Biochemical Systematics and Ecology, Vol. 18, No. 7/8, pp. 493-502, 1990. 0305-1978/90 $3.00+ 0.00 Printed in Great Britain. © 1991 Pergamon Press plc.

Plasticity and Genotypic Variation in some MenthaX verticillata Hybrids

MASSIMO MAFFEI Department of Morphophysiology-Botany, University of Turin, Viale P.A. Mattioli, 25. 1-10125 Turin, Italy

Key Word Index--MenthaX verdcillata; Lamiaceae; essential oils; monoterpenes; morphology; phenotypic plasticity; genotypic variation; two-way ANOVA; multivariate analysis.

Abstract--Leaf and flower oil terpene composition and several plant morphological characteristics of 17 MenthaX verticillata hybrids were analysed during two growing seasons (1988 and 1989). The data obtained were used to study the phenotypic plasticity, the genotypic variation and the genetic variation for phenotypic plasticity. All plants showed high levels of pheno- typic plasticity for both oil chemical and morphometrical parameters. Higher degrees of genotypic variation were found among the plants for oil components while a higher phenotypic plasticity was observed for morphological parameters. Temperatures and rainfall data were collected during the growing seasons and correlated to the data obtained from plant oil and morpho- logy. Low levels of phenotypic plasticity and high degrees of genotypic variation were found to form outliers in the population of M.X verticillata hybrids. The results obtained confirm a significant effect of environmental conditions on the physiology and morphology of the genus Mentha.

Introduction The genus Mentha is of great economic import- ance owing to the essential oil produced in specialized tissues located in the aerial parts of the plant [1]. For many years scientists and mint growers have collaborated in a joint effort to study the environmental conditions which affect the yield and composition of the essential oils produced. For this reason many anatomical and physiological studies have been carried out on this genus to achieve the biochemical rationale for the relationship between structure and function on one side and environmental inter- action on the other. The results of these investi- gations led to the deciphering of the various bio- chemical pathways [2] as well as patterns of morphological and biochemical interactions [3- 6]. For growers, who found striking differences in harvest yield and oil composition with changes in weather conditions, the interaction with the envi- ronment was one of the first problems needing to be solved. Many authors have described the physiological and morphological responses of some Mentha species to changes in environ- mental conditions [7-12], but they rarely pointed

(Received 11 June 1990)

out the importance of the study of the plastic responses in this genus. A plastic response is typical of an individual organism able to modify its physiology/morphology with changes in environmental conditions [13].

The present report, as part of a breeding pro- gram on the genus Mentha started by T. Sacco in early 1960 [14 and refs therein], is intended for the study of the phenotypic plasticity and geno- typic variation in some M.Xverticillata hybrids grown in a natural (not controlled) environment during two growing seasons (1988 and 1989). The M.×verticillata hybrids under study have been obtained from a seed population of M.X verticillata clone 7303 [15].

Numerous clones were obtained and each one exhibited different morphological and oil chemical characteristics. It was interesting to determine whether clonal lines, with evident differences in the morphological and oil chemical genotypical characteristics, may exhibit different patterns or degrees of phenotypic plasticity as well as genetic variation for phenotypic plasticity. Even though the genetic differentiation and the phenotypic plasticity strategies of a plant species for survival are very hard to generalize [16] the present report gives a preliminary study on the effect of the natural environment and the related

493

494 M. MAFFEI

plastic response on some M.X verticillata hybrids grown in a temperate climate.

Results Six plants for each of the 17 M.Xverticillata hybrids were analysed during the two years and the results obtained were processed in order to study the plastic response and the genotypic variation. Morphological and oil chemical data were treated both separately and together in a design in which the independent variables (SH, LBL, etc.) were placed in columns and the M.X verticillata hybrids in rows. The general MIN/ MAX value results will show the multiple hybrids nature of the design. Student's t-test will check for difference significance between the two years, the ANOVA will be used to evaluate plasticity and variability, whereas PCA and MANOVA to differentiate and partition the vari- ables and the M.× verticillata hybrids as described in the experimental section.

The results obtained on the mean values, the minima and the maxima values, as well as the coefficient of variation of all the parameters con- sidered (for both morphological and oil chemical characteristics) from all M.Xverticillata hybrids during the two years studied are given in Table 1.

