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How plant regeneration from Mentha × piperita L. and Mentha × citrata Ehrh. Leaf protoplasts affects their monoterpene composition in field conditions
J. Plant Physiol. Wll.
149. pp. 481-488 (1996)
How Plant Regeneration from Mentha x piperita L. and Mentha x citrata Ehrh. Leaf Protoplasts Affects their Monoterpene Composition in Field Conditions 1 2 MARIE-HELE:NE CHAPUT , HAN SAN , LOUIS DE Hys2, EMMANUEL GRENIER3, 1 1 HELENE DAVID , and ALAIN DAVID 1
Laboratoire de Biotechnologie et Physiologie Vegetaies, Facuite des Sciences Universite de Picardie Jules Verne, 33 rue Saint-Leu, 80039 Amiens Cedex, France
2
InstitU( du Tabac de Bergerac, Domaine de la Tour, 24100 Bergerac, France
3
Departemenr Mamematiques et Systemes d'information, Institut Superieur Agricole de Beauvais, BP 313, 60026 Beauvais Cedex, France
Received Ocrober 23, 1995 . Accepted March 8, 1996
Summary
A procedure to regenerate plants from leaf protoplasts of two micropropagated hybrid species of mint, Mentha x piperita L. and Mentha x citrata Ehrh., has been developed in order to determine whether the in vitro treatment could influence the monoterpene composition. Purified protoplasts were first plated in liquid media containing 3.5 ~mol/L BA, 1.25 ~mol/L zeatin, 2.5 ~mol/L NAA and 2.25 or 5.5 ~mol/L 2,4-0 as growth regulators to induce initial divisions. In these conditions, high percentages of protoplast-derived cells divided, especially for M X piperita clones (ADF ranging from 17% to 31 % at day 6). Reduction of both medium osmolality and 2,4-0 concentration resulted in a sustained development of microcalli for both species. Calli were then transferred onto solidified regeneration media. The first regenerated shoots of M. X piperita were observed 3 months after protoplast isolation on media containing 8.9 ~mol/L and 13.2 ~mol/L BA or 1.8 ~mol/L and 4.5 ~mol/L TOZ. Regeneration frequency did not exceed 4 %. Regeneration medium sequences were required for shoot regeneration on M. X citrata calli. At first, they were cultured on a 1.8 ~mol/L TOZ containing-medium for 1 week. They were then transferred onto media supplemented with various concentrations ofTOZ or BA. The highest frequencies of shoot regeneration were around 10 %. Shoot morphology was affected by TOZ for both species, but not by BA. In a field trial, the amount of menthone, menthol and carvone in the regenerated plants of M x piperita vulgaris was compared with that of the control. Results showed a decrease in the amount of menthone and menthol and an increase of carbone levels in all protoplast-derived plants.
Key words: Mentha X piperita L., Mentha x citrata Ehrh., mint, protoplast, plant regeneration, essential oil monoterpenes. Abbreviations: ADF = absolute division frequency; BA = 6-benzylaminopurine; 2,4-0 = 2,4dichlorophenoxyacetic acid; IAA = indolacetic acid; NAA = 1-naphthaleneacetic acid; PAR = photosynthetically active radiation; TOZ = thidiazuron.
