CA2466653C - Media compositions for faster growth of polygonatum cirrhifolium royle - Google Patents

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.

Description

MEDIA COMPOSITIONS FOR FASTER GROWTH OF POLYGONATUM
CIRRHIFOLIUMROYLE

Technical Field 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.1, Chowkhamba Sanskrit Santhan, Sloka 120-122).
These attributes are ascribed to the presence of steroidal saponins and polysaccharides in the rhizoinatous 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 biochemistfy 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. III, 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, Encyclop. 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. (Ghee, 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, apt 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 Polygonatuin 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 Cirrhifoliurn 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 (GA3) 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 1.0mg/l, 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 2.0mg/1, Naphthalene acetic acid (NAA) about 1.0mg/1, and Gibberellic acid (GA3) ranging between 5, to 20 mg/l.
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 Polygonatuna Cirrhifoliurn 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 KN03, O.44g/ 1 CaCl2.
2H20, O.37g/1 MgSO4. 71120, 0.17g/1 KH2P04, 37.25 mg/l Nat EDTA, 27.8 mg/l FeSO4.7H20, 0.83 mg/1KI, 6.2 mg/l H3 B03, 22.3 mg/1 MnSO4. 41120, 8.6 mg/l ZnSO4. 7H20, 0.25mg/l Nat MoO4. 2H20, 0.025 mg/l CuSO4. 5H20, 0.025 mg/I CoC12.6H20, supplemented with 250 mg/l myo-inosital, 0.5mgl/l nicotinic acid, 0.5mg/l pyridoxine HCI, 3mg/l thiamine HCI, 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 100 mg/1.
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 5 explants producing leaves, about 3.4 mean number of leaves per secondary explant, and about 6 ems mean leaf length, said composition consisting of MS basal culture medium, 6-benzyl-aminopurine (BAP) ranging between 3 to 6 mg/l, and Naphthalene acetic acid (NAA) about 1.0mg/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/1, Naphthalene acetic acid (NAA) about 1.0mg/1, and Gibberellic acid (GA3) ranging between 15 to 20 mg/l.
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 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 10 distilled water.

In still another embodiment of the present invention, sterilizing the rinsed seeds with 0.1%
mercuric chloride (HgC12) for 3 minutes.

In still another embodiment of the present invention, placing the sterilized seeds in sterile disposable plastic Petri plates (10 x 2 cm) containing semi-solid culture medium with 7.5%
15 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 HCI.

In still another embodiment of the present invention, sterilizing the medium for 20 minutes at 121 C andl5 lb. 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 GA3 into the medium after filter sterilization using 0.22 m 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 100 mg/1.

In still another embodiment of the present invention, incubating the one set of said first medium culture at 30 C under continuos 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 firs'.
y.
culture uie and viwur diuriali t ~e 301G200V/' dark conditions.
a.rTiperatuLure regime offJ, continuos tl1t1UT2S.
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 Cirihifolium 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-20TH

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 (Hg02) for 3 minutes.
In still another embodiment of the present invention, placing the sterilized seeds in sterile disposable plastic Petri plates (10 x 2 cm) containing semi-solid culture medium.

In still another embodiment of the present invention, incubating the parafilmTM-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 with IN NaOH or 1N HCI.
In still another embodiment of the present invention, sterilizing the medium for 20 minutes at 121 C andl5 lb. 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 1.0mg/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 2.0mg/l, Naphthalene acetic acid (NAA) about 1.Omg/l, and Gibberellic acid (GA3) ranging between 5 to 20 mg/l.
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 m 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 2.0mg/l, Naphthalene acetic acid (NAA) about 1.0mg/1, and Gibberellic acid (GA3) ranging between 5 to 20 mg/l.

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 Polygonatuin cirrhifolium Royle by placing seeds on a culture medium containing 2.2g/I NH4NO3, 2.0g/l KNNO3, O.44g/ 1 CaC12. 2H20, O.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/l Nat EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/lKI, 6.2 mg/l H3 B03, 22.3 mg/l MnSO4. 4H20, 8.6 mg/l ZnSO4. 7H20, 0.25mg/l Nat MoO4. 2H20, 0.025 mg/I CuSO4. 5H20, 0.025 mg/l CoC12.6H20, 250mg/l myo-inositol, 0.5 mg/l nicotinic acid, 0.5mg/l pyridoixine HCI, 3mg/l thiamine HCI, 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 continuos 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 5 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 10 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.1 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 C l , , 1 after 1-'I days ox : ~ , _ anion at. room tc_i~pcrature (290 2 C,, o ghly washed under tap water with 1-2 drops of Tween-20TH for hours; rinsed thrice in distilled water.

