Title of Invention

"A SYNERGISTIC MEDIUM COMPOSITION FOR MICROPROPAGATION OF PLANTS"

Abstract

The present invention relates to a synergistic medium composition for micropropagation of plants, in particular the medium is for the production of tissue cultured plants of Bixa orellana L. The establishment of interactions among different groups of phytohormones, and growth regulators alone or in combinations are responsible for certain interrelations existing among cells, tissues and organs. So there seems to be consensus that the success in inducing differentiation depends upon the type of plant part ("explant"), the physiological condition of the explant and physical and chemical milieu of explant during culture. Due to this, the science of tissue culture has been directed to optimize the physiological conditions of source plant, the type of explant, the culture conditions and the phytohormones used to initiate tissue culture. This substantiates the fact that development of a new process for proliferation of plants by tissue culture is not obvious.

Full Text

The present invention relates to a synergistic medium composition for Micropropagation of plants, in particular the medium is for the production of tissue cultured plants of Bixa orellana L. The invention is aimed at development of A synergistic medium formulation for production of viable Micropropagated plants with reference to the Bixa orellana. Creation of novel germplasm through tissue culture techniques and gene transfer holds great potential for improving the quality, resistance to diseases etc. Plant regeneration by tissue culture techniques is well established. A wide variety of plant species has been successfully regenerated in vitro via organogenesis or somatic embryogenesis. Organogenesis leads to organ formation i.e. shoot (or root), which can be isolated to induce development of roots (or shoots) to produce full plants while somatic embryogenesis leads to the development of somatic embryos (embryos developed without genetic fertilization) which have both shoot and root initially and are capable of developing into whole plants. Although the ability of individual parts of plants and cells to regenerate into complete plants (called totipotency) is a well known phenomenon, each plant or plant part requires specialized studies to invent the conditions that allow such regeneration. Some of the factors controlling growth and differentiation of such cultures have been determined. The establishment of interactions among different groups of phytohormones, and growth regulators alone or in combinations are responsible for certain interrelations existing among cells, tissues and organs. So there seems to be consensus that the success in inducing differentiation depends upon the type of plant part ("explant"), the physiological condition of the explant and physical and chemical milieu of explant during culture. Due to this, the science of tissue culture has been directed to optimize the physiological conditions of source plant, the type of explant, the culture conditions and the phytohormones used to initiate tissue culture. This substantiates the fact that development of a new process for proliferation of plants by tissue culture is not obvious. Another major area where innovativeness is required in tissue culture, is identifying the plant part that efficiently responds to the culture conditions and leads to prolific regeneration. Not all plant parts of a given species are amenable to efficient regeneration. It is a complex combination of the explant selected identified for regeneration, physiological state of the explant, growth conditions and growth regulators that determines success of a plant in tissue culture. Different explants from a given plant usually show entirely different and often unpredictable response to growth conditions for proliferation. No general principles can be applied to achieve regeneration. In each case, identification of the explant and identification of the culture conditions are innovative steps in the development of a tissue culture method for regeneration of a plant part into a number of plants. Similarly application of tissue culture techniques for the production and biosynthesis of useful plant constituents has been exploited for the production of secondary metabolites from excised root culture, callus and by crown gall tissue in a number of plants. (West F R. Jr and Mike E S 1957. Synthesis of atropine by isolated roots and root callus cultures of belladona, Botan.Gaz. 119:50-54; Klein R M 1960, Plant tissue culture: a possible source of plant constituents, Econ. Botany 14: 286-289). For example cell suspension and callus cultures of Mentha piperita & M. spicata