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THE GENETIC BASIS OF ACYCLIC OIL CONSTITUENTS IN MENTHA CITRATA EHRH. MBRRITT J. MURRAY AND DAVID E. LINCOLN A. M. Todd Co., Kalamazoo, Michigan 49005 Received November 17, 1969 HE strong lavender odor of the Bergamot mint, Mentha citrata Ehrh., is due Tto two principal oil constituents, linalyl acetate and linalool, that make up 84-90% of the oil (TODDand MURRAY1968). These chain or acyclic compounds are characteristic of M. citrata and some of its hybrids, whereas the major oil components of all other species of the genus Mentha are cyclic compounds. In Figure 1, the acyclic oil constituent linalool has been considered the pre- cursor of alpha-terpineol which produces either the 2-oxygenated-p-menthanes (spearmint odored having carvone and dihydrocarvone) or the 3-oxygenated- p-menthanes (the nonspearmint odored having piperitenone, pulegone, and men- thone) . The opposite view, that the acyclic compounds were derived secondarily ACYCLIC TYPES LZI AS CITRAL AND LINAWOL REITS EM A LINEAR OR ACYCLIC DESIGN INTERMEDIATE 1x1 >I-OXYGENATED SERIES U BELOW \ CYCLIC INTERMEDIATE IYI >3-OXYOENATID SERIES AS BELOW AOOCCt-$ I-OXYGENATED SERIES LINALYLY ACETATE CARMNE DIHYDROCARVONE \ A A MCNTHONE 3-OXYOMATED SERIES OER*?IYL PYROPI!OSPn4TE XERYI. PYROPHOSPHATE FIGURE1.-A very abbreviated diagram of monoterpene synthesis of the principal Mentha oil constituents, illustrating the differences in three biogenetic designs relative to the origin of cyclic compounds from acyclic ones. Genetics 65 : 457471 July 1970. 45 8 M. J. MURRAY AND D. E. LINCOLN from the cyclic ones, has not been postulated. Several biogenetic designs have been developed to account for the origin and derivation of the constituents of Mentha oils (REITSEMA1958; FUJITA1960a.b, 1961; KATSUHARA1966; HEFEN- DEHL, UNDERHILLand VON RUDLOFF1967; BURBOTTand LOOMIS1967; and LOOMIS1967). The separation of Mentha species into those having acyclic, 2- oxygenated, or 3-oxygenated oil constituents (HEGNAUER1953; REITSEMA1954, 1958) has been elaborated and refined by SHIMIZU(1963), but none of these designs explains why the cytogenetically advanced species M. citrata (2n = 96) with an octoploid chromosome number is characterized by the chemically basic acyclic oil constituents. MURRAY(1960a,b) has shown that the segregation of specific genetic factors determines oil composition, and that this segregation may bear no direct relationship to chromosome number. The genus Mentha is divided into two subgenera. The subgenus Pulegium has species with a short, rock garden-like habit, poorly differentiated stolons, axillary flower spikes, a high pulegone content of the oil, and a basic chromosome number of 9, 12, and possibly 10. The subgenus Menthastrum, of sole interest in this research, has species characterized by a perennial habit, well developed stolons, and a basic chromosome number of 12. In the one evolutionary line, the axillary flowered species (section uerticillatae) has no known species with 24 somatic chromosomes, but M. japonica Makino has 48 somatic chromosomes (IKEDA1961). M. arvensis L. and M. arvensis L. var. piperascens Briq. (cultivar) have 96 somatic chromosomes. The other evolutionary line of descent has the terminal spike-flowered species (section spicatae) which consists of M. longi- folia (L.) Huds. (M. syluestris L.) and M. rotundifolia L. (2n = 24)) and also M. spicata L. and M. crispa L. (2n = 48). The section capitatae has two species, M. citrata Ehrh. and M. aquatica L., characterized by globose or capitate termi- nal flower spikes, and 96 somatic chromosomes. All species of the subgenus Menthastrum can hybridize and have produced sterile interspecific hybrids as, for example, M. niliaca Jacq. em Briq. (2n = 36), M. spicata L. native spear- mint cultivar (2n = 36), M. piperita L. (2n :72), and M. cardiaca Baker (2n = 72) . While these hybrids are highly sterile, their colchicine-induced polyploid strains are fertile. Cytologically, the somatic chromosome number of M. aquatica was found to be 96 by RUTTLE(1931), JUNELL(1942), GRAHAM(1954), LOVE and LOVE (1956), MORTON(1956), IKEDA(1961), GADELLAand KLIPHUIS (1963), and BAQUARand REESE(1965). RUTTLE(1931 ) and IKEDA(1961) have shown that fertile M. aquatica has 48 bivalents. On the basis of extensive cytological work with M. aquatica hybrids, IKEDAconcluded that M. quatica had four distinct genomes designated as RaRa SS X,X, X,X,. Two strains of M. quatica with 60 somatic chromosomes reported by IKEDA(1961) are different from the strains studied in this paper and would appear to be F, M. aquatica x M. longifolia hy- brids. An early report of 36 somatic chromosomes for M. aquatica was coiisidered erroneous by RUTTLE( 1931 ) . Taxonomically and morphologically, M. citrata has been considered a glabrous variety of M. aquutica L. by DEWOLF(1954), or as a variety of M. piperita L. by OIL CONSTITUENTS IN MENTHA 459 HEGI(1931) and CLAPHAM,TUTIN, and WARBURG(1952), or as a mespecies described by EHRHARTand followed by BRITTONand BROWN(1913), SMALL (1933), FERNALD(1950), and many