© 1996 by Gustav Fischer Verlag, Stuttgart
482
MARlE-HELENE CHAPUT, HAN SAN, loUIS DE HyS, EMMANUEL GRENIER, HELENE DAVID, and ALAIN DAVID
Introduction
Aromatic components are widely encountered in plants, such as those belonging to Apiaceae, Myrtaceae or Lamiaceae. They represent an important group of natural products with industrial interest. They are used as perfume (lavender), as flavouring agents in food (mint, basil, rosemary), as drugs (thyme, eucalyptus) and in cosmetics (mint, borage). Essential oils of mint are also of great use for the aromatic industry (tooth-paste and sweet aromas). Each mint species is characterized by its essential oil content (Bricout et al., 1978), which confers its medicinal or culinary qualities. Linalool and linolyl-acetate are the major terpenes in Mentha X citrata Ehrh. , carvone in Mentha spicata (spearmint) and menthol in Mentha X piperita L. (peppermint), which also contains high amounts of menthone and/or menthyl-acetate. Due to the sterility of many mint species, conventional breeding methods are often difficult. So in vitro techniques, such as protoplast culture or protoplast fusion, are expected to provide a new possibility for increasing genetic variability and also a means of transferring desirable traits to plants. However, the validity of this approach depends on the ability of the regenerated plants to produce high yields of essential oil of suitable quality. Bricout and Paupardin (1975) and Bricout et al. (1978) showed that tissue culture could modify the production and/or the quality of essential oils in Mentha X citrata and in Mentha X piperita. Recently, Sato et al. (1993) obtained shoot regeneration from peppermint protoplasts, but the terpene composition of regenerated plants was not studied. This paper reports on the successful culture and plant regeneration of leaf protoplasts of two hybrid species of mint, Mentha X pipenta L. and Mentha X citrata Ehrh. Regenerated plants of Mentha X piperita were also assessed in the field for oil composition. Analysis was carried out using some of the terpenoid components as markers of a putative somaclonal variation.
Materials and Methods
Plant material Two sets of clones were used [Mentha x piperita L.. MitchamMilly ssp. vulgaris (MPV) and Hongrie ssp. Sylvestris (MPS); Mentha x citrata Ehrh., Bergamote-Latour (MCBL) and Lavanduliodora Men 154 (MCL»), both provided by the National Institute of Medicinal, Aromatic and Industrial Plants (91490 Milly-La Foret. France). Stems of each clone were surface-sterilized by dipping in «Mercryl-lauryJe» solution for 2 min, followed by a 45 min treatment with a 15 % (v/v) solution of commercial hypochlorite (Domestos). After three rinses with sterile distilled water, stems were divided into single node cuttings of 1 cm in length. which were transferred in vitro Onto a MS basal medium (Murashige and Skoog, 1962) containing 2 % (w/v) glucose. 0.05 % (w/v) casein enzymatic hydrolysate. 0.01 % (w/v) L ascorbic acid (as an antioxidant agent) and solidified with 0.25 % (w/v) Phytagel (Sigma). The pH was adjusted to 5.S before autoclaving at 11S·C for 20 min. Cultures were placed in a growth chamber at 23·C, with 16 hI day illumination (60 Ilmol. m -z. s-1 PAR) . When axillary buds had elongated and produced a stem with 4 to 5 nodes, this stem was
used for a new generation of microcuttings. Cycles of micropropagation occurred at 4-week intervals on the same medium without ascorbic acid.
Protoplast isolation Lamina from 4-week-old plants were slightly scarified and placed onto a 6-mL filter-sterilized enzyme solution composed of cell and protoplast washing (CPW) salts (Frearson et aI., 1973). O.S % (w/v) cellulase RIO (Yakult, Tokyo. Japan). 0.05 % (w/v) pectolyase Y23 (Sheishin. Tokyo. Japan), 9.1 % (w/v) mannitol and 0.05% (w/v) 2(N-morpholino) ethane sulfonic acid (MES) buffer at pH 5.6. Lamina were then incubated overnight in the dark at 31 .c. At the end of the digestion period, the mixture was gently shaken (30 rpm) for 10-15 min and then passed through a nylon sieve (SO Ilm) to separate released protoplasts from cellular debris and undigested tissue. Protoplast suspension was washed twice by centrifugation at 62 gn for 6 min in a CPW solution containing 0.25 mollL mannitol and 0.125mo1/L NaCl (washing solution). Yield and viability of protoplasts were evaluated from 4 independent experiments. Yield represented the number of purified protoplasts per gram of fresh tissue and viability was assessed with fluorescein diacetate (Widholm, 1972) and expressed as the percentage of purified protoplasts.