In still another embodiment of the p,:;selo '-'ven icn it w u' _ Ly 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 (10 x 2 crn).

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/1 NH4NO3, 2.0g/I
K1.NI03, 0.44g/ 1 CaCl2. 2H2O, 0.37g/l MgSO4. 7H2O, 0.17811 KH2PO4 , 37.25 mg/l Na,, EDTA., 27.8 mg/l FeSO4.7H20, 0.83 mg/1KI, 6.2 mg/1 H3 B03, 22.3 mg/l MnSO4. 4H20, 8.6 mg/1 ZnSO4.
7H20, 0.25mg/1 Nat MoO4. 2H20, 0.025 mg/1 CuSO4. 5H20, 0.025 mg/l CoC12.6H20.

In still another embodiment of the present invention, this medium was supplemented with 250 mg/I myo-inosital, 0.Snigl/l nicotinic acid, 0.5mg/1 pyridoxine HCl, 3mg/l thiamine HCI, 2mg/l glycine, 30g/1 sucrose, 7.5 g/l agar and 2, 10, 25, and 50mg/1 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, parafilmTM-sealed petri dishes were incubated in two separate growth chambers at a temperature of 30- C ( 2 C;
dark) and diurnal temperature of 30120 C ( 2 C; dark) at 50-60% relaltive humidity (RH).

In still another embodiment of the present invention, observations regarding gennination 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 medduum containing altered levels of gibberellic acid 5, 25, 50 and 100 mg/l.

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 1N HCl prior to adding agar.
In still another embodiment of the present invention, medium was sterlized for 20 minutes at 121 C (15 lb. psi pressure).
In still another embodiment of the present invention, GA3 was incorporated after filter sterlization (0.22 m 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 Polygonatuin 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/1 NH4NO3; 1.9g/l KNO3, O.44g/ 1 CaC12. 2H20, O.37g/l MgSO4.
7H20, 0.17g/l KH2PO4, 37.25 mg/l Nat EDTA, 27.8 mg/l FeSO4.7H20, 0.83 mg/1c, 6.2 mg/l H3 B03, 22.3 mg/l MnSO4. 4H20, 8.6 mg/l ZnSO4. 7H20, 0.25mg/1 Nat MoO4. 2H20, 0.025 mg/l CuSO4. 5H20, 0.025 mg/l CoC12.6H20, 100mg/l myo-inositol, 0.5 mg/l nicotinic acid, 0.5mg/I pyridoxine HCI, 0.1mg/l thiamine HCI, 2mg/l glycine, 30g/I sucrose, 7.0g/1 agar, supplemented with 1-6mg/1 of 6-benzyl-aminopurine (BAP) and 0.1-1.0mg /1 naphthalene acetic acid (NAA) in set 1 and 3mg/l of BAP, 1.Omg/1NAA and 1-20 mg/l 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/1 KNO3, O.44g/ 1 CaC12. 2H20, O.37g/l MgSO4. 7H20, 0.17g/l, KH2PO4, 37.25 mg/l Nat. EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/1KI, 6.2 mg/l H3 B03, 22.3 mg/i MnSO4. 4H20, 8.6 mg/l ZnSO4. 7H20, 0.25mg/l Nat MoO4. 2H20, 0.025 mg/l CuSO4.
5H20, 0.025 mg/1 CoC12.6H20, 100mg/1 myo-inositol, 0.5 mg/1 nicotinic acid, 0.5mg/l pyridoxine HCI, 0.1mg/1' thiamine HCI, 2mg/l glycine, 30g/l sucrose, 7.0g/l agar supplemented with 1-6mg/l of BAP and 0.1- 1.0mg /1 of NAA. Set 2: MS basal culture medium containing 1.65g/l NH4NO3; 1.9g/1 I~NO3, 0.44g/ 1 CaC12. 2H20, 0.37g/1 MgSO4.
7H20, 0.17g/l KH2PO4, 37.25 mg/I Nat EDTA, 27.8 mg/1 FeSO4.7H20, 0.83 mg/1KI, 6.2 mg/l H3 B03, 22.3 mg/l MnSO4. 4H20, 8.6 mg/1 ZnSO4. 7H20, 0.25mg/1 Nat MoO4.
2H20, 0.025 mg/l CuSO4. 5H20, 0.025 mg/l CoC12.6H20, 100mg/l myo-inositol, 0.5 mg/l nicotinic acid, 0.5mg/1 pyridoxine HCI, 0.lmg/1 thiamine HCI, 2mg/l glycine, 30g/l sucrose, 7.0g/1 agar and supplemented with 3.0mg/1 of BAP, 1.0mg/1NAA and 1-20mg/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 lb. psi pressure) for 20 minutes.
GA3 was incorporated after filter sterilization (0.22 pm pore cell, milipore filter to cooled 5 (40-45 C) autoclaved medium. BAP and NAA were added prior to autoclaving.
Medium was dispensed in culture tubes (25x150mm, BorosilTM) and conical vials (100 ml, BorosilTM) as 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 GA3 on the in 10 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.cir-rhifo1iu727, indicated by the release of rosette of 15 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 its 20 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 continuos multiplication of the species with no dormancy period for commercial cultivation.