During 1988 and 1989 the most variable parame- ters were SB, LIL, LIF, MOL, MOF, IML, IMF, MLL, MLF, CAL and CAF. During 1988 TN had a greater coefficient of variation than in 1989.

The correlation matrix, calculated on all the parameters taken into consideration and obtained by grouping the two years, indicated a positive correlation between LBL and SH (0.724). Positive correlations were also found between LIF and IMF and between LIF and MLF (0.518 and 0.544, respectively); MLL was positively corre- lated to LIF, MOF, IMF and MLF (0.789, 0.489, 0.528 and 0.796, respectively), while MFL was positively correlated to MFF and MOF (0.909 and 0.524, respectively). Negative correlations were found between SB and LBL (-0.676). MFF was negatively correlated to LIF, IMF, MFL and MLL (--0.818,--0.602,--0.711 and -0.965, respec- tively), while MFL was negatively correlated to MOF, MLL, MLF and LIF (--0.484, --0.952, --0.883 and --0.772).

The Student's t-values obtained from the pair- wise comparison of the morphological data along with the coefficient of correlation of the character states expressed in the two years are shown in Table 2. In general there were highly significant differences between the data

TABLE 1. MEAN VALUES, MINIMA (Min) AND MAXIMA (Max) VALUES AND COEFFICIENT OF VARIATION (CV) OF THE MORPHOLOGICAL AND OIL

CHEMICAL PARAMETERS FROM ALL MENTHAX VERTICILLATA HYBRIDS DURING 1988 AND 1989

Mean Min Max CV

Para meters 1988 1989 1988 1989 1988 1989 1988 1989

SH 51.08 67.32 10.72 30.52 87.84 115.29 40.56 34.36

LBL 28.13 41.57 1.34 3.07 75.40 94.81 59.69 56.26

LBN 13.04 16.08 1.79 7.76 44.08 46.50 38.83 28.51

SB 2.49 2.36 0.52 0.25 12.45 10,40 73.82 75.47

LA 17.76 12.61 4.54 5.44 55.81 33.47 58.85 48.47

LS 1.72 2.21 0.96 0.88 7.18 12.01 39.71 48.60

DW 25.90 26,64 18.79 18.55 31.37 36.25 12.46 12.61

TN 16940.40 10870.60 7857.00 4663.00 72030.00 23531,00 77.04 36.01

TD 8.62 7.96 3.22 1.00 16.64 18.08 33.94 42.15

TF 72.83 85.86 34.56 36.00 128.47 131.00 29.05 29.52

LIL 11.88 11.00 2.69 1.86 36.38 35.55 77.26 79.05 LIF 14.86 15.36 4.18 2.30 46.00 43.28 73.66 72.30 MOL 6.34 6.22 0.00 0.00 48.00 45.39 223.69 223.86

MOF 4.33 3.22 &00 0.00 25.86 31.20 182.10 243.11

MFL 23.71 22.51 0+00 0.00 44.99 45.60 62.27 60.75

MFE 39.32 37.81 0.00 0.00 73.49 74.25 58.50 59.71 IML 4.20 3.75 0.00 0.00 50.10 40.35 283.88 270.77

IMF 3.42 3.00 0.00 0.00 35.62 35.00 270.35 264.17

MLL 9,39 11,50 0.00 &00 36.00 38.69 138.23 108.62

MLF 9.72 5.08 0.00 0.00 36.65 14.78 106.39 98.21

CAL 3.83 3.93 0.00 0.00 45.88 49.20 298.17 304.30 CAF 6.14 6.26 0.00 0.00 56.00 55.00 273.31 272.62

PLASTICITY AND GENOTYPIC VARIATION IN MENTHA 495

TABLE 2. STUDENT'S t VALUES AND COEFFICIENTS OF CORRELATION (R) FROM THE PAIRWlSE COMPARISONS BETWEEN 1988 AND 1989 MOR-

PHOLOGICAL DATA OF MENTHA X VERTIClLLATA HYBRIDS

SH* LBL LBN SB LA LS DW TN TD TF MH (0.73) 0.52) (0.21) (0,31) (0.89) (0.32) (0.10) (0.80) (0.84) (0.72)