were reported to enable the production and biosynthesis of secondary metabolites (Lin and Staba 1961, Peppermint and spearmint tissue cultures, callus formation and submerged culture, Leoydia 24:139-145; Wang and Staba 1963, Peppermint and spearmint Tissue culture II: Dual-Carboy culture of spearmint Tissue. Jour of Pharmaceutical Science 52:1058-1062). The seeds of B/'xa orellana plant have a high content of an orange-red pigment, which consists of mainly of the apocarotenoid bixin and has been used for many years as a bread due and in vegetable oils and drinks (Dendy D.A.V. 1966, Annatto, the pigment of the Bixa orellana. East African Agric. Forest. J. 32(2): 126-132.). Demand for natural dyes continues to increase bixin is again being regarded in large quantities on the international market. Its economic importance warrants further study of B.orellana to obtain crops with greater pigment production., higher disease resistance and improved agronomic characteristics that will optimize field management. Bixin, the first cis-carotenoid to be isolated from natural sources, is the only one found in B.orellana. (Mercdante A.Z., Steck A, Pfander H, 1997, Isolation and identification of new apocarotenoids form annatto (Bixa orellana ) seeds. J. Agric. Food Chem. 45: 1050-1054). The chemical nature of this pigment, as well as that of other isoprenoids present is well known form studies o seed extracts (Mercdante A.Z., Steck A, Pfander H, 1997, Isolation and identification of new apocarotenoids form annatto (Bixa orellana ) seeds. J. Agric. Food Chem. 45: 1050-1054). Commercial plantations of this important species is raised through seedlings (Aparnathi K, Lata R, Sharma R. 1990, Annatto (Bixa orellana L): It s cultivation, preparation and usage. Intern. J. Trop Agric. 8: 80-88). Since the bixin or annatto yield generally varies from sample to sample, and from plant to plant, high yielding lines should be selected and vegetatively propagated for commercial plantations. In vitro techniques offer the possibility of rapid clonal propagation of important plants allowing production of true-to type and genetically stable progenies (Hu C.Y. and Wang P.J. 1983, hand book of plant cell culture, vol.1 (Evans, P.K. ed.) Mac Millan, New York, pp 177-227). In vitro regeneration of Bixa orellana may be used for the production of high yielding and productive genotypes and could accelerate tree-breeding programs. One major area where innovativeness is required in tissue culture, is identifying the plant part that efficiently responds to the culture conditions and leads to prolific regeneration. Not all plant parts of a given species are amenable to efficient regeneration. It is a complex combination of the explant selected identified for regeneration, physiological state of the explant, growth conditions and growth regulators that determines success of a plant in tissue culture. Different explants from a given plant usually show entirely different and often unpredictable response to growth conditions for proliferation. No general principles can be applied to achieve regeneration. In each case, identification of the explant and identification of the culture conditions are innovative steps in the development of a tissue culture method for regeneration of a plant part into a number of plants. B/xa orellana Linn.(Fam. Bixaceae) is an evergreen bush or small tree found native in tropical America. It is widely cultivated in many other tropical countries including southern India. The present invention deals with a tissue culture process for the development of a large number of plants from a specified part of B/xa orellana plant. The process of the present invention opens up new possibilities for producing highly efficient B/xa orellana plants with improved vegetative growth by using modern techniques of agrobiotechnology. The commercial extract of seeds of this plant is a rich source of the orange-red edible dye 'bixin' (annatto) and in purified form it is used in colouring butter, cheese, ice creams, bakery products, edible oils, food stuffs and also for cosmetic and pharmaceutical applications (Anonymous ,1948, The Wealth of India, Raw materials.Vol.1, CSIR, New Delhi, India, pp.196., Jondiko IJO, Pattenden G 1989, Terpenoids and apocarotenoids from seeds of B/xa orellana. Phytochemistry. 