J. Essent. Oil Res.

, 17, 66-70 (January/February 2005)

Variation in the Amount of Yield and in the Extract

Composition Between Conventionally Produced and
Micropropagated Peppermint and Spearmint

A. Aflatuni*
MTTAgrifood Research Finland,North OstrobothniaResearch Station, FIN-92400 Ruukki, Finland
J. Uusitalo and S. Ek
University of Oulu, Laboratoryof Mass Spectrometry, Departmentof Chemistry, FIN-90014 Oulu, Finland
A. Hohtola
University of Oulu, Departmentof Biology/Botany, FIN-90014 Oulu, Finland

Abstract
The quality and quantity of oil obtained from three different peppermint Mentha x piperitaL. origins and the
spearmint Mentha spicata L. were compared in micropropagated and conventionally propagated plants in northern
Finland (64°40'N) in 1997-1998. Each block comprised M. piperitapeppermint plants from three different origins;
namely, the United States, Bulgaria and Egypt (Black Mitcham), and a spearmint, M. spicataof Egyptian origin. The
micropropagated plantlets and conventionally propagated plants were transferred to the experimental field in June.
In the first year, there were no differences in the dry leaf yield between the micropropagated plants and their
conventionally produced counterparts, although the former had a higher leaf/stem ratio. In the second year, the dry
leaf yield of micropropagated plants, which were left in 1997 in field and developed from the underground stolons,
was higher than that of conventionally propagated plants.
The percentage of oil was higher with conventionally propagated plants, but only in the first year. Significant
differences were observed between mint origins in the different propagation methods. Moreover, conventionally
propagated plants showed a higher menthol percentage. There was no significant difference in the isomenthone
content of the plants.
In spearmint, the dry yield was higher in the second year with micropropagated plants, but there were no signifi-
cant differences in the percentage of carvone between the two propagation methods.

Key Word Index

Menthapiperita,Menthaspicata,Lamiaceae, essential oilcomposition, menthol, menthone, propagationmethods,
in vitro propagation.

Introduction Thepropagation material, used mostlyinthe multiplication

There are many reasons for the vegetative propagation of
mint. As a hybrid, peppermint seldom produces seeds that are
ableto germinate. As aconsequence, itis propagated exclusively
from its vegetativeparts, i.e., green shoots, underground stolons
and rooty turions. In addition, high costs and the plant's high
demand for manual labor disallowlarge-scale propagation with
green cuttings, which is appropriate only for the fast propaga-
tion of improved clones. Plantations are usually established
using underground stolons, although the peppermint also is
propagated by means of 8-10 cm high turions, developed from
underground stolons (1).
of peppermint, consists of stolons grown in the soil (green) or
on the surface (white). The satisfactory development ofstolons
during intensive cultivation is hindered by several known and
unknown factors. The mass of the root system increases with
the age of the plant, whereas the quantity of living stolons
decreases. In the cold climate conditions of northern Finland,
the best method for the conventional propagation of mints is
to cut stolons from the field before frost in autumn and store
them in a cool storage in moist peat during winter. Towards
the end of April they are transferred to a greenhouse for pre-
liminary growing. The plants are transplanted into a field as
early as possible after the frost is over (2).

*Address for correspondence Received: November2002

Revised; March 2003
1041-2905/05/0001-0066$6.00/0-© 2005 Allured Publishing Corp. Accepted: April 2003