M-17 7.36 29.81 3.51 5.47 13.33 1,761" 19.84 62.91 1.30t 0.25t M-20 7.99 4.63 10.52 4.40 25.01 6.97 12.25 2.49f 1.63t 1.151" M-21 8.40 2.96 0.231" 5.72 9.79 2.201" 12,58 22.69 4.18 2.57 M-23 7.53 1.541" 4,66 0.831" 5.24 18.14 8.87 28.45 0.751" 5.73

M-24 0.421" 188.68 10.46 0.421 19.45 10.32 0.961" 7.93 1.391" 10.61 M-25 9.85 24.21 5.65 8.85 32.37 1.331" 4.38 112.01 1.481" 2.98

M-26 13.57 8.31 16.36 6.03 4.30 0.661" 1.02t 4.79 0.151" 0.011" M-30 3.69 4.79 2.101. 2.601" 36.66 14.30 6.16 38.85 2.65 9.03 M-31 4.98 1.76t 7.12 3.49 9.55 9.19 2.90 30.18 5.04 0.021.

M-32 16.24 4.58 2.001" 2.38t 53.08 1.481" 3.84 1.521" 2.65 5.45 M-38 0.191" 0.451" 0.731" 1.491. 1.051" 0.841" 5.99 2.101" 6.32 14.41

M-50 7.11 17.97 4.02 11.42 31.18 7.49 2.161" 168.93 8.37 9.77 M-53 1.211" 5.44 4.38 0.431" 22.63 12.99 0.471" 44.24 9.75 5.98

M-55 18.52 15.81 14.97 0.541" 1.921" 41.63 4.30 83.40 6.58 6.07 M-56 2.54 26.63 1.651" 2.96 0.431. 7.79 6.27 3.47 0.321" 0.29t

M-58 12.48 11.33 14.85 4.81 9.95 6.42 10.63 31.43 1.071" 3.84 M-60 5.84 2.82 5.68 3.75 8.49 4.76 1.66t 86.32 0.831" 1.361.

*For legend see Figs 1 and 2.

tNo significant differences (P>0.05); MH~M.Xverticillata hybrids.

obtained during 1988 and 1989. M-38 showed no significant differences for most parameters with the exception of DW, TD and TF. DW, LBN, SB, LS and LBL showed very low correlation between the two years.

The two-way ANOVA (Table 3) performed on the two-year data revealed that both the M.× verticillata hybrids (M) and environment (E) were significant factors for the parameters taken into consideration (P<0.01). The MXE inter- action was not significant in only one case (LBN). The eigenvalues from PCA indicated that two components provided a good summary of the morphological and dry weight values. The princi- pal component (PC) 1 (Factor 1) explained the 25% of total variance, while the PC 2 (Factor 2) explained the 23%. Figure 1 depicts the scatter plot of the morphological parameters on the axes of the first two PCs. The plant dimensions (LA, SH, LBL, LBN) were grouped together in the right upper quadrant, while the plant (SB) and leaf (LS) ratios were located in two other quadrants. TF, TN and TD lay in the upper left, upper right and lower right quadrants, respectively.

Student's t-values and the coefficients of correlation obtained from the pairwise compari- son of the oil data during 1988 and 1989 are shown in Table 4. As for morphological data, and

also for the oil data, there were in general highly significant differences among the data from the two years.

Menthone, menthofuran, isomenthone and menthol were not calculable for M-21 and M-32 which in turn were the only M.×verticillata hybrids for which carvone was calculable. All oil components showed a high correlation, for both flowers and leaves, between the two years.

The oil component two-way ANOVA (Table 5) showed interaction values for M, E and M×E which were always highly significant. The eigen- values of the PCA indicated that the two first PCs accounted for 38 and 22% of the total variance. Figure 2 shows the scatter plot of the oil compo- nents, from both flowers and leaves, on the axes of the first two PCs (Factor 1 and Factor 2). Menthone and isomenthone were grouped together and located, along with menthofuran, in the upper right quadrant. Menthol and carvone (which was grouped with limonene) were located in opposite quadrants.