28: 3159-3162). There are many folklore medicinal uses of annatto (Kirtikar KR, Basu BD, Jain SK ,1975, B/xa orellana. Indian medicinal plant, Vol.1, 2nd edn. Delhi, India: Singh Publishers: 216-218., Duke JA ,1986, Isthmian Ethno botanical Dictionary, Scientific Publisher, Jodhpur, India, pp.25., De Feo V ,1992, Medicinal and magical plants in the northern Peruvian Andes. Fitoterapia 63: 417-440). Being a safe, economical and easy-to-use product, among naturally occurring colorants, annatto ranks second in economic importance. Apart from this the awareness and preference for natural colors increased annatto's value recently (Mercdante A.Z., Steck A, Pfander H, 1997, Isolation and identification of new apocarotenoids form annatto (Bixa orellana ) seeds. J. Agric. Food Chem. 45: 1050-1054). Commercial plantations of this important species is raised through seedlings. Since the bixin or annatto yield generally varies from sample to sample, and from plant to plant, high yielding lines should be selected and vegetatively propagated for commercial plantations. In vitro techniques offer the possibility of rapid clonal propagation of important plants allowing production of true-to type and genetically stable progenies (Hu C.Y. and Wang P.J. 1983, hand book of plant cell culture, vol.1 (Evans, P.K. ed.) Mac Millan, New York, pp 177-227). In vitro regeneration of Bixa orellana may be used for the production of high yielding and productive genotypes and could accelerate tree-breeding programs. The main objective of the present invention is to provide a simple process for large scale tissue culture based micropropagation of Bixa orellana. To date, four reports were available for regeneration of Bixa orellana through tissue culture. But the processes described earlier are not very efficient. The starting materials (explant) used in the earlier processes were different. For example, these processes have shoot buds, axillary buds and leaf pieces as the starting material. In this respect scanty reports on tissue culture of Bixa orrellana have been published. Even profuse callusing from the base of the explant without organogenic which normally hinders growth of the shoots is another drawback. Studies on the regeneration of Bixa orellana L. plantlets from axillary bud of seedlings (Ramamurthy, N., R.Savithramma, P.M.Swamy 1999, Multiple shoot induction and Regeneration of Japhara (Bixa orellana L.) through axillary bud derived callus cultures . J. Plant Biol. 26(3): 231-235; Marie Claire D'Souza and Madhuri Sharon. 2000 , In vitro clonal propagation of annatto (Bixa orellana L.) In Vitro Cell Dev Biol. -Plant 37: 168-172.), through intervening seed callus (. Sha Valli Khan P.S , E. Prakash and K.R. Rao. 2001, Callus induction and plantlet regeneration in Bixa orellana L,. an Annatto yielded tree. In Vitro Cell Dev-- Plant . 38: 186-190. ), and hypocotyls (De Paiva Neto, V.B., T.R. da Mota, W.C.Otoni. 2003, Direct organogenesis from hypocotyl-derived explants of annatto (Bixa orellana ). PI. Cell tissue and Organ Culture 75: 159-167. 2003) have been reported. These reports deal with the multiplication of shoots from pre-existing meristems in axis of leaves or through callus and up to 3-4 shoots could be obtained from nodal explant of Bixa orellana with poor reproducibly. Because leaf based protocols, however, are not efficiently reproducible and produce only a few shoots per explant. Although differentiation of shoots from callus was observed, the efficiency was extremely low with only 20-30% response. High frequency multiple shoot formation in Bixa orellana is not reported so far. So an efficient, simple, in vitro regeneration protocol is prerequisite for this plant. As conventional vegetative propagation of B/xa orellana plant is having limited in its potential for large scale propagation of elite plants, an efficient clonal propagation method of B/xa orellana has been sought which was not yet reported earlier for this plant. The present invention deals with a tissue culture process for the development of a large number of plants from a specified part of B/xa orellana plant. The process of the present invention opens up new possibilities for producing highly efficient B/xa orellana plants and also for hairy root cultures for secondary metabolites by using modern techniques of agro-biotechnology along with possibility of improving the color content of the seeds for value addition in food industry. Here, we report a feasible Micropropagation protocol of B/xa orellana using nodal sections as explants. The main object of the present invention is to provide a synergistic medium composition for Micropropagation of plants. Another object of the present invention is to investigate a case-by-case basis of the type of plant growth regulators and the amount of plant growth regulators that induce regeneration. Because the response to medium, hormones and growth conditions differs from plant species to species and variety to variety. Thus inventing conditions for efficient regeneration of plants, requires developing specialized knowledge about a given plant. Yet another object of the present invention is to develop a culture medium to obtain in vitro rooting of shoots. Still yet another objective of the present invention is to provide an improved growth and yield of color of seeds of B/xa orellana which provides better economic value. Accordingly the present invention relates to a synergistic medium composition for Micropropagation of plants which comprises; i) Medium A for induction of shoots which comprises i. NH4NO3 ii. KNO3 iii. H3BO4 iv. KH2PO4 v. Kl vi. Na2MoO4.2H20 vii. CoCI2 2H2O viii. CaCI2.2H2O ix. MgSO4.4H2O x. ZnSO4. 