Patent Grant
PP16474
The present invention relates to a new and distinct mint plant of the genus Mentha, species piperita, designated the variety ‘Cim Indus’.
This variety was selected through screening, field trial and analysis of monoterpene constituents of the essential oil of the germplasm. This variety possesses the characters of producing high amount of menthofuran ranging between 22 to 30% of total oil content, high amount pulegone ranging between 9.0 to 18% of total oil content, with essential oil content ranging between 0.32 to 0.40% of the total oil content.
Menthofuran (3,6-dimethyl-4,5,6,7-tetrahydrocoumarone) is one of the major constituent for aroma of the essential oil extracted from the leaves of Mentha piperita. Because any other compound has not duplicated the aroma effect, menthofuran is important in the formulation of certain standardized essential oils, such as peppermint oil. However, menthofuran is an expensive compound of limited availability as the plants produce 0 to 6% menthofuran (U.S. Plant Pat. No. PP11,788). Literatures are available for the chemical synthesis of menthofuran to substitute the naturally available menthofuran (U.S. Pat. No. 4,240,969) to reduce cost of production. Also the acceptability of synthetic menthofuran is a limiting factor in determining the cost of the essential oil mixture containing synthetic components in aroma industry.
Considering the importance of menthofuran in aroma industry under ‘New Millennium Indian Technology Initiative (NMITLI) programme’ launched by Council of Scientific and Industrial Research (CSIR), India, during 2001, a systematic approach was undertaken to evaluate the existing germplasm of M. piperita at CIMAP and breed for genetic enhancement towards high menthofuran biosynthesis in the essential oil. Systematic breeding experiments to allow open pollination followed by single seed progeny selection by chemotypic evaluation for enhanced constituent (menthofuran) led to development of this chemotype the variety ‘Cim Indus’.
The main object of the present invention is to develop a new and distinct mint plant.
Another main object of the present invention is to develop a novel mint plant through screening, field trial and analysis of monoterpene constituents of the essential oil of the germplasm.
Yet another object of the present invention is to develop a plant producing high amount of menthofuran.
Still another object of the present invention is to develop a plant producing high amount pulegone.
Still another object of the present invention is to develop a plant producing high herbage.
Still another object of the present invention is to develop a mint plant showing resistance against major plant disease conditions like leaf spot, rust, powdery mildew, lepidopteran pest Spilarctia obliqua.
The present invention relates to a new and distinct mint plant of Mentha piperita ‘Cim Indus’, selected through screening, field trial and analysis of monoterpene constituents of the essential oil of the germplasm, possessing the characters of producing high amount of menthofuran ranging between 22 to 30% of total oil content, high amount pulegone ranging between 9.0 to 18% of total oil content, with essential oil content ranging between 0.32 to 0.40% of the total oil content.
Accordingly, the present invention relates to a new and distinct mint plant of Mentha piperita ‘Cim Indus’, selected through screening, field trial and analysis of monoterpene constituents of the essential oil of the germplasm, possessing the characters of producing high amount of menthofuran ranging between 22 to 30% of total oil content, high amount pulegone ranging between 9.0 to 18% of total oil content, with essential oil content ranging between 0.32 to 0.40% of the total oil content.
The plant was developed at the CIMAP research farm, Lucknow, India and asexually propagated through suckers for further planting in the same farm. By appearance, this new variety is a typical plant of Mentha piperita. The difference between this new variety and the parent or other known varieties is in the herbage at menthofucan, pulegone and other essential components. Thus, this is a new and distinct mint plant of Mentha piperita variety ‘Cim Indus’, selected through screening, field trial and analysis of monoterpene constituents of the essential oil of the germplasm, possessing the following combination of characters:
The flowers are arranged in whorls and the inflorescence grow which vary in length. The flowers include:
The leaves have predominantly the carvone and menthofuran smell. This variety produces no fruit and no seeds.
The present invention is related to the development of a novel high menthofuran and pulegone producing chemotype which can also yield high amount of pulegone through proper harvest management. The plant chemotype was obtained through screening of the open pollinated seed progenies of the variety ‘Kukrail’. The invention is further related to the plant producing more herb yield leading to higher production of essential oil per unit area compared to the seed parent variety. The selected plant possesses the property of accumulating more menthofuran and pulegone at specific developmental stages and hence proper management prior to processing can yield high amount of these important phytochemicals for industrial use. This plant is unique and clearly distinct from all other existing varieties of Mentha piperita. The new plant type ‘Cim Indus’ can be propagated vegetatively through suckers and runners for commercial cultivation.