The scatter plot of both morphological and oil chemical parameters on the axes of the first two PCs is represented in Fig. 3. The total variance explained by the PC1 (Factor 1) accounted for the 24%, whereas the variance of the PC2 (Factor 2) was 18%. DW was grouped with the plant

496 M. MAFFEI

TABLE 3. MEAN SQUARE VALUES OF THE MORPHOLOGICAL PARAMETERS FROM TWO-WAY ANOVA PERFORMED ON ALL M.X VERTICILLATA HYBRIDS

M.× verticillata hybrids Environment MXE Error Dep. var. (M) (E)

SH 2692.97 (6)* 4615.74 (1) 145.97 (6) 33.34 (68) LBL 4031.23 (6) 3418.39 (1) 755.74 (6) 27.10 (68) LBN 99.23 (6) 116.42 (1) 15.67t (6) 12.18 (68) SB 53.21 (6) 21.71 (1) 21.19 (6) 0.80 (68) LA 1908.60 (6) 569.76 (1) 188.11 (6) 2.10 (68) LS 0.60 (6) 3.47 (1) 0.16 (6) 0.05 (68) DW 49.23 (16) 42.12 (1) 57.11 (16) 2.68 (166) TN 8.18×10 ~ (16) 1.75x109 (1) 3.19x108 (16) 1.02X106 (166) TD 99.22 (16) 19.80 (1) 8.91 (16) 1.40 (166) TF 5457.52 (16) 8014.48 (1) 949.66 (16) 33.32 (166)

*Degrees of freedom. tNo significant difference (P>0.05).

1.0

0.5

¢~1 n- O I-- 0 O ,¢ g,

-0.5

-1.0

LBN

-1.0 -0.5 O 0.5 1.0

FACTOR 1

FIG. 1. SCATTER PLOT OF THE MORPHOLOGICAL PARAMETERS ON THE AXES OF THE FIRST TWO PRINCIPAL COMPONENTS (Factor 1 and Factor 2). Circles denote the location of the data from 1988 and 1989. SH=stem height; LBL=lateral branch length; LBN=lateral branch number; SB=ratio between SH and LBL; LA=leaf area; LS=ratio between the longer and the shorter axis of the leaf; TN=leaf trichome number; TD--leaf trichome density; TF=flower trichome number; DW=plant dry weight.

dimensions (LA, SH, LBL, LBN), the leaf ratio (LS) and isomenthone (IM). Once again TF, TN and TD were completely separated. TF was associ- ated with menthofuran, TN with limonene and carvone, while TD was located in the upper right quadrant showing no association with oil corn-

ponents. MF, ML and CA were still located in different quadrants, while IM and MO no longer showed close associations.

The differentiation among M.× verticillata hybrids was also studied by canonical discrimi- nant analysis of the PC scores of individual

PLASTICITY AND GENOTYPIC VARIATION IN MENTHA 497

TABLE 4. STUDENT'S t VALUES AND COEFFICIENTS OF CORRELATION (R) FROM THE PAIRWlSE COMPARISONS BETWEEN 1988 AND 1989 OIL DATA OF THE MENTHAX VERTIClLLATA HYBRIDS DURING 19~8 AND 1989

Limonene Menthone Menthofuran Isomenthone Menthol Carvone L* F ** L F L F L F L F L F

MH (0.95) (0,91) (1.00) (0.86) (0.95) (0.93) (0.99) (0.97) (0.88) (0.83) (1.00) (1.00)

M-17 8.31 11.21 28.84 10.06 19.43 3.28 32.38 3.54 26.71 11.69 NC NC M-20 4.48 1.761" 22.59 9.38 2.30t 143.17 29.71 NC 25.70 16.42 NC NC M-21 3.40 2.77 NC NC NC NC NC NC NC NC 9,84 9.60 M-23 9.84 10.66 6.23 8.14 2.26t 10.11 11.22 33.65 5.75 182.87 NC NC M-24 0.32t 0.881" NC NC 49.19 6.86 1.941" 13.61 21.89 3.15 NC NC M-25 36.97 1.971" 8.82 21.64 31.27 10.99 2.23t 21.41 18.84 35.81 NC NC M-26 28.64 1.471" 35.03 9.33 6.29 66.88 2.83 141.05 35,61 6.16 NC NC M-30 12.39 16.27 NC 0.17t 11.18 3.96 NC 9.36 495.83 25.91 NC NC M-31 23.17 197.76 NC 205.70 35.99 61.04 NC 16.50 7.93 20.18 NC NC M-32 11.36 12.13 NC NC NC NC NC NC NC NC 6.43 5.65 M-38 3.28 12.04 5.01 98,29 25.08 20.61 17.80 1.611" 27.76 49.11 NC NC M-50 18.20 64.99 NC NC 9.32 2.76 NC NC 7.49 154.48 NC NC M-53 5.21 3.28 NC 58.21 23.18 55.72 4.78 20.55 NC 11.44 NC NC M-55 8.98 21.99 NC NC 28.68 20.29 NC NC 7.49 154.48 NC NC M-56 1.83 24.82 NC NC 7.38 61.26 NC NC 21.81 18.16 NC NC M-58 108.03 12.65 NC NC NC 6.62 NC NC 364.79 7.15 NC NC M-60 3.94 55.48 1.311" 42.05 17.89 12.41 NC 10.65 28.20 14.39 NC NC

