Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-03-08
2001-11-27
Campell, Bruce R. (Department: 1661)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S468000, C435S469000, C435S470000, C800S293000, C800S294000
Reexamination Certificate
active
06323394
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to a tissue culture process for producing a large number of viable mint plants in vitro. The process of the present invention employs specified pieces of stem of the mint plant as the starting material and identifies media and culture conditions for producing a large number of plants. Such plants can be used for micropropagation, selection of mutants, production of plants with altered levels of endogenous secondary metabolites and for genetic engineering.
BACKGROUND OF THE INVENTION
Various species of mint plants are grown primarily in India, China and Japan commercially for mint oil, menthol, vitamins and other metabolites that are valuable to the pharmaceutical industry. These are used in aromatic oils and herbal medicines. The present invention deals with a tissue culture process for the development of a large number of plants from a specified part of mint plant. The process of the present invention opens up new possibilities for producing somaclonal and physiological variants and for genetic improvement of mints by modern techniques of agrobiotechnology.
Mints are of interest globally because of their valuable secondary metabolites, especially mint oils and menthol for the industry. Since it is a vegetatively propagated crop, mutation techniques have largely been applied to improving characteristics like disease resistance, yield, metabolites and oil content. However, it is possible to apply tissue culture techniques for the improvement of mints by the selection of somaclonal variants and genetic engineering. The main objective of the present invention is to provide a simple process for large scale tissue culture based micropropagation of mints. Another objective of the present invention is to provide a powerful tool for the isolation of physiological variants, somaclones and mutants and for genetic manipulation of mints.
Plant regeneration by tissue culture techniques is well established. A wide varied 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 a 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.
One major aspect that has to be investigated on a case-by-case basis is the type of plant growth regulators and the amount of plant growth regulators that induce regeneration. Besides, chemical composition of the medium, temperature and other culture conditions play an important role in the induction of organogensis and somatic embryogenesis and their maturation to healthy fertile plants thereof. 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.
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.
To date, regeneration of many species and cultivars of Mentha has been reported 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 utilized axillary buds, leaf pieces and embryos as the starting material. In this respect several reports on tissue culture of mints have been published. Some of these are also related to the establishment of cell suspension cultures and callus, and are listed below for convenience and reference. Application of tissue culture techniques for the production and biosynthesis of useful plant constituents has been exploited for the production of alkaloids 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 ballandona, Botan.Gaz. 119:50-54; Klein R M 1960, Plant tissue culture: a possible source of plant constituents, Econ. Botany 14: 286-289 ). 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). Such cell suspensions were later reported to biotransform certain precursors into monoterpenes (Aviv D and Gulan E 1978, Biotransformation of monoterpenes by Mentha cell lines: Conversion of pulegone to isomenthone. Planta Medica 33;70-77; Aviv D, Krochmal E, Dantes A and Gulan E. 1983, Biotransformation of monoterpene by Mentha cell lines conversion of pulegone-substituents and related unsaturated &agr;-&bgr; ketones. Planta Medica 47:7-10). Triterpenes were reported to be produced by callus tissue of
Mentha arvensis
(Karasawa D and Shimzu S 1980, Triterpene acids in callus tissue from
Mentha arvensis
var. Piperascens.Mol.Agric.Biol Chem.44: 1203-1205). Small quantities of menthol were also detected in suspension cultures and callus cultures with the help of GLC TLC.(Bhaumik C and Dutta P C 1982, Menthol in static and suspension cultures of
Mentha arvensis
Lin: var piperascens Holmes: Indian Drugs (July) 387-388). These reports did not aim at the regeneration of plants from the above said cultures.
Multiplication of shoots from axillary buds of Mentha spp was reported by tissue culture techniques (Rech E L and Pires M J P 1986. Tissue culture propagation of Mentha spp. by the use of axillary buds. Plant Cell Reports 5:17-18, Revishankar G A and Venkatraman L V 1988. Rapid multiplication of plants from cultured auxillary buds of
Mentha piperita.
Philippine Jour. of Science 117:121-129). These reports deal with the multiplication of shoots from pre-existing meristems in axis of leaves,
Bhat Savithri
Gupta Shiv Kumar
Kumar Sushil
Tuli Rakesh
Birch & Stewart Kolasch & Birch, LLP
Campell Bruce R.
Council of Scientific & Industrial Research
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