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. Genetic data for M. citrata x NI. aquatica hybrids: Strain 1 of M. aquatica is true breeding for a menthofuran odor (Table 2). Self-pollination of the fertile clone, or sib crosses between the male-sterile clone and the fertile clone produce all menthofuran-odored individuals. When Strain 1 of M. citrata is hybridized with Strain 1 of M. quatica, 1,151 hybrids were lavender odored like the M. citrata parent and 1,117 were mentho- furan odored like the M. quatica parent. This l : l ratio seems to indicate that the M. citrata parent is heterozygous for a single dominant gene (Zi). If this assump- tion is correct, the menthofuran-odored F, individuals should be true breeding for a menthofuran odor and have the genotype ii. It will be noted that 15 different menthofuran-odoredF, individuals were self-pollinated and gave 833 F, individ- uals having a menthofuran odor. All F, lavender-odored individuals should be heterozygous for the dominant gene (Zi) and should give 3:l ratios when self- pollinated. Ten lavender-odored F, individuals were self-pollinated and each F, OIL CONSTITUENTS IN MENTHA 461 TABLE 2

Genetic data from a study of M. citrata x M. aquatica Strain 1 hybrids

Number of progeny with Lavender Menthofuran Self or cross odor odor Ratio* Strain 1 M. aquatica L., fertile clone self-pollinated .... 264 0:all Strain 1 M. aquatica L., 9 S, strains self-pollinated .... 586 0:a11 Strain 1 M. aquatica L., male-sterile clone sibbed to fertile .... 101 0: all Data for Strain 1 of M. citrata Ehrh: M. citrata x M.aquatica Strain 1 1151 1117 1:1 F, (15 menthofuran-odored F, individuals) .... 833 0:all F, (10 lavender-odored F, individuals) 332 117 3: 1 F, (4 lavender-odored F, individuals) 745 232 3:1 F, (2 lavender-odored F, individuals) 361 103 3: 1 F, (5 lavender-odored F, individuals) 472 all: 0 F, (1 lavender-odored F, individual) 181 all: 0 M. aquatica x F, individual with duplicate genes 58 18 3:1 Data for Strain 2 of M. citrata: M. citrata x M. aquatica Strain 1 554 all: 0 F, (4 lavender-odored F, individuals) 166 59 3: 1 M. aquatica x 10 lavender-odored F, individuals 1154 1156 1:1 M. citrata x 2 lavender-odored F, individuals 118 all: 0 Data for Strain 3 of M. citrata: M. citrata x M.aquatica Strain 1 336 4 63: 1 M. aquatica x F, lavender-odored once-Culture 1 41.8 214 2: 1 M. quatica x Fl lavender-odored once-Culture 2** 117 47 3: 1 M. aquatica x F, lavender-odored once-Culture 3 73 68 1:1 M. aquatica x F, lavender-odored twice (3 cultures) 360 99 3: 1 M. aquatica X F, lavender-odored twice (7 cultures) 573 559 1:1 M. aquatica x F, lavender-odored thrice (1 1 cultures) 784 785 1:1 M. aquatica x F, lavender-odored thrice (2 cultures) 185 67 3: 1 Data for Strain 4 of M. citrata: M. citrata x M. aquatica Strain 1 24 8 3:1 M. aquatica x F, lavender-odored-Culture1 11 4 3: 1 M. aquatica x F, lavender-odored-Culture2 55 58 1:1 F, (M. citrata x M. aguatica) lavender-odored 30 11 3: 1 F, (M. citrata x M. aquatica) menthofuran-odored .. 66 0:all Data for Strain 5 of M. citrata: M. citrata x M. aquatica Strain 1 28 26 1:1 Data for Strain 6 of M. citrata: M. citrata x M. aquatica Strain 1 20 15 1:1 * None of the P values is significant. ** Additional data: 7 cultures, 1936 not : 624 menthofuran-odored, progeny had a 3: 1 ratio. The combined data were 332 lavender-odored to 11 7 menthofuran-odored. Further self-pollination of lavender-odored individuals pro- duced five F, strains which were homozygous for the dominant gene Z as demon- strated by a total F, progeny of 472 lavender-odored individuals. It is necessary to assume that the gene Z can OCCUT on two different homologous chromosome pairs, since one F, individual crossed with M. aguatica gave a 3:l ratio of 58 462 M. J. MURRAY AND D. E. LINCOLN lavender odored to 18 menthofuran odored. This F, individual with dupliacte genes and the genotype Zli,Z& could have been derived from allosynaptic pairing between the ZIZl pair of chromosomes and the i,i, pair of chromosomes. The complete genotype for M. aquatica is i,i,i&. Strains 5 and 6 of M. citrata are similar to Strain 1 in having a genotype Zlili,iz,whereas Strain 2 has the ho- mozygous genotype ZIZli,is and breeds true. Strain 4 has the genotype ZlilZ,i,. Extensive plant breeding cultures support these conclusions. These data are not cited here since we discarded all seedlings lacking vigor before smelling the herb- age of the remaining seedlings. Small progenies of Strain 3 hybrids with M. aquatica may have no mentho- furan-odored individuals and seem to indicate that Strain 3 has a homozygous genotype. The ratio of 336 lavender-odored to 4 menthofuran-odored individuals reported in Table 2 could be due to the segregation of several heterozygous loci of the gene Z (15:l P< .01; 31:l P= .04; 63:l Pz.6). A more plausible explanation is that the four menthofuran-odored individuals occurred as the result of occasional quadrivalent pairing or of allosyndetic pairing of bivalents of an ZlZIi,i2 genotype. The fact that duplicate gene ratios are found in subse- quent generations suggests that Strain 3 may have the genotype ZJ,Z2iz. Occa- sional quadrivalent pairing of the two homologous pairs of chromosomes with the genotype ZII,Z& would not produce gametes having a recessive genotype unless the gametes were deficient in one chromosome as a result of a 3-1 chromosome distribution. The ratio of 2 lavender-odored to 1 menthofuran- odored individuals in culture 1 may be explained if we assume that the plant used as the pollen parent was trisomic for one chromosome pair and had the genotype Z,ZIiIi&.A 2 Z : 1 i gametic ratio would be expected in Datura and other plants, since pollen with an extra chromosome is not functional although the egg cells may carry an extra chromosome. Four exceptional segregants in a progeny of 340 individuals indicates that quadrivalent pairing with a 3-1 chrom- osome distribution is infrequent. The data for hybrids between M. citrata and Strain 3 of M. aquatica of German origin need not be presented, since the data are similar to those sum- marized for Strain 1 of Dutch origin in Table 2. Self-pollination of Strain 3 of M. aquatica, and crosses between Strains 1 and 3 produced all menthofuran- odored individuals in a total progeny exceeding 5,000 individuals. Genetic data for M. citrata F, hybrids with 9 principal Menthastrum species: Strain 1 of M. citrata with the genotype ZliIi&was hybridized with the principal species of the subgenus Menthastrurn. The data in Table 3 show that one-half of the F, hybrids have a lavender odor. These data show that all non-lavender- odored species have the recessive genotype ii. Indeed, these species would not have large amounts of the cyclic ketones given in column 1 of Table 3 if they had the dominant gene I. The nonlavender-odored F, hybrids have the normal ketone composition which would be expected from genetic segregation of the strain (MURRAY1960b). Strain 2 of M. citrata with the homozygous genotype Z,Z,i& was also hy- bridized with the principal species of the subgenus Menthstrum. While the data OIL CONSTITUENTS IN MENTHA 463