*L=leaves; **F=flowers; NC~not calculable. tNo significant differences (P>0.05); MH--M.×verticillata hybrids.

TABLE 5. MEAN SQUARE VALUES OF THE OIL CHEMICAL PARAMETERS FROM TWO-WAY ANOVA PERFORMED ON ALL M.× VERTIClLLATA HYBRIDS*

M.× verticillata hybrids Environment M×E Error Dep. vat. (M) (E)

Limonene L 961.34 (16)$ 37.89 (1) 26.91 (16) 0.08 (166) Ft 1436.98 (16) 9.23 (1) 66.31 (16) 0.13 (166)

Menthone L 2442.01 (16) 0.58 (1) 1.58 (16) 0.05 (166) F 710.69 (16) 58.68 (1) 52.95 (16) 0.02 (166)

Menthofuran L 2435.55 (16) 76.42 (1) 68.96 (16) 0.13 (166) F 6196.98 (16) 123.48 (1) 228.00 (16) 0.25 (166)

Isomenthone L 1501.86 (16) 14.42 (1) 14.09 (16) 0.02 (166) F 894.56 (16) 8.73 (1) 22.83 (16) 0.01 (166)

Menthol L 1879.10 (16) 204.3 (1) 127.76 (16) 0.07 (166) F 671.07 (16) 1048.89 (1) 143.46 (16) 0.10 (166)

Carvone L 1689.55 (16) 0.51 (1) 1.59 (16) 0.02 (166) F 3541.83 (16) 0.76 (1) 2.53 (16) 0.02 (166)

*L=leaves; tF=flowers. ~Degrees of freedom.

M.×verticillata hybrids to discriminate popula- tions and subset of populations. Three canonical discriminant functions accounted for 80% of the total variance in the PC scores from 1988 and 1989 data for morphological and oil chemical parameters. Plots of the three canonical discrimi- nants (CANON 1, 2 and 3; Fig. 4) showed M-17, M-38 and M-53 as outliers. M-30 and M-31 sampled in 1989 formed a group population together, with M-20, M-23, M-24 and M-25

sampled in 1988 and 1989. M-32, M-21, M-56 and M-50 did not change their spatial position in the discriminant space during the two years, whereas M-38, M-53, M-17, M-26, M-58, M-60, M-30 and M-31 showed different locations in 1988 with respect to 1989.

The maxima values of the daily temperatures from June to August during 1988 and 1989 along with the rainfall data (expressed in ml) are shown in Fig. 5, The two-way ANOVA performed on the

498 M. MAFFEI

n- O I-- O < LL

1.0

0.5

-0.5

-1.0

-1.0 -0.5 0 0.5 1.0

FACTOR 1

FIG. 2. SCATTER PLOT OF THE OIL PARAMETERS ON THE AXES OF THE FIRST TWO PRINCIPAL COMPONENTS (Factor 1 and Factor 2). Circles denote the location of the data from 1988 and 1989, CA=carvone; Ll=limonene; IM=isomenthone; MO=menthone; MF=menthofuran; ML-menthol.

minima, mean values and maxima (data not shown) indicated insignificant differences between the two years for minima and mean values, whereas significant differences (P<0.05) were obtained for maxima values. The differen- ces between months were always highly signifi- cant (P<0.001), whereas the interaction year×month was only significant for maxima values. The two way ANOVA performed on rain values showed no significant differences for year, month and year×month interactions.

