USOOPP1203OP2 (12) United States Patent (10) Patent No.: US PP12,030 P2 Kumar et al. (45) Date of Patent: Aug. 7, 2001

(54) HYBRID MINT PLANT NAMED (30) Foreign Application Priority Data 6 9 NEERKALKA Jun. 3, 1998 (IN) ...... 1509/98 (75) Inventors: Sushil Kumar; Nirmal Kumar Patra; (51) Int. Cl." ...... A01H 5/00 Suman Preet Singh Khanuja, Ajit (52) U.S. Cl...... Plt./259 Kumar Shasany; Alok Kalra; Harikesh Bahadur Singh; Hemendra (58) Field of Search ...... Plt./259 Pratap Singh; Ved Ram Singh; Nareshwar Mengi; Hasan Tanveer; Primary Examiner Bruce R. Campell Arif Ali Naqvi, Vijay Pal Singh; Assistant Examiner Wendy C Baker Kambod Singh, all of Lucknow (IN) (74) Attorney, Agent, or Firm-Dorsey & Whitney LLP (73) Assignee: Council of Scientific and Industrial (57) ABSTRACT Research, New Delhi (IN) The present invention relates to the development of a new (*) Notice: Subject to any disclaimer, the term of this and distinct interspecific mint hybrid Neerkalka developed atent is extended or adiusted under 35 by sexual crossing between improved Mother plant C. 154(b) by Oda arvensis (cv Kalka) and pollen plant Mentha Spicata (cv a -- y yS. Neera), which hybrid is propagated vegetatively by Suckers

(21) Appl.ppl. No.: 09/145,2909 or Stem cuttings and is stable for commerical cultivation. (22) Filed: Sep. 2, 1998 1 Drawing Sheet

1 2 FIELD OF THE INVENTION expressing the “carvone type' mint oil characteristics of Mentha Spicata and possesses the phenotype tending The present invention relates to CW and distinct inter- towards another naturally occuring species believed to origi specific hybrid mint plant namely Neerkala which is devel- nate from the hybridization of M. arvensis and M. Spicata. oped by Sexual crossing between improved Mentha ar'elists 5 The hybrid plant is more commercially acceptable due to its (cv Kalka) and Mentha Spicata (cv Neera). The hybrid is more favorable argonomic traits. propagated vegetatively by Suckers or Stem cutting and is Stable for commercial cultivation. SUMMARY OF THE INVENTION BACKGROUND OF THE INVENTION 10 Accordingly, the present invention provides Stable com mercially cultivable hybrid mint plant Neerkalka providing of the mint species are cultivated world wide for high herb yield, high oil yield and oil type with their specific mint aroma, which find uses in the culinary, mentholtinge, which plant is developed by Sexual crossing antiseptic, confectionery and flavoring purposes. The dis- between improved Mentha arvensis (cv Kalka) and Mentha tinct taste and aroma originating from the essential oils of is spicata (cv Neera). different Species in Mentha genus determine their specific use. The natural croSS compatibility occurs between the DETAILED DESCRIPTION OF THE PRESENT Species although manual emasculation and pollination is INVENTION impeded due to extremely small size of the florets. Mentha cardiaca Seems to have originated as a naturally occuring 2O The interspecific hybrid Neerkalka has been developed species believed to originate from the hybridization of M. by the Applicants by pollinating Mentha arvensis (cv Kalka) arvensis and M. Spicata. Mentha arvensis which is culti- with pollen from Mentha Spicata (cv Neera). The Applicants vated for mint oil has several widely adopted cultivars with planted the parent plants Mentha arvensis (cv Kalka) and Superior agronomic traits. The variety 'Kalka is one of the Mentha Spicata (cv Neera) in alternate rows to favor cross best cultivars grown widely by the farmers of India. 