25 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 Polygolatunm cirrhifoliuln Roy:ie 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/1 myo-inositol, 0.5 mg/l nicotinic acid, 0.5mg/1 pyridoxine HCI, O.lmg/1 thiamine HCI, 2mg/l glycine, 30g/l sucrose, 7.0g/1 agar, supplemented with 1-6mg/1 of 6- benzyl-aminopurine (BAP) and 0.1-1.0mg /1 naphthalene acetic acid (NAA) or 3mg/l of BAP, 1.Omg/1NAA and 1-20 mg/l of gibberellic acid (GA3), 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.cirrhifoliunz 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. cirr hifoliurn at relative humidity of 50-60%.
Brief description of the Accompanying drawings Fig.1 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 Cirr hifoliunz 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 Polygonatuin Cirrhifoliuin Royle.
Fig 6. shows release of first foliage leaves and de novo differentiation of axillary buds from the rhizomatous base of plant species Polygonaturn Cirrhifolium Royle .
Fig 7 shows sprouting of axillary buds in plant species Polygonaturn Cirrhifolium Royle.
Fig 8. shows excised axillary bud with emergent leaves in plant species Polygonatum Cirr hifolium 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 Polygonatuni 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 CaC12. 2H20, O.37g/l MgSO4. 7H20, 0.17g/l ICH2PO4, 37.25 mg/l Nat EDTA, 27.8 mg/l FeSO4.7H20, 0.83 mg/lU, 6.2 mg/l H3 B03, 22.3 mg/l MnSO4.
4H20, 8.6 mg/l ZnSO4. 7H20, 0.25mg/l Nat MoO4. 2H20, 0.025 mg/1 CuSO4. 5H20, 0.025 mg/l CoC12.6H20, 250mg/l myo-inositol, 0.5 mg/l nicotinic acid, 0.5mg/l pyridoxine HC1, 3mg/l thiamine HC1, 2mg/l glycine, 30g/1 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 GA3 supplemented to modified MS culture medium on germination percentage and duration of germination at 30 C in dark GA3 concentration Percent germination Duration of germination (mg/1) 2 23.69 7.04* 24-61 10 66.18 4.77 19-53 25 98.53 3.82 11-30 50 97.11 4.01 7-23 *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 of 2.2g/l NH4NO3, 2.0g/1 KNO3, O.44g/ 1 CaC12. 2H20, O.37g/l MgSO4. 71120, 0.17g/1 KH2PO4, 37.25 mg/l Nat EDTA, 27.8 mg/l FeS04.7H20, 0.83 mg/1KI, 6.2 mg/l H3 B03, 22.3 mg/l MnSO4. 41120, 8.6 mg/l ZnSO4. 71120, 0.25mg/l Nat MoO4. 2H20, 0.025 mg/l CuSO4.
5H20, 0.025 mg/l CoC12.6H20, 250mg/l myo-inositol, 0.5 mg/l nicotinic acid, 0.5mg/l pyridoxine HC1, 3mg/l thiamine HC1, 2mg/l glycine, 30g/1 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/l Of GA3.