others. SHIMIZU,KARASAWA and IKEDA (1966) have included M. citrata as one of three major varieties of M. aquatica. While M. aquatica and M. citrata with 96 somatic chromosomes appear to be poorly differentiated species, it is evident that some lavender-odored clonal strains collected in the field probably have an allohexaploid chromosome number (213= 72), resemble M. piperitu, and are sterile F, M. citrata X M. viridis (M.spicata) hybrids as suggested by SACCO(1960). What is the genetic basis for the acyclic compounds found in M. citrata oil? MATERIALS AND METHODS All stock strains of Mentha species, varieties, inbreds, male-sterile strains, colchicine-induced polyploid strains, and sterile interspecific hybrids have been maintained by clonal, or vegetative, propagation using either stolons or rooted branches. The strains used in this genetic study are given in Table 1. Since many of the Mentha species are either highly heterozygous or male sterile, the polyploid strains used in this work were obtained from colchicine treatment without self-pollination. TWO- inch pieces of stolon were soaked in 0.2% aqueous solution of colchicine for 2 hr followed by storage in tap water for 20 hr before retreatment and planting. Colchicine treatment of a multi- cellular stolon meristem of a dicotyledonous plant results in mixcchimeral tissue of 2n and 4n cells which upon growth leads to periclinal chimeras of several kinds. Some periclinal chimeras have a 4n epidermis over a 2n inner core, others have a 4n outer layer of subepidermal and epi- TABLE 1 Origin of species and strains ~ ~~ ~~ ~~ Species Donor and immediate source Native origin M. citrata Ehrh. Strain 1 A. M. Todd collection Europe Strain 2 Mr. Norbert Mueller, Hermiston, Ore. Unknown Strain 3 Dr. C. A. Thomas, Beltsville, Md. and U. S. Plant Introduction Division Sofia, Bulgaria Strain 4 Prof. W. D. Loomis, Corvallis, Ore. Unknown Strain 5 Mr. G. A. Derksen, Vicksburg, Mich. Unknown Strain 6 Mr. Herbert Cooley, Allegan Co., Mich Unknown M. aquatica L. Strain 1 Prof. R. Hegnauer, Leiden, Holland Holland Strain 2 Dr. E. C. Stevenson, Beltsville, Md. Unknown Strain 3 Dr. S. R. Baquar, Karachi, Pakistan Germany M. spicata L. Missiones Prof. G. Fester, Santa Fe, Argentina Unknown Line 1 A. M. Todd collection Kew Gardens, England Native spearmint Cultivar Europe M. cardiaca Baker Cultivar Scotland M. piperita L. Mitcham strain cultivar Mitcham Co., England M. aruensis L. var. piperascens Briq. A. M. Todd collection Japan All other species A. M. Todd collection Europe 460 M. J. MURRAY AND D. E. LINCOLN dermal tissue with an inner 2n core, and a few have entirely 4n tissue. Vegetative propagation with selection for a &-yearperiod is usually necessary to obtain a polyploid strain and be certain that it is entirely 4n. The gas chromatographic assays of the essential oils of various Mentha citrata strains and M. citrata-M. crispa hybrids were carried out in the A. M. Todd Co. chemistry laboratory by ROBERTE. HUGHES;F. J. CRAMER;WILLIAM FAAS; P. MEULMAN;and D. E. LINCOLN.The most accurate quantitative data for linalyl acetate and linalool were obtained using a 1.833.05 m by 3.2 mm O.D. stainless steel column packed with 5% Ucon LB55OX saturated with Tween 20 on Gas Chrom Q with the injection block temperature kept below 200"G. A higher temperature fre- quently leads to the partial breakdown of linalyl acetate as shown by STELTENRAMPand CASAZZA (1967). TODDand MURRAY(1968) have published typical chromatograms of M. citrata oils. The ketone compositions of the species in Table 3 were originally determined in the early 1950's using chemical methods and thin-layer chromatography (REITSEMA1956, REITSEMAand VARNIS1956). Quantitative gas chromatographic assays for carvone, dihydrocarvone, piperitone, menthone, pulegone, menthol, and menthofuran are now routine in most essential oil laboratories. EXPERIMENTAL RESULTS Cytogenetic obseruations: M. citrata is closely related to M. aquatica. They are easily hybridized and their hybrids are perfectly fertile. The 96 somatic chromo- somes of Strain 3 of M. aquatica are shown in Figure 4 of BAQUARand REESE (1965). RUTTLE (1931 op. cit. p. 455, Figures 52 and 53) has illustrated an M. aquatica pollen mother cell (PMC) in diakinesis with 48 chromosome pairs and a second metaphase cell with 48 chromosomes. IKEDA(1961 op. cit. p. 21, Figure 219-1) has shown that M. aquatica may have 48 bivalent chromosomes. The six strains of M. citrata reported upon here are perfectly seed fertile but male sterile. While PMC meiosis cannot be studied in clonally propagated monogenic male- sterile strains, the genetic evidence that follows indicates that quadrivalent pair- ing is infrequent. Completely pollen-fertile strains of M. citrata occur in nature and one-half of the F, M. citrata (male-sterile clone) X M. aqmtica (pollen- fertile clone) hybrids are pollen fertile. Male-sterile strains were used in this ge- netic study and in related plant breeding work (MURRAY1969), since their use in hybridization makes emasculation of the seed parent unnecessary and avoids all possibility of self-pollination.