Discussion The plants, in response to alteration in their environment, may change the morphological and physiological characteristics in order to maintain or improve fitness. Phenotypic plasticity consists of specific responses, each of them caused by an environmental factor, and the differences may differ among different geno- types [16-18]. The behavior of the M.X verticillata hybrids, which have been the object of this study, indicated, in general, high levels of pheno-

typic plasticity (both morphological and physio- logical) and genotypic variation as well as genetic variation for phenotypic plasticity.

The most variable among all parameters taken into consideration were the oil components of both flowers and leaves. Their coefficient of variation, which could be considered the amount of genetic variation, indicated high oil variability, reflecting the different genotypic characteristics of the plants [15]. In contrast, a general low morphological genotypic variation was present among plants.

Different patterns of plasticity were found by analysing separately the responses (in the sense of Bradshaw [17]) of the morphological and the oil chemical parameters to changes in the environmental conditions.

The two-way ANOVA offered a powerful method for comparing the plastic responses of the M.× verticillata hybrids. Three major compo- nents of variation were derived: the variance due to the M.X verticillata hybrids (M), the variance due to the year, or environment, effect (E) and

PLASTICITY AND GENOTYPIC VARIATION IN MENTHA 499

U.

1.0

0.5

-0.5

-1.0

DW

O

-1 .O -0.5 O 0.5 1.0

FACTOR 1

FIG. 3. SCATTER PLOT OF BOTH MORPHOLOGICAL AND OIL PARAMETERS ON THE AXES OF THE FIRST TWO PRINCIPAL COMPONENTS (Factor 1 and Factor 2). Circles denote the location of the data from 1988 and 1989. For the legend see Figs 1 and 2.

I

100 ] cs 0 | - r RW r D ~

_zoO ~ Z g', ~

_20° [ --~"'-. O -300 I ~ A ~. lID -400 r ~ ~"

500 i O0

"~,~ _z0 0o _ 3 0 0 C a n o n ` ` = . ' - - - ^ . . . . 3

FIG. 4. SCATTER PLOT OF THE MENTHA× VERTICILLATA HYBRIDS ON THE AXES OF THE FIRST THREE CANONICAL FUNCTIONS. M-17 1988=A; M-17 1989=B; M-20 1988 and 1989, M-23 1988 and 1989, M-24 1988 and 1989, M-25 1988 and 1989, M-30 1989, M-31 1989, M-5O 1988 and 1989, M-55 1988 and 1989=C; M-21 1988 and 1989=D; M-26 1988=E; M-26 1989=F; M-30 1988=G; M-31 1988--H; M-32 1988 and 19~9=1; M-38 1988=L; M-38 1989--M; M-53 1988--N; M-53 1989--O; M-56 1988 and 1989=P; M-60 1988=R; M-60 1988=S.

the variance due to the interaction of M with E (MXE). The "M" variance reflected the genotypic variability, and resulted in differences in overall

values of the parameters considered. The "E" variance, depending on the level of plasticity in the population, could be used to estimate the overall phenotypic plasticity in the population. Finally, the variance associated with the "M×E" interaction reflected the differences in the pattern of response of M to the E variable; in other words the variation among genotypes in response to the environment [19]. The error variance was a further useful parameter used as a function of the replication factor.

In these experiments M, E and MXE were highly significant factors (P<0.01) for all parameters considered, with the exception only of MXE for LBN. In particular, some morpho- logical parameters (such as SH, LBN, LS, TN and TF) showed higher mean square values for the E variable than for the M variable, thus indicating higher degrees of plasticity with regard to geno- typic variability. On the other hand, the oil com- ponents always showing higher mean square values for the M variable (with the exception of menthol in flowers) than for the E variable, indicated higher degrees of genotypic variation.

500 M. MAFFEI

38

30

~ 26,

~ 22,

18

14

10

1988 1989

30 60 90 30 60 90 June July Aug. June July Aug.

-5OO

E

• 400

• 300

, 200

0

Days

, 100

m Temperatures

Rain

FIG. 5. PLOT OF THE TEMPERATURES (MAXIMA VALUES) AND RAINFALL DATA (expressed in ml) DURING THE 1988 AND 1989 GROWING SEASONS.