Table 2: Effect of different levels of GA3 supplemented to modified MS culture medium on germination percentage and duration of germination at 20/30 C in dark GA3 concentration Percent germination Duration of germination (mg/1) 2 12.41 6.17 32-73 10 18.06 4.32 34-72 46.00 5.11 27-63 20 50 62.12 3.66 23-58 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.Og/l KNO3, O.44g/ 1 CaC12. 2H20, O.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/l Nat EDTA, 25 27.8 mg/l FeSO4.7H20, 0.83 mg/1KI, 6.2 mg/l H3 B03, 22.3 mg/l MnSO4. 41120, 8.6 mg/l ZnSO4. 71120, 0.25mg/l Nat MoO4. 2H20, 0.025 mg/l CuSO4. 5H20, 0.025 mg/l CoC12.6H20, 250mg/l myo-inositol, 0.5 mg/l nicotinic acid, 0.5mg/l pyridoxine HC1, 3mg/l thiamine HC1, 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/l. 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/l of GA3 results in 100%
synchronization after 11 days of culture.

Table 3. Effect of different levels of GA3 supplemented to modified MS culture medium for The release of epicotyl, coleoptile radicle and days to synchronization.
GA3 concentration Percent seeds Days to release epicotyl, (mg/1) with epicotyl, coleoptile & radicle coleoptile &
radicle 23.71 2.00 21 10 25 78.23 3.11 18 50 100.00 0.00 14 100 100.00 0.00 11 The synegistic activity for the in vitro release of epicotyl dormancy in P.cirrhifoliuni indicated by the release of rosette of leaves and differentiation of de novo 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 -N03, O.44g/ 1 CaCl2. 2H20, 0.37g/l MgSO4.
7H20, 0.17g/l I-H2P04, 37.25 mg/l Nat EDTA, 27.8 mg/l FeSO4.7H20, 0.83 mg/1I J, 6.2 mg/l H3 B03, 22.3 mg/l MnSO4. 4H20, 8.6 mg/i ZnSO4. 7H20, 0.25mg/l Nat MoO4. 2H20, 0.025 mg/1 CuSO4. 5H20, 0.025 mg/l CoC12.6H20, 100mg/l myo-inositol,'0.5 mg/l nicotinic acid, 0.5mg/l pyridoxine HCI, 0.lmg/l 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 ling/l 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).

------------------------------------------------------------------------------------------------------------BAP Percentage secondary Mean number of Mean leaf length(cm) Concentrat- explants producing leaves per secondary ion (mg/1) leaves explant --------------------------------NAA(mg/1) NAA (mg/1) NAA (mg/1) 0.1 0.5 1.0 0.1 0.5 1.0 0.1 0.5 1.0 -------------------------------------------------------------------------------------------------------------1.0 4.70 14.81 25.92 2.00 2.25 2.33 2.11 1.76 2.71 10 0.13* 0.24 0.16 0.34 0.27 0.42 3.0 11.12 66.67 85.18 1.99 2.74 2.93 2.03 3.17 3.08 0.16 0.42 0.31 0.51 0.83 0.63 15 6.0 20.22 20.74 70.37 2.34 3.01 3.40 3.71 4.04 5.56 0.43 0.32 0.41 0.49 0.99 1.31 -------------------------------------------------------------------------------------------------------------*S.E.

Example 5: In this experiment there were 5 treatments with varying levels of GA3 (1-20 20 mg/1) in combination with 3mg/l of BAP and 1.0 mg/l of NAA supplemented to MS basal culture medium containing 1.65g/1 NH4NO3; 1.9g/I KNO3, O.44g/ 1 CaC12. 2H20, O.37g/l MgSO4. 7H20, 0.17g/l KH2PO4, 37.25 mg/l Nat EDTA, 27.8 mg/l FeSO4.7H20, 0.83 mg/1KI, 6.2 mg/l H3 B03, 22.3 mg/l MnSO4. 4H20, 8.6 mg/l ZnSO4. 7H20, 0.25mg/I
Nat MoO4. 2H20, 0.025 mg/I CuSO4. 5H20, 0.025 mg/1 CoC12.6H20, 100mg/1 myo-inositol, 0.5 mg/l nicotinic acid, 0.5mg/l pyridoxine HCI, 0.lmg/l thiamine HCI, 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/1) 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 GA3 in combination with 2mg/i of BAP and 1.Omg/l 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 weeks of culture).

GA3 concentration Percentage secondary- Mean number Percentage Remarks (mg/1) explants producing of axillary buds axillary axillary buds per secondary buds pro-explant ducing leaves 1. 16.67 2.70 0.70* 37.03 Epicotylar bulge swells-forms rhizomatous base 5. 70.83 6.44 1.04 46.58 --10. 87.50 9.06 2.00 66.22 --15. 100.00 11.67 1.96 77.12 --20. 100.00 8.96 2.01 97.03 Rhizomatous base, callus induction -------------------------------------------------------------------------------------------------------------*S.E.