7 H20 xi. CuSO4.5H20 xii. FeNaEDTA. xiii. Thiamine HCI xiv. Pyridoxine HCI xv. Nicotinic acid xvi. Glycine xvii. Myoinositol xviii. indole-3-acetic acid xix. 6-benzyladenine xx. phenylacetic acid xxi. Triacontanol xxii. sucrose xxiii. agar for gelling 1400-1800 mg/l, 1500-2500 mg/l, 3-8 mg/l, 100-250 mg/l, 0.4-1.0 mg/l, 0.1-0.5 mg/l, 0.01-0.04 mg/l, 300-360 mg/l, 150-250 mg/l, 5-10 mg/l, 0.01-0.05, 30-45 mg/l, 0.5-2.0 mg/l, 0.5-2.0 mg/l, 0.5-2.0 mg/l, 1.0-3.0 mg/l, 75-200.0 mg/l, 0.01 -1.0 mg/l 1-4 mg/l 1-7 mg/l, 0.002-0.02 mg/l 20,000- 40,000 mg/l, 6000-8000 mg/l ii) Medium B for elongation of shoots or shoot buds which comprises a) NH4NO3 1400-1800 mg/l, b) KNO3 1500-2500 mg/l, c) H3BO4 3-8 mg/l, d) KH2PO4 100-250 mg/l, e) Kl 0.4-1.0 mg/l, f) Na2Mo04.2H20 0.1-0.5 mg/l, g) CoCI22H2O 0.01-0.04 mg/l, h) CaCI2.2H2O 300-360 mg/l, i) MgSO4.4H2O 150-250 mg/l, j) ZnSO4. 7H2O 5-10 mg/l, k) CuSO4.5H20 0.01-0.05, I) FeNaEDTA. 30-45 mg/l, m) Thiamine HCI 0.5-2.0 mg/l, n) Pyridoxine HCI 0.5-2.0 mg/l, o) Nicotinic acid 0.5-2.0 mg/l, p) Glycine 1.0-3.0 mg/l, q) Myoinositol 75-200.0 mg/l, r) indole-3-butyric acid 0.01-1.0 mg/l s) 6-benzyladenine or 2-isopentenyladenine 1-4 mg/l t) Phenyl acetic acid 1-7 mg/l u) sucrose 20,000- 40,000 mg/l, v) agar for gelling 6000-8000 mg/l, iii) Medium C for in vitro rooting of elongated shoots, which comprises a) NH4NO3 1400-1800 mg/l, b) KNO3 1500-2500 mg/l, c) H3BO4 3-8 mg/l, d) KH2PO4 100-250 mg/l, e) Kl 0.4-1.0 mg/l, f) Na2MoO4.2H20 0.1-0.5 mg/l, g) CoCI22H2O 0.01-0.04 mg/l, h) CaCI2.2H2O 300-360 mg/l, i) MgSO4.4H2O 150-250 mg/l, j) ZnSO4. 7H2O 5-10 mg/l, k) CuSO4.5H20 0.01-0.05, I) FeNaEDTA. 30-45 mg/l, m) Thiamine HCI 0.5-2.0 mg/l, n) Pyridoxine HCI 0.5-2.0 mg/l, o) Nicotinic acid 0.5-2.0 mg/l, p) Glycine 1.0-3.0 mg/l, q) Myoinositol 75-200.0 mg/l, r) indole-3-butyric acid or indole-3-acetic acid or 1-naphthalene acetic acid or Phenylacetic acid 0.5-3.0 mg/l s) sucrose 20,000- 40,000 mg/l, t) agar for gelling 6000-8000 mg/l The novelty of the present invention is it provides for the first time an efficient media composition for micropropagation of Bixa orellana from the shoot tip with single node explant, giving a large number of plants. This is a potentially very useful in plant biotechnology for micro propagation, selecting variants and in the field of genetic transformation and agrobiotechnology. In the present invention : The shoot initiation from shoot tip with single node explants was obtained on MS medium containing, sucrose, biotin, kinetin/6-benzyladenine alone or in combination with triacontanol at varied concentrations after incubating at optimum growth conditions. Subsequently the primary shoots were elongated on medium comprising MS salts, vitamins, cytokinin 6-benzyladenine (BA), auxin indole-3-acetic acid, triacontanol, phenylacetic acid, sucrose, at varied concentrations and incubated at 16:8 photoperiod. Regenerated plantlets were rooted on an rooting medium containing modified MS salts, sucrose, indole-3-butyric acid, indole-3-acetic acid, naphthalene acetic acid, phenylacetic acid at varied concentrations. Seeds from matured dried fruits In vitro germination of seeds on MS basal medium Shoot tip with single node explarlts/shoot tips/nodal explants from seedlings Shoot proliferation on MS medium containing cytokinins+auxins/ triacontanol Elongation of primary shoot on MS medium containing IBA+BA In vitro plantlets rooted on. rooting medium fortified with IBA Hardening and field transfer The following examples are given by way of illustration of the present invention Example. 