TABLE 3 Inheritance data for M. citrata Strain I hybrids with othm species

Number of progeny with 2n M. citrata Ehrh. as seed parent Ketones of Lavender Not lavender crossed to following pollen parents: pollen parent1 odor odor 2n M. longifolia (L.) Huds. 2n = 242 PP I2 4* 4m M. longifolia (L.) Huds. P-P 78 80 4n M. rotundifolia L. 2n = 24 P-P 43 41 4m M. niliaca Jacq. em. Briq. 2n = 36 c-d 47 54 4n M. spicata L. cultivar Native Spearmint 2n = 36 c-d 89 76 2n M. spicata L. Line 1 strain 2n = 48 c-d 47 45 4n M. spicata L. Line 1 strain c-d 27 35 2n M. crispa L. 2n = 484-6 “B” type (IKEDA’61) c-d 67 69 4n M. crispa L. c-d 71 50* 2n M. spicata L. Line 1 S, strain 2n = 48 m-P 17 18 2n M. spicata L. Missiones strain 2n = 48 m-P 10 8 2n M. arvensis L. European strain 2n = 96 m-p 125 152 2n M. arvensis L. var. piperascens Briq. 2n = 96 m-p 57 65 4n M. arvensis L. var. piperascens Brig. m-P 9 9 2n M. cardiaca Baker cultivar Scotch Spearmint 2n = 72 c-d 18 17 2n M. pipen-taL. cultivar Mitcham variety 2n = 72 m-P 22 14 411 M. piperita L. m-P 57 55 2n M. aquatica L. strain 1 2n = 96 m-p 95 98 Total 891 890

1 c-d = carvone & dihydrocarvone; m-p = menthone & pulegone; p-p = piperitone oxide & piperitenone oxide. Opinions vary whether M. aquatica has the m-p ketones or none of these ketones. 2 Somatic chromosome number of the diploid or natural strain. * Significant deviation from 1:I ratio at 5% level. All other P values not significant. are not presented in a table, all F, hybrids have a lavender odor in progenies of 50-100 individuals. A large-scale study of crosses between Strain 2 and M. crispa or M. crispa S1’s has given two exceptional nonlavender kinds of individuals. The primary incidence of these exceptions probably does not exceed 1% in a total progeny of 10,000 individuals. The first major exceptional type has 60-90% limonene and cineole. The second major type may have some isopinocamphone but definitely has two major terpenes and one oxidized compound without appreciable amounts of linalool or linalyl acetate. This segregant may be similar to the isopinocamphone strain of M. aquatica described by SHIMIZU,KARASAWA and IKEDA(1966). Lincoln found 19 individuals of the first type and 18 of the second type. None of the exceptions was menthofuran odored. Assay data for parental strains: HANDAet al. (1964) have reported that M. aguatica oil has 0.9 % alpha-pinene, 2.3% beta-pinene, 6.4% limonene, 22.4% cineole, 0.8% piperitone, 1.8% pulegone, 51.3% menthofuran, 0.0% menthone, 2.6% menthol, 6.2% menthyl acetate, 0.3% carvone, 1.9% 3-octanol, and 0.3% 3-octyl acetate. HEFENDEHL(1967a) has shown that M. aguatica Strain 3 has 67.75% menthofuran, 1.0% alpha-pinene, 1.8% beta-pinene, 0.9% sabinene, 1.0% myrcene, 5.1 % limonene, 7.9% cineole, 1.6% cis-ocimene, 0.4% trans- 464 M. J. MURRAY AND D. E. LINCOLN ocimene 4- gamma-terpinene, 0.1 % p-cymene, 0.1 % 3-octanol, 0.05% octyl ace- tate, 0.3% linalool f sesquiterpene-KW, 5.1 % carophyllene, 0.7% sesquiter- pene-KW, 0.2% humulene, 2.5% cadinene, 0.1 % caryophyllenoxide, and 3.7% oxygenated sesquiterpenes to give a total of 100.3%. F. CRAMERof our laboratory has found that the oil of Strain 1 of M. aquatica has 0.24% alpha-pinene, 0.71 % beta-pinene, 5.6% limonene, 4.3% cineole, 4.57; piperitone, 83.0% mentho- furan, 0.8% L-menthol, and no measurable quantity of L-menthone, D-iso-men- thone, neomenthol, 3-octanol, menthyl acetate, or sabinene hydrate. These data show clearly that menthofuran is the principal oil constituent of M. aquatica and that the acyclic compounds are either absent or present in very small quantities. HANDAet al. (1964) have reported that M. citrata oil has 0.6% alpha-pinme, 0.9% beta-pinene, 1.1% limonene, 0.2% cineole, 4.2% piperitone, 3.0% piperi- tone oxide, 8.1 % pulegone, 0.1 % menthofuran, 32.4% linalool, 45.0% linalyl acetate, and 3.8% unaccounted. Small amounts (0.1-2.0%) of limonene and of cineole are found in all M. citrata parental and hybrid strains investigated at this time by F. CRAMER.All determinations were based on retention time using several columns, and are strong presumptive evidence that limonene and cineole actually exist in M. citrata oils. No menthofuran was found in the parental strains or in the M. citrata hybrid strains that are in commercial production. R. E. HUGHESof our laboratory has found that small amounts (0.1-1.5%) of isopinocamphone occur in the parental strains, with the highest amounts in Strains 2 and 3. Table 4 summarizes the assay data of the parental strains of M. citrata for linalool and linalyl acetate. Strain 1 had 42.0% linalool and 46.1 % linalyl ace- tate in 1960, but the oil from more mature herbage harvested in 1962 assayed