25 ing between them. The of Mentha arvensis were Similarly, Mentha Spicata cultivars released by Central dusted with the pollen collected from Mentha Spicata flow Institute of Medicinal and Aromatic Plants (CIMAP), erS at regular intervals. The Applicants intention was to Lucknow, India, are widely adopted by the farmers for combine the better growth, Smell and disease resis Superior Spearmint oil quality. One Such cultivar is Mentha tance characters of Japanese mint with the carvone Smell of spicata Neera or CIMAP/Neera which is distinctly dif- 3O Spearmint to create a novel oil quality which can be more ferent in its RAPD pattern from other available genotypes/ acceptable in pharmaceutical as well as confectionery indus varieties. It is known for its profuse flowering and Seed tries. The mint Species used as parents, were Selected from Setting habit. AS Such, the Applicants have combined the the varieties already released by CIMAP and well accepted characters of these two mint species (Mentha arvensis and by the farmers and industries. The plants were grown at Mentha Spicata) and developed a hybrid herb (Mentha Pantnagar, India, in plots of 5 mx5 m. As the hybrid can arvensisxM. Spicata) Neerkalka which has high yield, reproduce vegetatively by vigorous Suckers and the geno desirable oil quality for various herbal uses like chewing typic characters are firmly fixed, the genotype can be of gums, toothpaste etc. The Selected hybrid clone retains the immense importance as improved variety. Hence, the nov profuse growth habit of Mentha arvensis simultaneously elty of the invention is that the hybrid was developed from US PP12,030 P2 3 4 improved varieties of CIMAP which are well characterized upon maturation. Out of a total of 260 Seedlings raised from for their genotypes. The hybrid plant thus produced has the hybrid seeds borne on the Mentha arvensis (cv Kalka), more biomass and oil yield (carvone based) than the parent a single distinct plant nearing Mentha cardiaca phenotype Mentha Spicata and the natural hybrid Mentha cardiaca. The including oil aroma was observed and Subsequently hybrid plant Neerkalka' is the distinct hybrid developed by Selected. This Selected plant which was propagated and the inventors through sexual crossing between female parent multiplied vegetatively many times later, is the hybrid clone Mentha arvensis (cv Kalka) and pollen donor Mentha Spi of the present invention, designated as Neerkalka and cata (cv Neera) whereas Mentha cardiaca (Syn. Mentha referred as “hybrid hereafter. gracilis cv Cardiaca) is believed to have originated in nature Since the florets of Mentha arvensis and Mentha Spicata by natural crossing between Mentha arvensis and Mentha are very Small in size, the manual emasculation and polli Spicata. In other words, the rationale behind the present nation are extremely difficult to achieve. To overcome this invention was to combine the characters of two known mint problem of controlled pollination, the approach of develop Species available with them, in a directed manner to yield a ing hybrid Seeds was modified towards increasing the inci plant of high herb yield, high oil yield and Spearmint oil type dence of natural crossing between restricted parents only. with menthol tinge. For this purpose, the Selected parents Mentha arvensis (cv Accordingly, the invention provides a novel hybrid plant Kalka) and Mentha Spicata (cv Neera) were raised in alter Mentha arvensisXM. Spicata Neerkalka having the follow nate rows (2:1:Kalka:Neera) in the field from the geneti ing combination of characters: cally pure Suckers (maintained in the breeder's plot in a. The Said hybrid is a croSS between female parent isolation). The plants raised in this way were grown to Mentha arvensis (cv Kalka) and pollen donor Mentha flowering. Natural crossing between these parents was Spicata (cV Neera), allowed to occur but simultaneously the florets of Mentha b. The said hybrid is tolerant to spot, rust and arvensis were repeatedly dusted with Mentha Spicata pollen powdery mildew diseases, collected manually. Seeds were collected from Mentha c. The said hybrid contains both menthol and carvone in arvensis (Kalka) and bulked. Similarly, seeds from Mentha the , spicata (Neera) florets were collected. The bulked seeds were grown Separately in flat earthen pots during winter. A d. The said hybrid produces