1 Seeds were collected from the matured just dried fruits (capsule) of Bixa orellana L. (Pink color flowers producing plant variety), from 2 years old garden grown plant. Decontamination of seeds performed by removing from its surface any contaminant which is potentially harmful to the tissue culture process, by Pre-treatment of the seeds with 0.5% bavistin solution (fungicide) for 2 hrs on a rotary shaker at 90 rpm in dark at 25 -27 °C and thorough washing with sterile distilled water twice. Soaking of the above said seeds in warm water (65 °C ) for one and half hour and leaving it in the same water for 7 days at room temperature in dark to facilitate leaching out of carotenoids present below the seed coat into the water. Subsequent treatment to soaked seeds with 2-3 drops of Tween 20 and 1% NaOCI (v/v) for 15 minutes, to get rid of any fungi or bacteria that might have thrived during the soaking period, and followed by three washes of 5 minutes each with sterile double distilled water. Final treatment to seeds with 0.1% HgCI2 (w/v) for 5 min after which thorough wash with sterile water 5 times. The seedlings that emerged were separated after 8 weeks and the shoot tip with single node, shoot tip explants and nodal explants (1-1.5 cm) were used as explants. Later the explants were inoculated onto Murashige and Skoog medium (1962) containing macro salts and micro salts along with vitamins, the growth regulator BA at 2-4 mg/l concentration in combination with indole acetic acid at 0.001-1.0 mg/lconcentration, Triacontanol at 0.002-0.20 mg/l concentration, gelling agent agar (0.7%) or phytagel ( 0.2%) w/v. The pH was adjusted at 5.7 prior to autoclaving at 121.degree.0 C., 15 Ib/inch.sup.2for20 min. The explants were placed on the medium with the help of sterile forceps in laminar flow. Cultures were incubated at 25±2°C. in light (1000-10,000 lux ) 16 h photoperiod. Culturing continued till shoots initiating out of it. Initiation of shoots occurred within eight weeks time with a frequency of 40-80%. In the absence of cytokinin type growth regulators or in their presence at a low concentration (below 1.0 mg/l BA or 2iP), differentiation of shoots from explant could not occur. However, on medium containing BA (1.0 mg/l) along with IAA (0.01 mg/l) and TRIA (0.005mg/l) several shoots (8 to 9) were initiated in eight weeks time in culture along with callusing and many shoot buds. Again at higher concentration of BA(2.0 mg/l) along with IAA (0.001 mg/l) very few shoots were produced. For harvesting the shoots, the cultures were taken out of the culture vessels and shoots were cut with the help of a sharp scalpel blade in a laminar flow. The shoots were again cultured on shoot elongation medium containing Murashige and Skoog salts and vitamins, sucrose 3% w/v, auxin type growth regulator indole butyric acid (1-5 mg/l), BA (0.5-3.0 mg/l) and gelling agent agar 0.7% w/v. The pH was adjusted to 5.8 prior to autoclaving at 121°C., 15 Ib/cm2 for 20 min. After 8 weeks culturing at 16:8 hrs photoperiod maximum number of shoots were elongated on medium containing 1.5 mg/l of BA and 1 mg/l of IBA. Shoots were then separated aseptically under laminar flow and transferred to a culture medium containing Murashige and Skoog salts and vitamins, sucrose 3% w/v, auxin type growth regulator IBA/ lAA/NAA/PAA (1-5 mg/l), and gelling agent agar 0.7% w/v. The pH was adjusted to 5.7 prior to autoclaving at 121.°C., 15 Ib/inch 2 for 20 min. For promoting formation of roots, the cultures were incubated in the above medium at 25±2°C. in light (40 mico mol/m2 s) 16 hr. photoperiod. Culturing was continued till roots were formed. Well developed root system was formed within 4-5 weeks time on medium with 1.0 mg/l of IBA , when the plantlets were ready to transfer into soil. The plants were acclimatized for autotrophic growth, prior to transfer in soil. Table. 1 Influence of Triacontanol on callusing and shoot buds formation from single node shoot tip explant's base of Bixa orellana L. in vitro (Table Removed) acallusing from the base of explant common in all hormonal combinations except MS basal medium. Values represent the means ± the standard deviation. * significant, ** highly significant at p Table.2 Elongation of in vitro shoot buds of Bixa orellana under the influence of BA and IBA combination (Table Removed) The inoculum means the hard callus stump with 25-30 shoot buds Values represent the means ± the standard deviation. * significant, ** highly significant at p Table.3 In vitro rooting of B. orellana microshoots under the influence of auxins and PAA (after 30 days) ± SEM (Table Removed) Values represent the means ± the standard deviation. * significant, ** highly significant at p Example.2 Seeds were collected from the matured just dried fruits (capsule) of Bixa orellana L. (Pink color flowers producing plant variety), from 2 years old garden grown plant. Decontamination of seeds performed by removing from its surface any contaminant which is potentially harmful to the tissue culture process, by Pre-treatment of the seeds with 0.5% bavistin solution (fungicide) for 2 hrs on a rotary shaker at 90 rpm in dark at 25 -27 degree C and thorough washing with sterile distilled water twice. Soaking of the above said seeds in warm water (65 °C ) for one and