TABLE 4

Assay data of M. citrata strains by gas chromatography

Strain Linalyl acetate Linalool Minor constituents assayed Year percent percent percent Strain 1 2n 1960 46.1 42.0 11.9 1961 41.5 37.4 21.1 1962 58.1 33.9 8.0 3-year average 48.6 37.8 13.6 Strain 1 4n 1960 48.1 39.9 12.0 1961 47.0 36.6 16.4 1962 56.4 35.4 8.2 3-year average 50.5 37.3 12.2 Three-year average of years 1964, 1965, and 1966 Strain1 411 50.6 37.4 12.0 Strain 2 58.5 ' 30.0 11.5 Strain 3 58.7 29.5 11.8 Strain 4 58.1 31.9 10.0 Strain 5 60.0 27.4 12.6 Strain 6 55.5 35.4 9.1 OIL CONSTITUENTS IN MENTHA 465

TABLE 5

An array of gas chromatographic assays of 250 Strain 2 M. citrata x M. crispa hybrids

Class range Linalyl Total minor percent Midpoint acetate Linalool components 77.682.5 80 1 .. 72.677.5 75 10 .. .. 67.6-72.5 70 10 .. .. 62.6-67.5 65 28 .. .. 57.662.5 60 44 4 .. 52.657.5 55 43 6 .. 47.652.5 50 41. 14 42.6-47.5 45 37 27 .. 3 7 6-42.5 40 21 40 .. 32.637.5 35 8 40 .. 27.6-32.5 30 3 56 22.627.5 25 1 36 3 17.6-22.5 20 .. 20 19 12.617.5 15 .. 7 80 7.6-12.5 10 .. 123 2.6 7.5 5 .. .. 25 Total number .. 250 250 250 Mean of hybrids 53.82 f .65 34.28 f .62 12.04 i .26 Mean of Strain 2 M. citrata 58.5 2 3.5 30.0 f 3.0 11.5 & 2.1 Difference -4.68 k 3.56 f4.28 f 3.1 +.54 * 2.1