66/Journal of Essential Oil Research Vol. 17, January/February 2005

 Peppermint and Spearmint
Table I. Differences between conventionally and micropropagated peppermint and spearmint origins in dry matter
1997
Peppermint origin Conv. Microp. SEM
Egypt 3035 1919 165
Bulgaria 2214 1505 165
United States 2644 1601 165
Spearmint 3758 2482 165
Mean 2913 1877 116
Conv. = conventionally propagated; Microp. = micropropagated
Several papers (3,4) have reported the influence of in vitro
growth conditions on the secondary metabolite biosynthesis
of terpenes. Peppermint is micropropagated for a number of
reasons, including breeding (5) and the rapid multiplication of
elite plants (6). Although a number of micropropagation stud-
ies have focused on attaining a mint yield of high quality and
quantity, only a few have dealt with differences in the yield and
oil quality and quantity between conventionally propagated and
micropropagated mints grown under field conditions.
The primary goal of the present study was to assess the
impact of two specific propagation methods, referred to as
conventional propagation and micropropagation, on the yield
and oil quantity and quality ofpeppermint and spearmint after
the first year of cultivation in the field. Another objective was
to establish whether the epigenotic effects caused by tissue
culture observed in first year growth, disappear in the next
generation of growth.
Experimental
Plant material: The field experiments were conducted
at the North Ostrobothnia Research Station of the Agricul-
tural Research Centre of Finland, in Ruukki (64°40'N) during
1997-1998.
The experimentwas set-up in a randomizedcomplete block
design at two locations with four replications. The randomized
treatments were the propagation methods and the mint origins.
Two propagation methods, conventional and micropropagation,
were investigated. Each block comprised M. piperita(pepper-
mint) plants from three different origins; namely, the United
States, Bulgaria and Egypt (Black Mitcham), and a spearmint,
M. spicata, of Egyptian origin.
Micropropagation: Havingbeen sterilizedin 3.5% sodium
hypochlorite for 10 min, nodal cuttings were propagated on MS
medium (7) with 0.5 mg/L kinetin. The plants were grown at a
temperature of +22°C (16 h light, 8 h dark) at a light intensity
of about 50 W m-2. The shoots were then transferred into MS
multiplication medium supplemented with 0.5 mg/L 6-benzyl-
aminopurine (BAP) and 0.25 mg/lindole-3-butyric acid (IBA).
Roots were generated on WPM medium (8) containing 0.1
mg/L PIBA. Finally, following a two-month acclimatization in
a greenhouse, the micropropagated plantlets were transferred
to the experimental field on June 15.
Conventionally propagatedplants:The roots were lifted
from the field at end of October and stored in a cool store at
a temperature of approximately +10 to -1°C. In the middle of
Vol. 17, January/February 2005
Dry matter kg/ha
1998
P-value Conv. Microp. SEM P-value
<001 4201 6780 1076 0.11
<.01 2339 4747 1076 0.13
<.001 2903 5680 1076 0.08
<.001 1270 5382 1076 0.01
<.001 2678 5647 931 0.01
April, green stolons were cut from the roots and planted in a
plugtrayofthe same size as the micropropagatedplants (55x55
x 62 mm). After a preliminary growing period in a greenhouse,
the stolons were transplanted to the experimental field at the
same time as the micropropagated plants. Before transplanta-
tion, the micropropagated and conventionallypropagatedplants
were of about the same size. In 1998, crop was developed from
the underground stolons left in 1997.
The soil type was fine sand, and the soil was fertilized at
the rate of N 40, P 28, K 56 kg/ha before planting. The plot
size was 1 x 3 m = 3 m2 . The plants were transplanted to the
field by a potato planter such that each row comprised 15
plants at a distance of 20 cm from each other. The number of
replications was four.
In both years, the plants were harvested at the start of
flowering. In 1997, they were harvested on 13 August; in
1998, the peppermints were harvested on 25 August and the
spearmints on 9 September.
After the harvest, the fresh yield was dried (in +40 0C),
and random samples (2 x 200 g) were analyzed to assess their
dry matter content, ratio of dry leaves to stems and oil con-
centration.
Oil Isolation and Analysis
To isolate oil, mint leaves (50 g), dried at +30 0C, were mixed
with 700 mL of water. The mixture was then hydrodistilled
for 2 h at +100°C at normal atmospheric pressure, and its oil
percentage was measured. Next, 10 monoterpene components
were identified in the peppermints and five in spearmint. This
analysis involved both propagation methods and production
years. Of the identified components, this paper discusses the
seven most common ones (menthol, menthone, isomenthone,
neomenthol, pulegone, menthofuran and carvone).
The constituents ofthe oilwere analyzedusingGC/FID, and
peak identifications were based on studies with GC/MS using
a Perkin Elmer Ion Trap Detector. Sample components were
identifiedbymass spectra matchingwith aWileyNBS Registry
Mass Spectra Data Base. In addition, the identifications were
verified by comparing their retention time and mass spectrum
with a reference compound, whenever possible.
Sample preparation: Leaf samples were randomly col-
lected from different parts of plants and represented leaves
of all ages. The fresh samples were stored in a freezer. For
analysis, the leaves were thawed at room temperature for
10 min, then crushed in liquid nitrogen. A crushed sample
Journal of Essential Oil Research/67
 Aflatuni et al.