The M×E interaction can arise from differen- ces between the M.× verticillata hybrids in their response [13], which can be quantified by using the mean square values. For the morphological parameters, in all the significant means squares involving MXE the M component contributed only 50% to the differences, whereas for the oil data M was the main contributor.

With regard to morphological data, differen- ces among the M and E entries were consider- ably larger for TN, TF, LBL, SH and LA, whereas for the oil data menthofuran and carvone had greater mean square values than the other oil components. Thus, among the M.×verticillata hybrids, a greater role of phenotypic plasticity compared to genotyplc variation was more pro- nounced in the morphology than in oil composition.

The genetic correlation between the character states expressed in different environments quantifies the genetic variation in plasticity in terms of the genetic model of evolution [19]. As the correlation approaches +1 the potential of the character states for independent evolution goes to zero [19]. Thus, the high correlations found for all oil components and for LA, TN, TD, TF and SH involve expression of most of the same genes in the same way in each of the two environments [20], whereas the lower correla-

tion obtained for some of the morphological parameters (DW especially) permits a greater degree of independent evolution [19].

The frequency of the oil glands, plant dry matter and morphological development in peppermint are strikingly influenced by small temperature changes [9]. In Mentha arvenis var. piperascens, a plant which is closely related to the M. × verticillata hybrids, the oil composition is a function of mean daily temperatures during the growing season [9]. Temperature and rainfall changes affect the oil composition of many Mentha species [10-12]. Our results, probably in relation to the highly significant differences in the maxima temperatures experienced by the plants during the two years, confirmed this striking environmental influence. Changes in oil compo- sition with environmental changes are clearly a plastic phenomenon [21] which was shown by our result. The positive correlations among some oil components (i.e. limonene, menthofuran and menthone), as well as the close chemical rela- tionship between these monoterpenes, indicates both common biosynthetic precursors [22] and linkage of biochemical pathways between leaves and flowers, whereas, some negative correla- tions indicated a strong negative effect of menthofuran on both limonene (its precursor [23]) and the menthols.

PLASTICITY AND GENOTYPIC VARIATION IN MENTHA 501

The plot of oil components on the PCA factors allowed some biosynthetic interpretations: the spatial distribution of the oil components was in agreement with their biogenetic origin [23]. Menthone and menthofuran were grouped in the same quadrant indicating a common precursor (which is known to be pulegone [23]); menthol and carvone were located in opposite quadrants indicating a separation in the biochemical path- way (in fact these two compounds are the result of the P-450 hydroxylations of the common pre- cursor limonene in two different positions [23, 24].

The general plot of both morphological and oil chemical parameters showed a statistical linkage between menthofuran and flower trichome number in one quadrant, and between limonene, carvone and the leaf trichome number in the other. In peppermint the flowers produce a higher menthofuran percentage than leaves [4- 6]; this situation is in accordance with our results and could explain the close linkage observed.

The most divergent among the M. × verticillata hybrids were: M-38, which was characterized by the lowest plasticity among all hybrids for morphological parameters and by high percent- ages of menthone in the distilled oil [15]; M-53, which also had high percentages of menthone and M-17 which contained isomenthone [15]. Other M. × verticillata hybrids that might be con- sidered outliers were M-56 containing menthol and M-32 containing carvone [15]. However, an absence of morphological response does not necessarily mean that a plant lacks plasticity [13].

In conclusion, the results obtained on the M.Xverticillata hybrids confirmed a significant effect of environmental factors on the morphol- ogy and oil composition of this genus. However, the above mentioned response variability of the material has to be taken into account and caution is needed to avoid any misinterpretation given by the relatively low number of repetitions. Nevertheless, low levels of morphological plasticity and high degrees of oil genotypic varia- tion tended to form "stable" outliers. On the other hand, high levels of phenotypic plasticity involved a major reorganization of the relation- ships among characters [18]. A strong genetic differentiation, by representing a more long-term reaction, is less adaptive than a plastic response [16], but should be taken into consideration in

selecting new Mentha cultivars. In fact, in accord- ance with Bradshaw [17], there is a great interest in the way in which an individual can maintain stability in the face of environmental influences. So, the less plastic the response of a mint clone the more useful will be (in terms of economic importance) its breeding and selection.