half hour and leaving it in the same water for 7 days at room temperature in dark to facilitate leaching out of carotenoids present below the seed coat into the water. Subsequent treatment to soaked seeds with 2-3 drops of Tween 20 and 1% NaOCI (v/v) for 15 minutes, to get rid of any fungi or bacteria that might have thrived during the soaking period, and followed by three washes of 5 minutes each with sterile double distilled water. Final treatment to seeds with 0.1% HgCb (w.v) for 5 min after which thorough wash with sterile water 5 times. The seedlings that emerged were separated after 8 weeks and the shoot tip with single node, shoot tip explants and nodal explants (1-1.5 cm) were used as explants. Later the explants were inoculated onto Murashige and Skoog medium (1962) containing macro salts and micro salts along with vitamins, the growth regulator BA at 2.22-13.32uM concentration in combination with indole acetic acid at 0.057-5.7uM concentration, or MS medium with PAA at 2-15uM; BA at 2.22-31.08uM concentration, gelling agent agar (0.7%) or phytagel ( 0.2%) w/v. The pH was adjusted at 5.7 prior to autoclaving at 121.degree.0 C., 15 lb/inch.sup.2 for 20 min. The explants were placed on the medium with the help of sterile forceps in laminar flow. Cultures were incubated at 25±.2.degree. C. in light (1000-10,000 lux) 16 h photoperiod. Culturing continued till shoots initiating out of it. Initiation of shoots occurred within eight weeks time with a frequency of 40-100%. In the absence of cytokinin type growth regulators or in their presence at a low concentration (below 4.44 BA or 4.92uM 2iP), differentiation of shoots from explant could not occur. However, on medium containing BA (22.22uM) along with PAA (2 mg/l) almost 100% explants responded by producing several shoots (3-5) were initiated in eight weeks time in culture along with moderate callusing and many shoot buds. Again at higher concentration of BA (7 mg/l) along with PAA (14.68uM) very few shoots were produced. For harvesting the shoots, the cultures were taken out of the culture vessels and shoots were cut with the help of a sharp scalpel blade in a laminar flow. The shoots were again cultured on shoot elongation medium containing Murashige and Skoog salts and vitamins, sucrose 3% w/v, auxin type growth regulator indole butyric acid (2.22-8.88uM), BA (2.22-13.32uM) and gelling agent agar 0.7% w/v. The pH was adjusted to 5.8 prior to autoclaving at 121.degree. C., 15 lb/cm.sup.2 for 20 min. After 8 weeks culturing at 16:8 hrs photoperiod maximum number of shoots were elongated on medium containing 6.66uM of BA and 4.44uM of IBA. Shoots were then separated aseptically under laminar flow and transferred to a culture medium containing Murashige and Skoog salts and vitamins, sucrose 3% w/v, auxin type growth regulator IBA/ lAA/NAA/PAA (2-15uM), and gelling agent agar 0.7% w/v. The pH was adjusted to 5.7 prior to autoclaving at 121.degree. C., 15 lb/inch.sup.2 for 20 min. For promoting formation of roots, the cultures were incubated in the above medium at 25.±.2.degree. C. in light (40 .uM ol/m.sup.2 s) 16 hr. photoperiod. Culturing was continued till roots were formed. Well developed root system was formed within 4-5 weeks time on medium with 1 mg/l of IBA , when the plantlets were ready to transfer into soil. The plants were acclimatized for autotrophic growth, prior to transfer in soil. Table.4 Effect of BA and PAA on multiple shoot formation from single node shoot tip explants of Bixa orellana in vitro (after 60 days) ±SEM (Table Removed) Values represent the means ± the standard deviation. * significant, ** highly significant at p Example 3 Seeds were collected from the matured just dried fruits (capsule) of Bixa orellana L. (Pink color flowers producing plant variety), from 2 years old garden grown plant. Decontamination of seeds performed by removing from its surface any contaminant which is potentially harmful to the tissue culture process, by Pre-treatment of the seeds with 0.5% bavistin solution (fungicide) for 2 hrs on a rotary shaker at 90 rpm in dark at 25 -27 degree C and thorough washing with sterile distilled water twice. Soaking of the above said seeds in warm water (65 °C ) for one and half hour and leaving it in the same water for 7 days at room temperature in dark to facilitate leaching out of carotenoids present below the seed coat into the water. Subsequent treatment to soaked seeds with 2-3 drops of Tween 20 and 1% NaOCI (v/v) for 15 minutes, to get rid of any fungi or bacteria that might have thrived during the soaking period, and followed by three washes of 5 minutes each with sterile double distilled water. Final treatment to seeds with 0.1% HgCI2 (w.v) for 5 min after which thorough wash with sterile water 5 times. The seedlings that emerged were separated after 8 weeks and the shoot tip with single node, shoot tip explants and nodal explants (1-1.5 cm) were used as explants. Later the explants were inoculated onto Murashige and Skoog medium (1962) containing macro salts and micro salts along with vitamins, gelling agent agar (0.7%) or phytagel ( 0.2%) w/v. The pH was adjusted at 5.7 prior to autoclaving at 121.degree.0 C., 15 lb/inch.sup.2 for 20 min. The explants were placed on the medium with the help of sterile forceps in laminar flow. Cultures were incubated at25.