Claims (67)

Claims

1. A set of culture media for use in initiating in vitro induction of germination and release of epicotyl dormancy in a Polygonatum Cirrhifolium Royle plant, the set comprising:
a. a first medium for initiating germination of said plant, and for initiating release of epicotyl, coleoptile and radicle dormancy in a seed of the plant, said first medium comprising Murashige and Skoog (MS) basal culture medium modified to have a concentration of Gibberellic acid (GA3) ranging between 10 to 100 mg/L, b. a second medium for initiating release of first foliage leaf from epicotyl dormant explants, said second medium comprising MS basal culture medium modified to have a concentration of 6-benzyl-aminopurine (BAP) ranging between to 6 mg/L; and a concentration of naphthalene acetic acid (NAA) ranging between 0.5 to 1.0mg/L, and c. a third medium for initiating release of axillary buds from epicotyl dormant explants, said third medium comprising MS basal culture medium modified to have a concentration of 6- benzyl-aminopurine (BAP) of about 2.0mg/L, a concentration of Naphthalene acetic acid (NAA) of about 1.0mg/L, and a concentration of Gibberellic acid (GA3) ranging between 5 to 20mg/L, wherein the first medium, second medium, and third medium are used in the sequence first medium, second medium and then third medium, for initiating in vitro induction of germination and release of epicotyl dormancy in the Polygonatum Cirrhifolium Royle plant.

2. The set of claim 1, wherein the MS basal culture medium comprises either:

2.2g/L NH4NO3, 2.0g/L KNO3, 0.44g/L CaCl2.cndot.2H2O, 0.37g/L
MgSO4.cndot.7H2O, 0.17g/L KH2PO4, 37.25 mg/L Na2 EDTA, 27.8 mg/L FeSO4.cndot.7H2O, 0.83 mg/L KI, 6.2mg/L H3BO3, 22.3 mg/L MnSO4.cndot.4H2O, 8.6 mg/L ZnSO4.cndot.7H2O, 0.25mg/L

Na2MoO4.cndot.2H2O, 0.025 mg/L CuSO4.cndot.5H2O, 0.025 mg/L CoCl2.cndot.6H2O, supplemented with 250 mg/L myo-inosital, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine HCl, mg/L thiamine HCl, 2mg/L glycine, and 30g/L sucrose; or 1.65g/L NH4NO3, 1.9g/L KNO3, 0.44g/L CaCl2-2H2O, 0.37g/L MgSO4.cndot.7H2O, 0.17g/L KH2PO4, 37.25 mg/L Na2 EDTA, 27.8 mg/L FeSO4.cndot.7H2O, 0.83 mg/L KI, 6.2mg/L H3BO3, 22.3 mg/L MnSO4.cndot.4H2O, 8.6 mg/L ZnSO4.cndot.7H2O, 0.25mg/L

Na2MoO4.cndot.2H2O, 0.025 mg/L CuSO4.cndot.5H2O, 0.025 mg/L CoCl2.cndot.6H2O, supplemented with 100 mg/L myo-inosital, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine HCl, 0.1 mg/L thiamine HCl, 2mg/L glycine, and 30g/L sucrose.

3. The set of claim 1 or claim 2, wherein the first medium is for use for germination, wherein a percentage induction of germination of at least 65% is achieved within a time ranging between 7 to 23 days, and wherein the concentration of GA3 in the first medium is about 50mg/L.

4. The set of claim 1 or claim 2, wherein the first medium is for use for breaking epicotyl dormancy, so as to achieve about 100% release of epicotyl, coleoptile and radicle, and to achieve the release of epicotyl dormancy within a time ranging between 11 to 14 days, and wherein the concentration of GA3 in the first medium ranges between 50 to 100 mg/L.

5. The set of any one of claims 1 to 4, wherein the second medium is for use for initiating release of first foliage leaf from epicotyl dormant explants so that about 86% of secondary explants producing leaves, with a mean number of leaves per secondary explant of about 3.4, and a leaf length of about 6 cms, wherein said second medium has a concentration of BAP ranging between 3 to 6 mg/l, and a concentration of NAA
of about 1.0mg/L.