more biomass and oil in total of 260 seedlings could be raised from the seeds comparison to Mentha Spicata and Mentha cardiaca collected from the florets of Mentha arvensis, whereas about cultivars “Neera and MCAS 2, 290 seedlings were obtained from Mentha Spicata, which e. The hybrid genotype has a unique RAPD profile, and were raised Successfully in individual pots. The Seedling f. It has a pleasant Smell of both carvone and menthol from Mentha Spicata were almost alike in morphology as useful in medicinal and aromatic preparations. compared to the parental phenotype (Neera) and possessed In an embodiment, the present invention provides a similar carvone rich smell. All the seedlings from Mentha hybrid yielding Spearmint oil having the following ingredi arvensis except one, resembled the morphology of Mentha ents: Limolene (6.8-23.2%), Menthol (0.66-2.45%), Car arvensis (cv Kalka) and had menthol aroma. There was an vone (64.0-76.1%) and other unidentified fractions in the exeptional Single Seedling resembling the cv Kalka growth essential oil totaling to 100%. habit with a carvone rich aroma Supplemented with a mentholtinge, and thus having a pleasant novel combination BRIEF DESCRIPTION OF THE in the essential oil. The distinct plant showed morphology ACCOMPANYING DRAWINGS approaching towards another Species Mentha cardiaca but FIG. 1 is a photograph of a stem of the hybrid, “Neerkalka for growth properties, leaf size and herbage yield, it showing the Size and color of the . resembled Mentha arvensis (cv Kalka). The size and colour of the leaves in the hybrid are similar to Mentha arvensis (cv BREEDING HISTORY Kalka) whereas, the shape resembles the pollen parent Mentha Spicata (cv Neera) (Photograph #1). There are 2 The new hybrid clone of the present invention is the mint leaves at each mode, 4 at the apex, and the leaves are plant developed in planned breeding programs conducted at arranged in opposite decussate phyllotaxy. The aroma of the Central Institute of Medicinal and Aromatic Plants oil was predominantly like that of the pollen parent while the (CIMAP), Lucknow, India and its field station Pantnagar, menthol tinge it possessed was inherited from the female India. The early Steps of development, Such as crossing of parent; thus the plant with recombined character was named parent lines, were conducted in CIMAP, Lucknow. The field “Neerkalka’. The two parental species, the hybrid and trials were conducted at the field Station at Pantagar, India. Mentha cardiaca were grown (planting date 18 Jan., 1996) The plant of the present invention has been asexually from pure SuckerS Side by Side in different plots and were reproduced in the following manner. Generally, Mentha compared among each other for the morphological charac Species are cultivated through Suckers as normal mode of ters. The plants species were grown at 150:30:30 asexual commercial propagation. Suckers are the under (N:POKO) urea, SSP, MOP fertilizer dose and harvested ground plant part which give rise to Similar plantlets when 110 days after planting to compare the yield attributes. The planted in the field. The hybrid plant Neerkalka produces comparison of Some of the characters are presented in Table enough Suckers for asexual propagation. The Stems of the 1. The color codes are according to The R.H.S. Colour Chart plants were planted in the month of October in the field at published by The Roal Horticultural Society, 80 Vincent CIMAP. The plants were established which produced under Square, London SW1P2PE, 1995. ground Suckers. These Suckers were then planted in the main field in the month of January for trial. EVIDENCE OF UNIFORMITY AND STABILITY The breeding method involved is of cross pollination of Mentha arvensis (cv Kalka) florets with Mentha Spicata (cv The genotype Neerkalka has remained Stable and uniform Neera) pollen and the seeds thus obtained, were collected for its morphological characters and showed