±.2.degree. C. in light (1000-10,000 lux) 16 h photoperiod. Even after 60 days of culturing none of the shoot and nodal explants responded for shoot organogenesis. More over most of these explants turned to brown. Event the shoot tip explants not responded and wilted. So hormones incorporation is necessary for proliferation of shoots from nodal explants and also for growth of shoot tip. Example 4 Developed seedling based plantlets by sowing the fresh seeds of B. orellana in garden soil. Separation of 12-15 cm long seedlings and planting in soil with a distance of at least 2 months. Later Measurement of vegetative growth of the said micropropagated and filed grown plants and in vivo seedling based plants was done.Tissue cultured and normal field-grown plants started flowering 18 months after planting. The total colour content of Bixa seeds obtained from fruits of 70days old (just before turning to brown) were extracted in dark with 1:10 (w/v) chloroform. The extracts were evaporated in vacuo and resuspended with 1 ml of chloroform. The total colour content was estimated according to Me Keown G.G. and Mark F. 1962; The composition of oil soluble annatto food colours J.A.O.A.C. 45: 761-766). (Formula Removed) Where Asoo is absorption at 500nm A404 is absorption at 404nm V is final volume W is weight of the sample taken Table.5 Total colour content and morphological differences between tissue culture and normal field grown plants (Table Removed) Table.6 Total colour content and morphological features of tissue cultured field grown plants (Table Removed) After 21 months the control filed grown plants were with a shoot length (height) of 200 cm and 15 branches which was almost same but with marginal betterment with the tissue cultured field grown plant of the same age. From this study it is clear that the improvement of growth of a plant by using tissue cultured plants. So the 8/xa orellana plants can be developed by micopropagation and the improvement of the vegetative growth and yield of seeds with containing altered/unaltered levels of colour useful as food colourant. In accordance with the various aspects of this invention, an easy, efficient and rapid method is provided for inducing shoots at high frequency. The process of this invention provides differentiation and offers many advantages over the prior art. The reproducibility and rapidity clonal propagation and the chance in the level of flavour metabolites obtainable routinely by this process is expected to facilitate genetic transformation of 6/xa orellana via Agrobacterium and/or biolistic based transformation techniques. An additional advantage of this invention is that only one explant gives several shoots within one or subsequent step. Mass propagation as well as selection of mutants can now be expedited with the application of this invention. Influence of triacontanol in enhancing shoot multiplication and rooting of in vitro derived shoot tips of Capsicum frutescens and Decalepis hamiltonii W & A were studied earlier by the same laboratory (Reddy, BO, Giridhar P and Ravishankar GA 2002, The effect of triacontanol on micropropagation of Capsicum frutescens.L and Decalepis hamiltonii W & A. Plant Cell Tissue and Organ Culture , 71, 2002, 253-258. ). In both shoot multiplication and rooting phases, triacontanol was administered at 2-20ug I"1. Triacontanol fostered optimal multiple shoot formation, axillary shoot proliferation at 2 ug I"1 while the rooting was effective at 5 ug I"1. Triacontanol enhanced shoot growth and chlorophyll content of leaves and also influenced root induction and supported growth of the roots. This work reveals that triacontanol can be used as an effective growth regulator in the micropropagation of Capsicum frutescens and Decalepis hamiltonii; an endangered shrub of Deccan peninsular India (Reddy, BO, Giridhar P and Ravishankar GA 2002, The effect of triacontanol on micropropagation of Capsicum frutescens.L and Decalepis hamiltonii W & A. Plant Cell Tissue and Organ Culture , 71, 2002, 253-258.).. Similarly the phenylacetic acid in combination with 6-benzyladenine had