33.9% linalool and 58.1 % linalyl acetate. Despite this biological variation, strains selected for a high linalool content are always high in linalool. Strain 5 consistently has a high linalyl acetate and low linalool content. Assay data for Strain 2 M. citrata x M. crispa hybrids: Table 5 arrays the linalyl acetate and linalool values for 250 Strain 2 M. citrata X M. crispa hybrids. A few of these hybrids have nearly equal amounts of linalyl acetate and linalool, but the majority of the hybrids have more linalyl acetate than linalool. Almost half (109) of the strains have linalyl acetate values above 55% whereas only five strains have linalool values above 55%. The mean values for linalyl acetate, linalool, and the total of minor constituents for the 250 hybrids are not signifi- cantly different from those of the M. citrata parent (bottom of Table 5). The chromatogram published as Figure 3 by TODDand MURRAY(1968) clearly showed that 35 different minor mostly unidentified constituents are found in the oil of a given strain. The total amounts of the minor constituents vary from 4.0 to 24.7% in different hybrids. Grafting experiments: Reciprocal grafts were made between Strain 1 of M. citrata Ehrh. and the following species: M. longifolia (L.) Huds. (high piperi- tone), M. rotundifolia L. (high piperitenone), M. spicata L. Line 1 and M. crispa L. (high carvone), $1. spicata L. S, Strain 199 (high menthone), M. crisp SI Strain 213 (high pulegone), M. crispa S, Strain 214 (high piperitone), M. uruensis L. var. piperascens Briq. (high menthol), and M. aquatica L. (high menthofuran). 466 M. J. MURRAY AND D. E. LINCOLN The grafts were grown to maturity and the tops distilled and assayed by gas chromatography (F. CRAMERand P. MEULMAN). The small amounts of oil ob- tained from one to three grafts collected in Skellysolve B restricted the analysis to major components, especially in low yielding species like M. aquatica. While our 1960 data lacked the precision possible today, the results indicated that root- stocks with two to four basal leaves of the other species did not supply a translocatable enzyme that would allow the M.citrata scion (plant top) to produce ketones or other major oil constituents not found in the parental Strain 1. Conversely, there was no inhibitory effect of the nearly leafless M. citrala root- stock on the production of normal oil constituents in the other species used as scions.