consistency in US PP12,030 P2 S 6 performance for various quality attributes during its evalu 58% carvone in its essential oil. The Gas Liquid Chroma ation and vegetative multiplication till date. tography (GLC) studies of these oil samples showed no peak for either menthone, menthol or menthyl acetate. But the oil TABLE 1. samples of the hybrid contain menthol 0.66% to 2.45% and carvone 64% to 76% depending upon various states of Characteristics of cv Neera, cv Kalka, cv MCAS2 and the newly growth. Table 2 shows important constituents of the essen developed hybrid clone Neerkalka of Mentha. tial oil extracted from the hybrid and analysed by GLC. Hybrid M. spicata M. arvensis M. cardiaca Traits (Neerkalka) (Neera) (Kalka) (MCAS 2) TABLE 2

1. Plant height 56.8 + 2.13 52.4 + 2.10 97.0 + 2.16 48.6 + 2.20 Constituents % of total oil (cm) 2. Leaf length 7.42 + 0.40 2.34 + 0.16 9.90 + 0.37 6.31 + 0.32 1. Limolene 6.8-23.2 (cm) 2. Menthol 0.66-2.45 3. Leaf width 2.66 - 0.18 0.18 0.14 2.66 - 0.10 1.12 + 0.10 3. Carvone 64.O-76.1 (cm) 4. Leaf colour green green green green (137A) (137B) (137C) (137D) 5. Leaf shape green green green green (137A) (137B) (137C) (137D) TABLE 3 6. Hairiness hairs on the hairs on the hairs on the hairs on the veins, veins, veins, veins, Detailed Botantical Description: glabrous glabrous glabrous glabrous a. Stem shape: Quadrangular 7. Leaf margin finely sharp normal normal finely sharp b. Number of nodes serration serration serration serration 8. Stem colour green (144C) green (144C) green (144D) green (144C) with purple with purple with purple with purple Main branch: 29 -f- 4 pigments at pigments at pigments at pigments at Upper branch (29" node): 3 +f- O the base the base the base the base Middle branch (17" node): 6 -f- O (186C) (186D) (186C) (186C) Lower branch (8" node): 6 -f- O 9. Influore- racemose of idefinite racemose of racemose of c. Average length of internodes SCeCe. axillary aCeOSC axillary axillary Main branch: verticicil- verticicil- verticicil 1.1, 1.2, 1.3, 1.5, 1.5, 2.1, 2.2, lasters lasters lasters (from lower to upper nodes 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.8, O. whitish whitish whitish whitish in cm) 2.8, 2.9, 2.9, 2.9, 2.8, 2.8, 2.8, colour purple (69D) purple (69D) purple (69D) purple (69D) 2.7, 2.7, 2.6, 2.5, 1.9, 1.1 with purple d. Leaf apex shape: pointed (acute) tinge (73B) Leaf base shape: pointed 1. Single plant 184.4 it 734 - 3.27 208.O. 173.4 e. Leaf shape: Elliptical to Ovate with 4-10 finely weight (g) 2.11 4.77 3.05 sharp serrartions on either side. 2. Leaf: Stem 0.61 - 0.03 0.54 - 0.01 0.90 - 0.01 0.58 0.02 . Petiole length: 4-6 cm 3. Herbage 128 8O 165 94 g. Color of upper surface of leaf: Green group (137 A) h. Color of lower surface of leaf: Yellow green group (147 B) yield (Quintal/ i. Number of trichomes per leaf hectare) 804 4. Oil O.80 O60 O.80 O.64 (Avg. Trichomes X1000): content (%) j. Trichome ratio (lower leaf/ 215 5. Oil yield 102.4 48.0 132.O 6O2 upper leaf): (kg/hectare) k. Time to flowering: April–May (70 days after planting 6. Oil aroma carvone CaWOle menthol CaWOle first flowering detected) based with based based based l. Lastingness of Bloom: Continue till harvesting (110 menthol days after planting) Flower shape: Tubular tinge 1-2 mm 7. Growth semi prostate erect erect Pedicel length: Pedicel color: Yellow green group (146 C) habit prostate Calyx diameter: 1 mm, four fused Calyx color: Yellow green group (146 C) Corolla: Four, fused to a corolla tube The hybrid is resistant to leaf Spot, rust and powdery Corolla color: Purple group (76 D) mildew, when planted in the infected field. Only 0-4 plants Pubescence of corolla: smooth Number of anthers: Four, Ocididimetry, remain inside in the field of 2000 were observed to developed these the corolla tube diseases in Separate trials. These resistant characters are Color Designation: Red purple group (59A) apparently inherited from the parent