a positive influence on the proliferation of shoots from axillary buds of nodal explants of Decalepis hamiltonii. Even the combination of these two hormones may show adventitious shoot formation along with elongation of primary shoot as we reported earlier (Giridhar P, Vijayaramu D, Obul Reddy B, Rajasekaran, T and Ravishankar GA, 2003, Influence of phenylacetic acid 9on clonal propagation of Decalepis hamiltonii Wight and Am., an endangered shrub. In Vitro Cell Dev. Biol. -Plant 39: 463-467.) In accordance with the various aspects of this invention, an easy, efficient and rapid method is provided for inducing shoots at high frequency. The process of this invention provides differentiation and offers many advantages over the prior art. The reproducibility and rapidity clonal propagation and the change in the level of colour content of seeds obtainable routinely by this process is expected to facilitate genetic transformation of Bixa orellana via Agrobacterium and/or biolistic based transformation techniques. An additional advantage of this invention is that only one explant gives several shoots within one or subsequent step. Mass propagation as well as selection of mutants can now be expedited with the application of this invention. The main advantages of the present invention are: 1. The development of efficient medium for shoot regeneration from nodal/shoot tip / shoot tip with node explants. 2. In vitro rooting of regenerated shoots. 3. Healthy and efficient plants will be obtained by using this protocol. 4. According to this protocol a maximum of 6 months time is required to get tissue culture derived plants. We claim: 1) A synergistic medium composition for micropropagation of plants which comprises; i) Medium A for induction of shoots which comprises a. NH4NO3 1400-1800 mg/l, b. KNO3 1500-2500 mg/l, c. H3BO4 3-8 mg/l, d. KH2PO4 100-250 mg/l, e. Kl 0.4-1.0 mg/l, f. Na2MoO4.2H20 0.1-0.5 mg/l, g. CoCI22H20 0.01-0.04 mg/l, h. CaCI2.2H20 300-360 mg/l, i. MgS04.4H20 150-250 mg/l, j. ZnS04. 7H20 5-10 mg/l, k. CuSO4.5H20 0.01-0.05, I. FeNaEDTA. 30-45 mg/l, m. Thiamine HCI 0.5-2.0 mg/l, n. Pyridoxine HCI 0.5-2.0 mg/l, o. Nicotinic acid 0.5-2.0 mg/l, p. Glycine 1.0-3.0 mg/l, q. Myoinositol 75-200.0 mg/l, r. indole-3-acetic acid 0.01-1.0 mg/l s. 6-benzyladenine 1-4 mg/l t. phenylacetic acid 1-7 mg/l, u. Triacontanol 0.002-0.02 mg/l v. sucrose 20,000- 40,000 mg/l, w. agar for gelling 6000-8000 mg/l ii) Medium B for elongation of shoots or shoot buds which comprises a) NH4NO3 1400-1800 mg/l, b) KNO3 1500-2500 mg/l, c) H3BO4 3-8 mg/l, d) KH2PO4 100-250 mg/l, e) Kl 0.4-1.0 mg/l, f) Na2MoO4.2H20 0.1-0.5 mg/l, g) CoCI22H20 0.01-0.04 mg/l, h) CaCI2.2H20 300-360 mg/l, i) MgS04.4H20 150-250 mg/l, j) ZnS04. 7H20 5-10 mg/l, k) CuSO4.5H20 0.01-0.05, I) FeNaEDTA. 30-45 mg/l, m) Thiamine HCI 0.5-2.0 mg/l, n) Pyridoxine HCI 0.5-2.0 mg/l, o) Nicotinic acid 0.5-2.0 mg/l, p) Glycine 1.0-3.0 mg/l, q) Myoinositol 75-200.0 mg/l, r) indole-3-butyric acid 0.01-1.0 mg/l s) 6-benzyladenine or 2-isopentenyladenine 1-4 mg/l t) Phenyl acetic acid 1-7 mg/l u) sucrose 20,000- 40,000 mg/l, v) agar for gelling 6000-8000 mg/l, iii) Medium C for in vitro rooting of elongated shoots, which comprises a) NH4NO3 1400-1800 mg/l, b) KNO3 1500-2500 mg/l, c) H3BO4 3-8 mg/l, d) KH2PO4 100-250 mg/l, e) Kl 0.4-1.0 mg/l, f) Na2MoO4.2H20 0.1-0.5 mg/l, g) CoCI22H20 0.01-0.04 mg/l, h) CaCI2.2H20 300-360 mg/l, i) MgS04.4H20 150-250 mg/l, j) ZnS04. 7H20 5-10 mg/l, k) CuSO4.5H20 0.01-0.05, I) FeNaEDTA. 30-45 mg/l, m) Thiamine HCI 0.5-2.0 mg/l, n) Pyridoxine HCI 0.5-2.0 mg/l, o) Nicotinic acid 0.5-2.0 mg/l, p) Glycine 1.0-3.0 mg/l, q) Myoinositol 75-200.0 mg/l, r) indole-3-butyric acid or indole-3-acetic acid or 1 -naphthalene acetic acid or Phenylacetic acid 0.5-3.0 mg/l s) sucrose 20,000-40,000 mg/l, t) agar for gelling 6000-8000 mg/l 2. A synergistic medium composition for micropropagation of plants substantially as herein described with reference to the examples and the figures accompanying the specification.

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592-DEL-2004-Abstract-(02-06-2009).pdf

592-del-2004-abstract.pdf

592-DEL-2004-Claims-(02-06-2009).pdf

592-DEL-2004-Claims-(18-06-2009).pdf

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592-DEL-2004-Correspondence-Others-(02-06-2009).pdf

592-DEL-2004-Correspondence-Others-(18-06-2009).pdf

592-del-2004-correspondence-others.pdf

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592-del-2004-description (complete).pdf

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592-del-2004-form-18.pdf

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592-DEL-2004-Form-3-(02-06-2009).pdf

592-del-2004-form-3.pdf

592-del-2004-form-5.pdf