DISCUSSION AND CONCLUSIONS Dominance without dosage eljects: There is no marked dosage effect of the gene Z on chemical composition. The assay values of Strain 1 (Zjijizi2)and the unselfed polyploid Strain 1 (ZlilZlijizizi&)are very similar (Table 4). Strains 1, 5, and 6 with the genotype Zji1i9izare not consistently different from Strains 2 and 4 with the genotypes ZIZli& and ZlijZzis.The mean values of the assays of Strain 2 M. citrata x M. crispa hybrids Z2ilizizare not significantly different from Strain 2 M. citrata (ZIZIi&) assays. (Table 5). Mentha species and hybrids having linalool: The present research has shown that M. citrata strains and 50-100% of the M. citrata F, hybrids with the princi- pal specie; of the subgenus Menthastrum will have a lavender odor. M. citrata hybrids with M. aquatica or M. arvensis (2n = 96) and M. arvensis var. pipera- scens cultivar (2n = 96) are fertile. All other hybrids are highly sterile. SACCO(1960) reports that X M. citrata has high linalool and linalyl acetate but considers the species to be a hybrid between M. aquatica and M. uiridis. SACCO(1959) obtained from seed a new mint having 55% linalool and named the mint M. uiridis cultivar X lauanduliodora Sacco N. cult. KUBRAKet al. (1968) report that the main constituents of the oils of M. siluestris, M. Zongifolia L. sp. caucasia Huds., M. candicans, and M. citrata are 6040% linalool, 5-50% geraniol, and 1-2% sesquiterpene hydrocarbons. M. Mirennue Br. is also re- ported as having 42% linalool (cited by FUJITA1960b). These reports may in- dicat? that the gene Z has persisted in strains of fertile species having 24 or 443 somatic chromosomes. Sterile lavender-odored strains with about 72 somatic chromosomes and aptly described from morphological appearance as X M. piperita var. citrata apparently exist in nature and are certain to occur in any area where M. citrata and M. spicata co-exist and hybridize. If acyclic compounds are the precursors of cyclic compounds in Mentha oil biogenesis, residual small amounts of linalool might be expected in Mentha species having predominantly cyclic constituents. This is apparently true for all species that have been intensively studied. SMITHand LEVI (1961) and SHIMIZU (1963) have reported 0.1-0.8% linalool in M. aruensis oils. SMITH, SKAKUM and LEVI (1963) found 0.1-0.4% linalool and possibly trace amounts of linalyl acetate in the oils of M. cardiaca Gerard ex Baker (Scotch Spearmint cultivar), OIL CONSTITUENTS IN MENTHA 467 M. viridis L., and M. spicata L. (Native Spearmint cultivar), but they do not consider that either compound, known primarily from gas chromatographic retention time, has been positively identified. HEFENDEHL(1967a) found no linalyl acetate and possibly trace amounts of linalool in the oil of M. aQuatica L. Postulated efJects of the gene I on biogenesis of major oil constituents: The genetic data presented here for Strain 1 M. citrata hybrids with M. aquatica (Table 2) and with other species (Table 3) were known to REITSEMA(1958) when he postulated that a linear intermediate compound (X) gave either an acyclic compound (Z)or a cyclic intermediate compound (U).We have given in Figure 1 the two main bifurcations (forks) determined by genes in the REIT- SEMA design, but the design was far more important in ascribing a definite role to piperitenone and piperitone in the development of menthone and pulegone and in deriving menthofuran from pulegone. FUJITA(1960a,b, 1961) published a biogenetic design giving the possible derivation of nearly all known Mentha oil constituents. The very abbreviated summary in Figure 1 emphasizes the postulated origin of cyclic compounds from acyclic ones. Linalool was considered a basic oil constituent which gave either linalyl acetate or geraniol. In this design, geraniol or its geometric isomer nerol produced alpha-terpineol which was converted to limonene. Limonene was then converted to either carvone/dihydrocarvone (the 2-oxygenated compounds) or to isopiperitenone/piperitenone/pulegone/menthone (the 3-oxygenated com- pounds). One might postulate that the recessive gene i caused the conversion of linalool to geraniol and that the dominant gene I prevented the conversion. This biogenetic design by FUJITAcould explain the action of the gene I and the residual (0.1-0.8%) amounts of linalool apparently found in M. uiridis, M. spicata, M.aruensis var. piperascens, and M.aquatica. A biogenetic design by LOOMIS(1967) added several menthol isomers and differed from the FUJITAdesign in deriving piperitenone from terpinolene and terpinolene from alpha-terpineol. While there is general