6. The set of any one of claims 1 to 5, wherein the third medium is for use for initiating release of axillary buds from epicotyl dormant explants so that about 100% of secondary explants producing axillary buds, the number of axillary buds per secondary explant ranging between 9 to 12, and 77 to 98% of the axillary buds produce leaves, and wherein said third medium has a concentration of BAP of about 2.0mg/l, a concentration of NAA of about 1.0mg/l, and a concentration of GA3 ranging between 15 to 20 mg/l.

7. The set of any one of claims 1 to 6, wherein the compositions are for use for in vitro multiplication of the plant with no dormancy period.

8. The set of any one of claims 1 to 7, wherein the media are for use for germination of the plant so that a percentage induction of germination of at least 98% is achieved.

9. The set of any one of claims 1 to 8, wherein the media are for use so as to initiate germination and to achieve the release of epicotyl dormancy within at most 81 days.

10. The set of any one of claims 1 to 9, wherein the set is for use as a first step towards release of epicotyl dormancy.

11. The set of any one of claims 1 to 10, wherein the plant has a defined genotype.

12. The set of any one of claims 1 to 11, wherein the plant is obtained from a defined geographical region.

13. The set of any one of claims 1 to 12, adapted to optimize germination rates.

14. Use of the set of any one of claims 1 to 13, for multiplication of the plant.

15. The use according to claim 14, wherein the plant is for use to prepare a medicine.

16. A method for in vitro induction of germination in a Polygonatum Cirrhifolium Royle plant, said method comprising:
a. obtaining seeds from the plant, wherein the seeds are sterilized, b. placing the sterilized seeds in sterile Petri dishes (10 x 2 cm) containing a first culture medium comprising 7.5% agar, c. incubating the Petri dishes containing the sterilized seeds at a temperature ranging between 20 to 24°C and relative humidity (RH) ranging between 50 to 60%, wherein the Petri dishes are sealed with Parafilm.TM., d. identifying sterilized seeds with emerged hypocotyls, e. transferring first and second subsets of the sterilized seeds with emerged hypocotyls under aseptic conditions to first and second portions, respectively, of a second culture medium comprising Murashige and Skoog (MS) basal culture medium, 10 to 100 mg/L Gibberelic acid (GA3), and a pH of 5.8, f. incubating the first subset in the first portion of the second culture medium at 30°C under continuous dark conditions, g. incubating the second subset in the second portion of the second culture medium under diurnal temperature regime of 30/20°C and continuous dark conditions, h. identifying germinating seeds in the first and second subsets, i. transferring the germinating seeds to a third portion of the second culture medium, j. incubating the germinating seeds in the third portion at 20°C under 16 hours photoperiod in a growth chamber.

17. The method of claim 16, further comprising identifying a release of an epicotyl with an emergent coleoptile and a radicle in the germinating seeds in the third portion.

18. The method of either of claims 16 to 17, wherein the pH of the second culture medium is adjusted to 5.8 with NaOH or HCl,

19. The method of claim 18, wherein the NaOH or HCl is 1N.

20. The method of any one of claims 16 to 19, wherein the second culture medium is sterilized for 20 minutes at 121°C and 15 lb. psi pressure.

21. The method of claim 20, wherein the GA3 is incorporated into cooled, sterilized second culture medium after filter sterilization using 0.22 µm pore size filter.

22. The method of any one of claims 16 to 21, wherein the Petri dishes are macle of disposable plastic.

23. The method of any one of claims 16 to 22, wherein 65 to 98% of the seeds germinate between 7 and 53 days

24. The method of claim 23, wherein 78 to 100% of the seeds release an epicotyl, coleoptile, and radicle within 11 to 18 days.

25. The method of any of claims 16 to 24, wherein a percentage induction of germination of about 98% is achieved within 7 to 23 days.

26. The method of claim 25, wherein the second culture medium comprises 50mg/l GA3.

27. The method of any one of claims 17 to 26, wherein substantially 100%
release of the epicotyls, coleoptiles and radicles is achieved within 11 to 14 days,

28. The method of claim 28, wherein the second culture medium comprises 50 to mg/L GA3.

29. The method of any one of claims 16 to 22, wherein germination of seeds occurs in vitro within 81 days.

30. The method of any of claims 16 to 29, wherein the second culture medium further comprises naphthalene acetic acid or 6-benzyl-aminopurine.