Protoplast culture Purified protoplasts were initially cultured in the dark at 23 ·C and at a density of 40 x 103 protoplasts per mL. They were suspended in three different culture media: AE2. AE3. AE4. The basal medium contained 6.3 mmollL KN0 3 • 2.5 mmollL NH 4N0 3, 4.1 mmollL CaCI2> 0.75 mmollL MgS0 4, 0.6mmollL KH zP0 4 and MS micronutrients (Murashige and Skoog. 1962). 7.2 % (w/v) glucose, 0.025 % (w/v) casein enzymatic hydrolysate. 2.5 % (v/v) coconut water (Sigma), vitamins, sugars and organic acids as described by Kao (1977) and supplemented with 0.01 % (w/v) spermidine (AE2 and AE4 media) or 0.02 % spermidine (AE3 medium). Growth regulators were as follows: 3.5 IlmollL BA. 1.25 Ilmoi/L Zeatin. 2.51lmo1lL NAA with either 5.5 IlmollL 2,4-0 (AE2 and AE3 media) or 2.251lmo1lL 2,4-0 (AE4). The pH was adjusted to 5.7-5.S (KOH). Osmolality ranged from 4S0 to 510 mOsm (Kg H ZO)-I. Absolute Division Frequency (ADF) was defined as the number of divided protoplast-derived cells at day 6 per initial number of cultured protoplasts. It was estimated on the basis of three independent experiments. After 2 weeks of culture. microcalli were resuspended in AE5 medium, which was the same as AE4 but with the osmolality reduced to 300-330 mOsm (Kg H ZO)-1 decreasing the glucose concentration.
Shoot regeneration Two to three weeks later. when their development was sufficient in AE5 medium. calli were transferred onto solidified regeneration media. and exposed to a 16h photoperiod (60llmol.m-z.s-1 PAR). All regeneration media contained SH mineral salts (Schenk and Hildebrandt. 1972). vitamins (Morel and Wetmore. 1951). 2 % (w/v) sucrose. 0.61lmo1lL IAA and 0.25 % (w/v) phytagel (Sigma) . For the clones Mitcham-Milly (MPV) and Hongrie (MPS). the media were supplemented with 1.S. 4.5. 9.1 Ilmoi/L TDZ or 4.4. S.9. 13.2IlmoI/L BA (Table 4). For the clone Bergamote-Latour (MCBL), calli were first transferred onto a regeneration medium containing 1.Sllmol/L TDZ for 1 week. They were then submitted to different sequences of growth regulators as shown in Table 1.
Regeneration of plants from mint protoplasts; mono terpene composition
Table 1: Growth regulator composition of regeneration medium sequences tested for the culture of protoplast-derived calli of M. x citrata, clone Bergamote-Latour (MCBL). Regeneration Growth remedium gulators (~M) Medium 1a sequence
Growth regulators (~M) Medium 2~
RS 1 RS 2 RS 3 RS4 RS 5 RS 6 RS 7 RS8
3.6TDZ 3.6BA 3.6 BA (2 weeks) 3.6 BA (2 weeks) 1.8 BA (2 weeks) 3.7 Zeatin 4.6 Zeatin 4.6 Zeatin (2 weeks)
a
b
1.8TDZ 1.8 TDZ 1.8 TDZ 1.8 TDZ 1.8 TDZ 1.8 TDZ 1.8TDZ 1.8 TDZ
Growth regulators (~M) Medium 3
4.4 BA 8.8 BA
4.4 BA 2.3 Zeatin
Calli are cultivated for 1 week on medium 1 and then transferred onto medium 2. Unless otherwise stated, calli are maintained on medium 2.
The percentage of regenerating calli (number of calli bearing at least one shoot/number of transferred calli) was estimated after 2 months of culture on the regeneration media. Regenerated shoots were propagated in vitro as mentioned above, acclimatized in a greenhouse and finally transferred to the field. The chronology of the successive culture steps is shown in Table 2.