agreement in other regards, LOOMISdoes not believe that alpha-terpineol and its 2- or 3-oxygenated series of compounds with a cis configuration could be biologically synthesized from geraniol and linalool with a trans configuration. The question is whether linalool can exist in a correct stereoisomeric configuration to produce geraniol or nerol. The concept of two isomeric forms of linalool seems to have been advanced by KATSUHARA(1 966). SHIMIZU(1 963) derives 2- and 3-oxygenated oil constituents from geranyl pyrophosphate without citing intermediate compounds. The conversion series menthone to menthol to menthyl acetate and of pulegone to menthofuran proposed by REITSEMA(1958) have been accepted by FUJITA (1960) and LOOMIS(1967), but HEFENDEHL(1967a) does not believe that the present evidence conclusively proves that pulegone is converted to menthofuran in M. aquatica. There is also disagreement regarding the role and origin of piperitone and piperitenone. LOOMIS(1967) and others have ascribed a major role to piperitenone and postulated that it produces pulegone which produces either menthone or menthofuran, whereas piperitone derived from piperitenone 468 M. J. MURRAY AND D. E. LINCOLN produces a minor oil constituent, isomenthone. These assumptions are based on chemical structure and seem in direct contradiction to genetic evidence. First, piperitenone has seldom been identified in species other than M. rotundifozia, whereas piperitone is found in M. spicata, M. piperita, M. arvensis, and all strains having pulegone and menthone. Secondly, strains of M. spicata with pulegone and menthone as principal ketone constituents have the genotype cc AA or cc Aa, whereas the basic species M. longifolia and strains or SI segregants of M. spicata with large amounts of piperitone have the double recessive geno- type cc aa (MURRAY1960b). However, genetic evidence is not conclusive for a several step process unless each of the genes controlling an individual conver- sion is heterozygous allowing its recognition and study. The series alpha-terpineol to limonene to carvone to dihydrocarvone proposed by FUJITA(1960b) was accepted by LOOMIS(1967) but FUJITAassumed that limonene produced either carvone or piperitenone. The position of limonene in the LOOMISdesign (1967) would seem to make it impossible to have limonene in species having 3-oxygenated compounds, yet limonene has been identified in M. piperita, M. aquatica, and M. aruensis var. piperascens. There is no question that linalool produces its ester linalyl acetate, but the role of linalool in a bio- genetic design needs further study. The rapid incorporation of label in three to five minutes and the small amounts of precursor compounds make tracer work with I4C exceedingly difficult (REITSEMAet al. 1961; BATTAILEand LOOMIS1961 ; HEFENDEHL,UNDERHILL and VON RUDLOFF1967). BURBOTTand LOOMIS(1969) have also shown that there may be rapid loss of labeled monoterpenes in pepper- mint. These critical comments indicate that the biogenesis of Mentha oil con- stituents is not well established at this time. Our present work suggests that a more accurate understanding of the biogenetic sequences in oil synthesis will be attained in part from careful chemical analysis of oils for minor constituents and in part from determining the genetic basis for major differences in oil composition. To conclude, the apparent effect of the dominant gene I on biogenesis is to largely but not totally prevent the conversion of linalool to cyclic compounds resulting in an accumulation of linalool and its ester, linalyl acetate. One cannot assume that the conversion of linalool -+ geraniol + alpha-terpineol + limonene and cineole is completely prevented, since M. citrata clearly has small amounts of limonene and cineole and possibly of piperitone and pulegone. The recessive gene i seems to allow the rapid and nearly complete conversion of linalool to cyclic compounds with only very residual quantities of .01-0.8% linalool likely to be found in most species. Grafting experiments did not provide evidence that the genotypes Zi or ii influ- ence the production of a translocatable enzyme. Similar experiments performed by HEFENDEHL(1967b) forced him to conclude that the enzymes involved in oil synthesis were formed in the leaves and not influenced by rootstocks and roots. BATTAILE,BURBOTT and LOOMIS( 1968) have recovered enzymes from appro- priate whole plant extracts of M. piperita that will cause the conversion of pulegone to menthone, isomenthone, and menthol, but they apparently have not studied the conversion of linalool. OIL CONSTITUENTS IN MENTHA 469