31. The method of any one of claims 16 to 30, wherein the , prior to placing the sterilized seeds in sterile Petri dishes (10 x 2 cm) containing a first culture medium comprising 7.5%
agar, the seeds are washed thoroughly for 2 hours under tap water with 1-2 drops of Tween-20 and rinsed with distilled water.

32. The method of any one of claims 16 to 31, wherein rinsed seeds are sterilized with 0.1 % mercuric chloride (HgCl2).

33. A method of inducing release of epicotyl dormancy in Polygonatum cirrhifolium Royle, comprising:
a. preparing a culture medium comprising Murashige and Skoog (MS) basal culture medium, 6-benzyl-aminopurine (BAP) ranging between 3 to 6 mg/l, and Naphthalene acetic acid (NAA) ranging between 0.5 to 1.0mg/l, and a pH of 5.8.

b. transferring at least one secondary explant consisting of an epicotyls with emergent coleoptiles and a radical obtained from germinating seeds under aseptic conditions, into a container containing the culture medium, c. sealing the container with Parafilm.TM., and d. inclubating the Parafilm.TM.-sealed containers at. 20°C under a 16 hr photoperiod with light intensity of 2000 lux, e. identifying the release of a first foliage leaf from the explant.

34. The method of claim 33, wherein the pH of the culture medium is adjusted to 5.8 with NaOH or HCl,

35. The method of claim 34, wherein the NaOH or HCl is 1N.

36. The method of any one of claims 33 to 35, wherein the culture medium is sterilized for 20 minutes at 121 °C and 15 lb. psi pressure.

37. The method of any one of claims 33 to 36, wherein the light intensity of 2000 lux is provided by cool, white fluorescent tubes of 40 watts.

38. The method of any one of claims 33 to 37, wherein the percentage of the at least one secondary explants producing leaves ranges between 65 and 86%, the mean number of leaves per secondary explant ranges between 2.7 and 4, and the mean leaf length ranges between 3 an 6 cm.

39. The method of any one of claims 34 to 38, wherein substantially 86% of the at least one secondary explants produce at least one leaf, the mean number of leaves per secondary explant is 3.4, and the mean leaf length is 6 cm.

40. The method of claim 39, wherein the culture medium comprises about 1.0mg/l NAA.

41. The method of any one of claims 33 to 40, wherein the culture medium further comprises Gibberelic acid.

42. A method of inducing the release of epicotyl dormancy from a differentiated de novo axillary bud and release of foliage leaves in Polygonatum Cirrhifolium Royle, the method comprising:
a. preparing a culture medium comprising Murashige and Skoog (MS) basal culture medium, 6- benzyl-aminopurine (BAP) of substantially 2.0 mg/L.

naphthalene acetic acid (NAA) of substantially 1.0mg/L, Gibberelic acid (GA3) ranging between 5 and 20 mg/L, and a pH of 5.8, b. transferring at least one secondary explant consisting of an epicotyl with an emergent coleoptile and radical obtained from germinating seeds under aseptic conditions, into a container containing the culture medium, c. sealing the container with Parafilm.TM., and d. inclubating the Parafilm.TM.-sealed containers at 20°C at 50-60%
relative humidity under a 10 to 16 hr photoperiod with light intensity of 2000 lux, e. subculturing the at least one secondary explant every four weeks on the culture medium, and f. identifying the differentiated de novo axillary bud and release of foliage leaves from the at least secondary explant.

43. The method of claim 42, wherein the pH of the second culture medium is adjusted to 5.8 with NaOH or HCl,

44. The method of claim 43, wherein the NaOH or HCl is 1N.

45. The method of any one of claims 42 to 44, wherein the second culture medium sterilized for 20 minutes at 121°C and 15 lb. psi pressure.

46. The method of claim 45, wherein the GA3 is incorporated into cooled, sterilized second culture medium after filter sterilization using 0.22 µm pore size filter.

47. The method of any one of claims 42 to 46 wherein the percentage of the at least one secondary explants producing axillary buds ranges between 70 and 100%, the mean number of axillary buds per secondary explant ranges between 6 and 12, and the percentage of axillary buds producing leaves ranges between 45 and 98%.

48. The method of any one of claims 42 to 47, wherein substantially 100% of the at least one secondary explants produce axillary buds, the number of axillary buds per secondary explant ranges between 9 and 12, and the percentage of axillary buds producing leaves ranges between 77 and 98%.