M. arvensis (cv Kalka). Stigma: bifid Till date the hybrid has shown stability in morphological Ovaries: Bicarpellary, syncarpous characters, herbage and oil yield. The genotype has aggres Color of stigma: Violet group (85 C) Sive Suckers, growing under the Soil Surface to provide Color of ovaries: Yellow green group (151 A) protection from adverse weather conditions and mechanical damage. The above examples are only illustrative in nature and The plant is propagated vegetatively and large amount of should not be construed to limit the scope of the invention. planting materials (Suckers) become available in a short period of time. This can be grown in monoculture and can Statement of Distinction be fitted into different cropping patterns in which case ploughing with disk harrow is needed to destroy the Suckers As evident from morphology, the hybrid is distinct from and make the land Suitable for the Subsequent crop. the parents Mentha arvensis (cv Kalka) and Mentha Spicata Essential oil Samples were prepared from the parents as (cV Neera) and the natural Species Mentha cardiaca. well as the hybrid of the present invention. The oil of female The essential oil of the hybrid contains menthol and parent M. arvensis contains 80 to 82% menthol but no carvone where as those of the parents contain either menthol carvone or carvol. The pollen parent M. Spicata contains O CWOC. US PP12,030 P2 7 8 The hybrid is Superior in growth habit in comparison to MAP-Medicinal and Aromatic Plants natural Species M. cardiaca. The hybrid also has a higher biomass and oil yield. The co-dominance of the Polymerase Chain Reaction The essential oil of the hybrid has predominantly a (PCR) amplified bands with printer MAP 03 in the hybrid carvone Smell with menthol tinge which is special and unique. from M. arvensis (Kalka) and M. Spicata (Neera) is clear. So RAPD profile analysis shows codominant polymorphic the hybrid of the present invention is distinct, unique, novel bands in the hybrid from Kalka and Neera when the genomic and can be used for different medicinal and aromatic pur DNA is amplified with primer MAP 03. poses. The hybrid has better morphological and economical The hybrid contains 2n=72 chromosomes in comparison traits and at present is available only with CIMAP. to the parents Mentha arvensis (cv Kalka) 2n=96 and Mentha Spicata (cv Neera) 2n-48. In addition to the characteristics features described in Randomly Amplified Polymorphic DNA Analysis table 1, the novel hybrid has the following features: The RAPD patterns of the hybrid are completely different (a) No. of leaves at each node=2. from those of the parents as well as the M. cardiaca which is thought to be the natural hybrid of M. arvesnsis and M. (b) No. of leaves at the apex=4. Spicata. The hybrid of the present invention was developed (c) Phyllotaxy: Opposite deccusate by crossing M. arvensis (cv Kalka) and M. Spicata (cv Neera) and is thus unique and novel. The following primers (d) Fertilizer dose (Kg/ha): 150:30:30 (N:P:K)-Urea, were used to develop a unique and distinct RAPD profile of SSP, MOP the hybrid (Table 4). (e) Maturity:110 days (Neerkalka & M. Spicata pollen TABLE 4 parent, 120 days Mother parent: M. arvensis) Primers Nucleotide Sequence (f) Oil odour: “Neerkalka: Carvone based with menthol tinge, Kalka: Menthol based and Neera; Carvone 1.MAPO1 5'AAATCG GAG C3' 2.MAP O2 5GTC CTA CTC GC3' based. 3.MAPO3 5GTC CTTAGC G3 4MAPO4 STGC GCG ATC G3 (g) Stem thickness: 4-5 mm (at 5" internode, standard S.MAP OS SAAC GTA CGC G3 method) 6.MAP O6 5'GCA CGC CGG A3' 7...MAP 07 SCAC CCT GCG C3' (h) Agroclimatic conditions: Temp. 18-37 C. 8.MAP 08 SCTA TCG CCG C3 (Maximum.) 8–22 C. (Minimum). 9.MAP 09 SCGG GAT CCG C3' 1OMAP 10 5'GCG AATTCC G3 We claim: 11.MAP 11 5'CCCTGC AGG C3' 1. A new and distinct variety of interspecific mint plant, as 12MAP 12 SCCAAGCTTG C3 herein described and illustrated.

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