Since mint growers have to fight continuously against changes in environmental conditions, the evaluation of the phenotypic plasticity and genetic variation of phenotypic plasticity of the cultivated genotypes might be considered a use- ful and important tool for selecting stable plants. Besides, selection for plasticity has become a major focus of most of the modern breeding pro- grams [13].

Experimental Seventeen Mentha× verdc///ata hybrids were obtained from a seed population of M.×vertici//ata clone 7303 as described previously [15]. The plants were propagated vegetatively for four years in experimental plots of the Otto Botanico of the UniversiW of Turin, Italy. Measurements were performed dur- ing the last two years of growth (1988 and 1989). Six plants for each hybrid were tested. Measurements and oil extractions were performed at full bloom.

The morphological analysis used the following parameters: SH=stem height, calculated in centimetres from ground to the tip of the main stem inflorescence; LBL=length of the longest lateral branch, calculated in centimetres from the principal stem to the tip of the lateral branch inflorescence; LBN~lateral branch number, which was the same as leaf pair number; SB=ratio between SH and LBL, which was indicative of the plant shape; LA=leaf area, expressed in square centimetres; LS~ratio between the longer axis and the shorter axis of the leaf, which was indicative of the leaf shape; TN=total leaf glandular trichome number, calculated on both sides of the most expanded, but not senescent leaves; TD~leaf peltate trichome density, expressed as number of glandular trichomes per square centimetre, calculated on both sides of the most expanded, but not senescent leaves; TF=total glandular trichome number per flower, calculated on the flower calix since the petals of the plants under study always lack trichomes; DW=dry weight of the plant, calculated from a mixture of leaves of different size, stems and flowers randomly collected from the plants.

The oil chemical composition of both leaves and flowers was estimated after distillation in a Likens and Nickerson apparatus.

The extracting solvent was a mixture (5:1) of diethyl ether and hexane, and steam distillation-extraction was continued for 1 h. The extract was washed several times with distilled water and then dried by passing over a column packed with anhydrous MgSO 4. The solvent was evaporated by a gentle stream of N 2. For all analyses, distillation was performed on the most expanded, but not senescent leaves, and on the flowers borne on the tip of the main stem.

The essential oils were analysed using a gas chromatograph

502 M. MAFFEI

with a flame ionization detector. Separation was accomplished by a 30-m fused silica column coated with Carbowax 20M with the following operating conditions: injection temperature: 230°C, hydrogen carrier gas flow: 1.5 ml/min; temperature program: 3 rain isothermal at 60°C, then a linear temperature rise of 3°C/min to 180°C. The compounds were identified using gas chromatography-mass spectrometry equipped with a 50-m fused silica column coated with Supelcowax 10. The helium carrier gas flow rate was 1.5 ml/min. Mass spectros- copy was performed at 70 eV. The injector temperature was 230°C and the ion source temperature was 250°C.

The oil analyses used as parameters for flowers (F) and leaves (L) the following oil components: limonene (LIF, LIL), menthone (MOF, MOL), menthofuran (MFF, MFL), iso- menthone (IMF, IML), menthol (MLF, MLL) and carvone (CAF, CAL).

Temperatures and rainfall data were collected daily from 1 June to 31 August, which was considered the optimum growth period for all the plants under study.

The statistical analyses were performed on a Macintosh IIx computer by using a "Systat" multi-statistics program. The difference significance between the mean values of the Mentha hybrid parameters during the two years was estimated by using Student's t-test. In order to partition the component variation in the plant behavior within each parameter, a two-way ANOVA was performed with environment (E, standing either for temperature, rainfall, etc.) as one factor, the M. x verticillata hybrids (M) as another and the various parameters (both morphological and oil chemical) as the dependent variable. Principal component analysis (PCA) was calculated with a vari- max rotation of the component Ioadings. The differentiation of the parameters was studied by examining the pattern and structure of their distribution, whereas the differentiation of the M. × verticillata hybrids was estimated by canonical discrimi- nant analysis. For the latter, the categories considered were: the two years (E) and the 17 M.Xverticillata hybrids (M); the model was represented by all the dependent vari- ables~constant+E+M+EXM. The post-hoc test considered only the M effect.

Acknowledgements--The author is grateful to G. Doglia and F. Chialva for their technical assistance during GC-MS analyses; to T. Sacco for supplying the plants and to G. Berta and V. Filipello for the use of the Macintosh IIx computer.

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