49. The method of claim 48, wherein the culture medium comprises GA3 ranging between 15 to 20 mg/L.

50. A method of in vitro cultivation of Polygonatum comprising the steps of:
a. contacting a Polygonatum seed with a first medium comprising Murashige and Skoog (MS) basal culture medium and gibberellic acid (GA3) present in an amount from about 10 mg/L to about 100 mg/L, b. upon emergence of a hypocotyl of a seedling, contacting said seedling with a second medium comprising MS basal culture medium, 6-benzyl-aminopurine (BAP) present in an amount from about 3 mg/L to about 6 mg/L, and naphthalene acetic acid (NAA) present in an amount from 0.5 mg/L to about 1.0 mg/L, and c. upon emergence of a first foliage leaf of said seedling, contacting said seedling with a third medium comprising MS basal culture medium, BAP present in an amount of about 2.0 mg/L, NAA present in an amount of about 1.0 mg/L, and GA3 present in an amount from about 5 mg/L to about 20 mg/L.

51. The method of claim 50, wherein GA3 in the first medium is present in an amount from about 50 mg/L to about 100 mg/L.

52. The method of claim 50, wherein GA3 in the third medium is present in an amount from about 15 mg/L to about 20 mg/L.

53. The method of any one of claims 50 to 52, wherein NAA in the second medium is present in an amount of about 1.0 mg/L.

54. The method of any one of claims 50 to 53, wherein the first, second, and/or third medium further comprises agar.

55. The method of any one of claims 50 to 54, wherein the MS basal culture medium of the first, second, and/or third medium has a pH of about 5.8.

56. The method of any one of claims 50 to 55, wherein said contacting steps are performed at a temperature is from about 20°C to about 24°C.

57. The method of any one of claims 50 to 56, wherein said contacting steps are performed at a relative humidity about 50% to about 60%.

58. The method of any one of claims 50 to 56, wherein said Polygonatum is contacted with said first medium in the absence of light.

59. The method of any one of claims 50 to 58, wherein said Polygonatum is contacted with said second and/or third medium under illumination of about 2000 to about 3500 lux.

60. The method of any one of claims 50 to 59, wherein the primary explant is contacted with the second medium less than 60 days after said seeds are contacted with the first medium.

61. The method of any one of claims 16 to 60, wherein the MS basal culture medium comprises either:

2.2g/L NH4NO3, 2.0g/L KNO3, 0.44g/L CaCl2.cndot.2H2O, 0.37g/L
MgSO4.cndot.7H2O, 0.17g/L
KH2PO4, 37.25 mg/L Na2 EDTA, 27.8 mg/L FeSO4.cndot.7H2O, 0.83 mg/L KI, 6.2mg/L

H3BO3, 22.3 mg/L MnSO4.cndot.4H2O, 8.6 mg/L ZnSO4-7H2O, 0.25mg/L Na2MoO4 .cndot.2H2O, 0.025 mg/L CuSO4.cndot.5H2O, 0.025 mg/L CoCl2.cndot.6H2O, supplemented with 250 mg/L myo-inosital, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine HCl, 3mg/L
thiamine HCl, 2mg/L glycine, and 30g/L sucrose; or 1.65g/L NH4NO3, 1.9g/L KNO3, 0.44g/L CaCl2.cndot.2H2O, 0.37g/L MgSO4-7H2O, 0.17g/L KH2PO4, 37.25 mg/L Na2 EDTA, 27.8 mg/L FeSO4.cndot.7H2O, 0.83 mg/L KI, 6.2mg/L H3BO3, 22.3 mg/L MnSO4.cndot.4H2O, 8.6 mg/L ZnSO4-7H2O, 0.25mg/L
Na2MoO4-2H2O, 0.025 mg/L CuSO4.cndot.5H2O, 0.025 mg/L CoCl2-6H2O, supplemented with 100 mg/L myo-inosital, 0.5 mg/L nicotinic acid, 0.5 mg/L pyridoxine HCl.
0.1 mg/L thiamine HCl, 2mg/L glycine, and 30g/L sucrose.

62. The method of any one of claims 16 to 61, for in vitro multiplication of the plant species with no dormancy period.

63. The method of any one of claims 16 to 62, wherein the plant has a defined genotype.

64. The method of any one of claims 16 to 63, wherein the plant is obtained from a defined geographical region.

65. The method of any one of claims 16 to 64, wherein the method is adapted to optimize germination rate of the plant.

66. The method of any one of claims 16 to 65, for multiplication of the plant.

67. The method of claim 66, wherein the plant is for use to prepare a medicine.