‘Kukrail’ is a released variety of CIMAP which is maintained along with the germplasm of CIMAP in the field systemically every year. Every year in the month of October, the twigs are planted in small sized plots (3 m×3 m) for generation of enough planting material for planting in the main field during the month of January. Open pollinated seeds are collected from different genotypes every year and analyzed for monoterpene constituents in the essential oil. ‘Cim Indus’ also known as CIMAP/MP20 is such a genotype selected from open pollinated seed progenies of the variety ‘Kukrail’. Mentha piperita is propagated vegetatively through runners. With the NMITLI initiative the runners generated from the seed plots were planted in 5 m×5 m plots during the month of January 2001, following normal agronomic practices with the objective to screen genotypes rich in menthofuran in the essential oil. Replicated samples from each genotypes were taken from the field by planting multiplied runners in the month of January 2001, 2002 and 2003 for 3 consecutive years in RBD fashion and different growth and yield characteristics were recorded (Table 1). For yield trials the replicated plots were prepared by adding only FYM 1.5 ton per ha. The three-year averages of herb yield, essential oil yield and the variations in major essential oil components are detailed below for the genotype (CIMAP/MP20 compared to the CIMAP released varieties of Mentha piperita ‘Kukrail’, ‘Tushar’, ‘Pranjal’. ‘Pranjal’ bears the U.S. Pat. No. 14,090.
If menthofuran is aimed the genotype can yield highest amount of natural menthofuran than any other variety released and reported which is the case for pulegone also.
Oil Extraction and GLC Analysis
Oil samples from the field grown plants were extracted by hydrodistillation using Clevenger's apparatus and weighed to record the yield. Over ground shoot samples were collected from the whole plant selected randomly from the middle of the row of each replicated plots at different days after planting (35. 55, 75, 95, 115 days after planting). Shoots collected from individual genotypes were bulked for each treatment plot and essential oil was distilled from all the replicates taking 500 g of bulked shoots containing leaves. The final analysis of all the essential oil samples was accomplished on Varian CX-3400 using a 30 m×0.25 mm Supelcowax-10 column. The injector and detector temperature were maintained at 200 and 225° C. respectively, with oven temperature programmed from 60 to 200° C. at the rate of 7° C. min−1 increase, with initial and final holds of 2 and 5 minutes respectively. Hydrogen gas was used as carrier at the rate of 1 ml min−1 and an aliquot of the sample was injected with a split ratio of 1:50. Data were processed in the electronic integrator Varian 4400 and the identification was based on retention time of authentic samples of 1-menthol (Takasago, Japan) and retention indices calculations (Jennings W & Shibamoto T (1980) Qualitative analysis of flavour and fragrance volatile by capillary GC, Academic Press Inc., New York.).
piperita genotype CIMAP/MP20 at different stages of growth.
The genotype CIMAP/MP20 has a characteristic oil profile which expresses differentially at different stages of growth. The menthofuran content was found to be higher at 75 days stage which decreased during 95 days and again increased during harvesting time 115 days. Corresponding menthol content in the essential oil content was found to be negatively correlated to the menthofuran content at corresponding stages of growth.
Pulegone content increased after 75 days and was stabilized after 95 to 115 days (Table 2). The comparative monoterpene profiles for different components is presented in Table 3 during harvesting time (115 days stage).
Trichome Analysis of the Genotype CIMAP/MP20
Monoterpenes are known to be cytotoxic to plant tissues, inhibiting respiration and photosynthesis by drastically affecting the mitochondria, golgi bodies etc and decreasing cell membrane permeability (Brown J T, Hegarty P K & Charlwood B V (1987) The toxicity of monoterpenes to plant cell cultures. Plant Science 48:195-201.). Monoterpenes are either sequestered in the plants in specialized structures like glandular hairs in Pelargonium (Brown J T & Charlwood B V (1986) Differentiation and monoterpene biosynthesis in plant cell cultures. In; Morris P, Scragg A, Stafford A and Fowler M (eds) Secondary Metabolism in Plant Cell Cultures. Cambridge University Press, Cambridge, 1986, p.68.), trichomes in Mentha or stored in the form of non-toxic glycoside derivatives in vacuoles e.g. Rosa spp.