Table II. Differences between traditionally and micropropagated peppermint and spearmint origins in dry leaf matter and leaf stem/
ratio
Dry matter kg/ha
1997 1998
Peppermint origin Conv. Microp. SEM P-value Conv. Microp. SEM P-value
Egypt 1305 1270 95 0.80 2052 3063 440 0.12
Bulgaria 869 1031 95 0.24 1213 2157 440 0.15
United States 1040 1101 95 0.66 1421 2782 440 0.04
Spearmint 1654 1566 95 0.52 636 2224 440 0.02
Mean 1217 1242 66 0.80 1331 2557 370 0.06

Leaf/stem
1997 1998
Peppermint origin Conv. Microp. SEM P-value Conv. Microp. SEM P-value
Egypt 0.8 2.0 0.1 <.001 1.0 0.9 0.1 0.59
Bulgaria 0.6 2.2 0.1 <0.001 1.3 0.9 0.1 0.04
United States 0.6 2.2 0.1 <.001 1.2 1.0 0.1 0.38
Spearmint 0.8 1.7 0.1 <.001 0.9 0.7 0.1 0.40
Mean 0.7 2.0 0.1 <.001 1.1 0.9 0.1 0.12

Conv. = conventionally propagated; Microp. = micropropagated

of 180-200 mg was accurately weighted and 1.0 mL of
methylene chloride containing styrene (304 ng/l.L) as an
internal standard was added. The samples were extracted
in an ultrasonic bath for 5 min and filtered. Turbid extracts
were dried with anhydrous Na2SO4 before introduction to
a gas chromatograph.
GC/FID analysisformints planted in 1998: Gas chroma-
tographywas performed on aPerkin ElmerAutosystem XL gas
chromatograph usinghelium as acarrier gas in constant pressure
mode (37 psig). The temperature of the injector and the detec-
tor (FID, flame ionization detector, hydrogen flow 45 mL/min,
air flow 450 mL/min) were 250°C and 300°C, respectively. The
column was a fused-silica column coated with RTX-1701 (30 m
x 0.32 mm x 0.25 lAm). The injected sample volume was 1 jL,
and the split-flow was adjusted to 13 mL/min.
Statistical methods: Randomized complete block designs
with four blocks were set-up at two locations.
The measurements were repeated for each plot during the
two-year study. These measurements were correlated and the
correlation was taken into account. The covariance structure
for the repeated measurements was selected after comparing
all biologically sensible structures using Akaike's information
criterion (9).
The assumptions for normality and constancy of error
variance were checked by graphical methods; namely, plots
of residuals for constancy of error variance and box-plots for
normality of errors. The parameters were assessed by the
REML estimation method using the SAS system for Windows,
release 8.2, and the MIXED procedure.
Results and Discussion
Transplantedplants:Afterthepreliminarygrowingperiod
in the greenhouse before transplantation, the micropropagated
plants were mostly of the same height and very uniform in
shape. Their growth was vigorous and straight, which made
them easier to handle and plant with a potato or a cabbage
planter than the conventionally propagated plants, which were
of varying height with abundant creeping shoots.
Dry yield, dry leaf yield and leaf/stem ratio: Although
the dryyield of 1997was significantlyhigherwith conventional
propagation (Table I) than with micropropagation, the dryleaf
yield failedto exhibit asignificant difference (Table II). In 1998,
however, the dry yield and the dry leaf yield of plants that were
micropropagated in 1997 and developed from underground
stolons in 1998 were both higher than those of conventionally
produced plants.The significant lower yield of biomass from
micropropagated mints in 1997 could be interpreted as the
effect of epigenetic or somaclonal variations caused by tissue
culture. The differences were significant in peppermint and
spearmint of US origin.
Randhawa et al. (10) reported that the application of
growth regulators (GA) did not increase herb and oil yields in
M. piperita, but in the case of M. spicata, a lower concentra-
tion of GA and IAA gave a significantly higher total yield than
that of the control. According to Cameron et al. (11), a sharp
decrease in average fruit size and weight is observed in straw-
berries when micropropagated plants are used directly for fruit
production. However, research has not established a difference
in total fruit production or fruit quality between the progeny
of micropropagated and conventionally obtained plants.
The leaf/stem ratio in dry matter was significantly higher
in the first year, but was reduced to an insignificant level in
the second year (p < 0.001). In 1997, micropropagated mints
of different origin contained more leaves than conventionally
propagated mints, but in 1998 the differences were no longer
significant (Table II).
Oil content and composition: Conventionally propa-
gated plants had a significantly higher percentage of oil than
micropropagated plants in the first year (Table III, mean). In
the second year, however, these differences were considerably
smaller. Similar results have been reported by Holm et al. (12),

68/Journal of Essential Oil Research Vol. 17, January/February 2005

 Peppermint and Spearmint

Table IlIl. Effect of traditional and micropropagation methods on the oil percentage of the various peppermint and spearmint
origins
Oil % in 1997 Oil %in 1998
Peppermint origin Conv. Microp. SEM P-value Conv. Microp. SEM P-value

