plant Polygonatum Cirrhifolium Royle in only 81 days in vitro. 4. Media compositions for faster growth of Polygonatum Cirrhifolium Royle substantially as herein described with reference to the foregoing examples.

Documents:

01838-delnp-2003-abstract.pdf

01838-delnp-2003-claims.pdf

01838-delnp-2003-correspondence-others.pdf

01838-delnp-2003-description (complete).pdf

01838-delnp-2003-drawings.pdf

01838-delnp-2003-form-1.pdf

01838-delnp-2003-form-18.pdf

01838-delnp-2003-form-2.pdf

01838-delnp-2003-form-3.pdf

1838-DELNP-2003-Abstract-(22-10-2008).pdf

1838-DELNP-2003-Claims-(22-10-2008).pdf

1838-DELNP-2003-Correspondence-Others-(22-10-2008).pdf

1838-DELNP-2003-Description (Complete)-(22-10-2008).pdf

1838-DELNP-2003-Form-1-(22-10-2008).pdf

1838-DELNP-2003-Form-2-(22-10-2008).pdf

1838-DELNP-2003-Form-3-(22-10-2008).pdf

1838-DELNP-2003-Others-Document-(22-10-2008).pdf

1838-DELNP-2003-PCT-210-(22-10-2008).pdf

1838-DELNP-2003-PCT-409-(22-10-2008).pdf

1838-DELNP-2003-Petition-137-(22-10-2008).pdf