So the number of trichomes at different developmental stages of the genotype CIMAP/MP20 and its variation in different leaves (both the upper and lower surface) situated at different level (0 level: leaf at the tip, 1 level: next leaf down to 0 level, 2 level: next leaf down to 1 level, 3 level: next leaf down to 2 level, 4 level: next leaf down to 3 level,) were characterized and finally all the trichome at different levels of the leaves were averaged, calculated per centimeter square leaf area. A peak trichome density was observed from 75 to 95 days in all the leaves at different levels except the leaf at the tip. At 0 level the leaves are at active developmental stage which may be cause for steady rate for trichome formation (Table 4).
Taxonomic description of the peppermint plant CIMAP/MP20 are as given below:
The colour codes are in accordance with The R.H.S. Colour Chart published by The Royal Horticultural Society, 80 Vincent Square, London SW1P 2PE,1995. The genotype CIMAP/MP20 was named and referred as ‘Cim Indus’ in this specification.
DNA Isolation and PCR Amplification Reactions
DNA was isolated from leaf tissue essentially according to the protocol described previously (Khanuja S P S, Shasany A K, Darokar M P & Sushil Kumar (1999) Rapid Isolation of PCR Amplifiable DNA from the Dry and Fresh Samples of Plants Producing Large Amounts of Secondary Metabolites and Essential oils by Modified CTAB Procedure. Plant Molecular Biology Reporter 17: 74.) and pooled DNA (equal amount from 20 individual plants of a genotype in a field) constituted the samples for polymerase chain reactions (PCRs) which were carried out in 25 μl volume.
A reaction tube contained 25 ng of DNA, 0.2 unit of Taq DNA polymerase, 100 μM each of dNTPs, 1.5 mM MgCl2 and 5 p mol of decanucleotide primers. The amplifications were carried out using the DNA Engine thermal cycler (MJ Research, USA) following the protocol of Khanuja et al. (Khanuja S P S, Shasany A K, Srivastava A & Sushil Kumar (2000). Assessment of genetic relationships in Mentha species. Euphytica 111: 121-125.). The amplified products were loaded in 1.2% agarose gel containing 0.5 μg ml−1 of ethidium bromide and photographed by Polaroid system. Twenty decamer primers procured from Operon Technologies, USA (OPA) were used to detect polymorphism in the selected genotype. The similarity matrix obtained after multivariant analysis using Nei and Li's coefficient (Nei, N. & W. Li, 1979.
Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. (USA) 76: 5269-5273.) is shown in Table 5. These similarity coefficients were used to generate a tree for cluster analysis using UPGMA method (
Uniformity and Stability
Like any other Mentha piperita genotype this genotype is also planted vegetatively through runners and suckers. No variation of any kind was observed in this genotype for the last 3 years of trial maintaining the quality of oil and phenotype. The RAPD analysis of random plant samples in different years of trial also did not show any variation in profiles for this genotype indicating the stability of this genotype.
Disease and Pest Resistance
The incidence of lepidopteran pest Spilarctia obliqua and fungus mint rust (Puccinia sps), leaf spot and mildew were not detected in the field continuously for 3 years in the genotype CIMAP/MP20.
Metabolic Regulation of Menthofuran Biosynthesis
The genotype CIMAP/MP20 was rich in pulegone and menthofuran. In the biosynthetic pathway Geranyl pyrophosphate is converted to limonene which in turn is transformed into isopiperitenol, followed by pulegone. Pulegone is converted to menthone followed by menthol. Menthol and Menthone are the main constituents of the essential oil of Mentha piperita. In one branch of the pathway pulegone is converted to menthofuran. In this genotype the monoterpene pulegone is biosynthesized at an accelerated rate and the reaction favour more towards the menthofuran synthesis than menthol. In the initial stage (up to 55 days stage) of growth of this genotype the reaction favours for the production of menthol at a reduced rate from pulegone with less accumulation of pulegone and menthfuran. But at later stage as the plant matures the reaction favours accumulation of more menthofuran and pulegone and instead the biosynthesis of menthol decreases. This indicate the role of regulatory proteins in the monoterpene metabolism and the importance of this genotype for the isolation of such protein for future modification of metabolic pathway modification.
| Number | Date | Country | |
|---|---|---|---|
| 20050150027 P1 | Jul 2005 | US |