Egypt 2.2 1.3 0.11 <.005 2.0 2.0 0.09 0.84

Bulgaria 2.1 1.5 0.11 <.005 2.0 2.5 0.09 <.05
United States 1.8 1.7 0.11 0.23 2.1 1.7 0.09 0.02
Spearmint 1.8 1.3 0.11 0.01 1.6 1.5 0.09 0.32
Mean 2.0 1.6 0.10 0.02 1.9 1.9 0.04 0.99

Conv. = conventionally propagated; Microp. = micropropagated

who foundthatthe oil content ofconventionallypropagatedpep-
permint ranged from 1.1-2.6%, while the corresponding figures
for micropropagated peppermints varied from 0.9-1.9% in the
first year. According to Ravishankar and Venkatarman (13), in
the rapid multiplication ofpeppennint, the oil content of original
plants and those grown in vitro were found to be 0.85% on a
freshweightbasis. Alsoplants raisedintissue culturehaveshown
an oil proffle similar to that of the parent plant (14).
Significant differences in oil content between the various
mint origins and the two propagation methods were observed
in 1997 and 1998 (P = 0.04 and p < 0.01). In both years, the
conventionally propagated peppermint of US origin contained
a higher oil percentage than micropragated.
In experiments conducted by Holm et al. (12), micro-
propagated peppermints had a higher menthol content dur-
ing the first year. In the second year, however, the results
obtained by the two methods were practically identical.
The amount of menthone, menthol and carvone in plants
regenerated from the protoplasts of M. piperita vulgaris
decreased when compared with the control. On the other
hand, an increase has been observed in the carvone levels of
all protoplast-derived plants (5). The present study showed
no differences between the various propagation methods
in the carvone percentage of spearmint (in 1997, P = 0.25,
and in 1998, P = 0.40).
There was no significant interaction between the study
years and the propagating methods in the percentage of iso-
menthone (P = 0.16) and neomenthol (P = 0.44). Also, mint
origin failed to produce a significant difference (isomenthone
P = 0.52, neomenthol P = 0.27 in 1997; isomenthone P = 0.31,
neomentholP = 0.69 in 1998) betweenthepropagation methods
(Table IV). Holm et al. (12) reported a higher isomenthone
content in conventionally propagated peppermint plants in the
first year, but the difference disappeared in the following year.
On the other hand, plants produced by both methods showed
an almost identical neomenthol content in the first year, but
in the second, the neomenthol content of micropropagated
peppermints was slightly higher.
In 1997, the menthofuran content of conventionally
propagated mints of Egyptian, Bulgarian and US origin was
0%, 2.1% and 0.9%, respectively, but in micropropagated
peppermints menthofuran was not detected at all. In 1998,
menthofuran was not produced regardless of the propaga-
tion method. In research conducted by Holm et al. (12), the
menthofuran content of micropropagated plants was found
to exceed that of conventionally produced plants in the first
year. In the second year, however, the amounts were about
the same.
Pulegone was not detected in isolated oils. In this study,
the oil content of the various mints was determined only from
the leaves. This serves to explain the low menthofuran content
and the absence of pulegone, as these substances are usually
found in high amounts in flowers, sometimes comprising over
50% of flower oil (15).
In conclusion, the results of the experiment have clearly
establishedthatinthe firstyeartherewere no differences in the
dryleafyieldofmicropropagatedandconventionallypropagated
plants, although the leaf/stem ratio of micropropagated plants
was significantly higher. In the second year, however, the dry
leaf yield of micropropagated plants was higher than that of
their conventionally propagated counterparts.
This study establishes that the quality and quantity of
peppermints grown by the conventional and the micro-
propagation method depends on the origin of the plants
and varies from year to year. The quality of conventionally
propagated plants was significantly higher in the first year,
but the difference was reduced to an insignificant level
during the next year.
Micropropagation is a feasible method when rapid multi-
plication is of essence, for example, when there is a shortage
of plant material or elite plants. In northern conditions, poor
overwintering may occasionallyproduce an insufficientnumber
of seedlings. Thus, micropropagatedplants, beingmoreuuniform
in size and shape, are easier to transplant than conventionally
propagated ones.
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Vol. 17, January/February 2005
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TITLE: Variation in the Amount of Yield and in the Extract

Composition Between Conventionally Produced and
Micropropagated Peppermint and Spearmint
SOURCE: J Essent Oil Res 17 no1 Ja/F 2005
WN: 0500108202022

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