SUMMARY The herbage odor of water mint Mentha aquatica (2n = 96) is that of pure menthofuran since the principal oil constituent of this species is 40-80 % mentho- furan. This 8n species has the genotype ililizizand breeds true for a menthofuran odor. The acyclic oil constituents, linalool and linalyl acetate, that constitute 84-90% of the oil of the related 8n lavender-odored species M. citrata Ehrh. are caused by the dominant gene I. A study of M. aquatica X M. citrata hybrids shows that Strains 1, 5, and 6 of M. citrata have the genotype ZIiliziz,Strain 2 the genotype ZIZIiziz, and Strain 4 the genotype ZliIZ2iz. Strain 3 may have the geno- type ZIZIZziz. Since bivalent pairing usually occurs, disomic 1: 1 and duplicate gene 3:l segregating testcross ratios can be obtained.-When Strain 1 of M. citrata is hybridized with M. rotundifolia and M. longifolia (2n = 24), M. spicata and M. crispa (2n=48), and M. aruensis, M. aruensis var. piperascens, and M. aquatica (2n = 96), one-half of the F, interspecific hybrids have a lavender odor. Almost all Strain 2 M. citrata F, interspecific hybrids with other species have a lavender odor.-The apparent effect of the gene Z on oil biogenesis is to largely but not completely prevent the conversion of linalool to cyclic compounds. As a result, linalool and its ester, linalyl acetate, are accumulated in large amounts while only small amounts of cineole, limonene, and the oil constituents piperitone and pulegone derived from limonene are made. In most biogenetic designs, limonene is considered to produce either carvone/dihydrocarvone or piperitenone / piperitone/ pulegone / menthone / menthol / menthyl acetate with menthofuran derived from pulegone. The gene Z thus largely prevents the for- mation of the cyclic ketones and their specific alcohols and esters that characterize most species of the subgenus Menthastrum whose basic chromosomenumber is 12.

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