Field trial Shoots of Mentha x piperita vulgaris were randomly isolated from ten calli designated from A to] (two shoots per callus, e.g. AI , A2; Bl, B2; .. .), rooted, and the plants transferred to greenhouse conditions. They were cultivated from April to May 1994 in pots containing 0.2 L ofTriohum Tray substrate (Klasmann-France). Plants were watered daily. Two blocks were established in the field on May 17th (SW France). Both of them contained one replicate of each callus-derived plant, obtained by vegetative propagation (twenty plants), and six control plants originating from the same M. x piperita vulgaris genotype grown in natural conditions. Plants were randomly distributed within each block (interval between plants = 2 m) and fertilised with ammonium nitrate (60 kg/ha) . Plants were harvested 120 days after transfer to the field.
483
Oil composition analysis Leaves of each plant of M x piperita vulgaris were frozen at -18 ·C, then Iyophilised and ground into powder (particle size 0.5 mm) . Two grams of leaf powder were extracted with 25 mL of 95 % ethanol. The extraction was carried out on an orbital shaker for 2 hours at 20·C. Then the suspension was filtered and the filtrate analysed immediately. Two microliters of filtrate (ethanolic solution) were analysed by gas chromatography in a glass column, external diameter 7 mm, internal diameter 2 mm, length 2 m packed with SE 30 5 % on chromosorb. The chromarographic conditions were: gas pressure (nitrogen) 2.5 bars; injector temperature 200 ·C, column temperature 50·C (isotherm), FlO detector temperature 200 .c. The retention time of the three compounds analysed (Aldrich products) were: (-)-menthone 4 min 0 s; DL-menthol 4 min 15 s; (- )-carvone 5 min 30 s. Calibration was effected with solutions containing the three compounds (menthone, menthol and carvone) and anethol (retention time 6 min 36 s) as the internal standard. The areas of the peaks were calculated by an integrator SP 4290. The quantity of each compound is expressed in mg per g.d.w.
Statistical analysis Data was subjected to analysis of variance. Comparison between the mean of each callus-derived plant and the mean of the control were performed using Dunnett's two-tailed test. The effect of blocks was removed from the error component. When testing the difference between plants derived from the same callus, the variation within calli was distinguished, leading to a partially hierarchical model. The use of a logarithmic scale was necessary to normalize the distribution of residuals for menthone and menthol amounts. However, as the transformation did not affect the conclusions. results are presented from the original data.
Results
Protoplast isolation For each clone, numerous green protoplasts were easily released when lamina were treated with an enzyme solution containing 0.8 % cellulase RIO and 0.05 % pectolyase Y23. At the end of the digestion period, very little cellular debris
Table 2: Chronology of the successive steps from protoplast to plants transferred in greenhouse conditions. Time
Culture Step
day 0
Isolation and culture of protoplasts in media AE2. AE3, AE4 (darkness. 23 ·C. density = 40x 103 p/mL; osmolality 480-510 mOsm (KgH 2 0)-I)
=
day 6 week 3 week 5/6 week 13/14 week 21
Estimation of ADF for each clone Transfer of protoplast-derived colonies into AE5 medium (osmolality = 300/330 mOsm (KgH 20)-I) Transfer of calli onto the regeneration media (16h photoperiod. 60 ~mol . m- 2 . s- 1 PAR. 23 ·C) Estimation of the percentage of regenerating calli Micropropagation of regenerated plants Transfer to the greenhouse
Growth Regulators and polyamines in culture medium AE2: AE3: AE4:
BA 3.5 3.5 3.5
AE5:
BA 3.5
Zea 1.25 1.25 1.25
NM 2.5 2.5 2.5
NM Zea 2.5 1.25 see Tables 1 and 4
~M)
2,4-0 5.5 5.5 2.25
Sper 39 78 39
2,4-0 2.25
Sper 39
484
MARIE-HEd:NE CHAPUT, HAN SAN, LOUIS DE HyS, EMMANUEL GRENIER, HELENE DAVID, and ALAIN DAVID
Table 3: Yield, viability and diameter of leaf protplasts. Means and SE were calculated from 4 independent experiments. Yield: Number of purified protoplasts per gram of fresh tissue; viability: percentage of fluorescent protoplasts; N.D: not determined.
Mentha x citrata
Mentha x piperita
Species clone
MitchamMilly
Hongrie
Bergamote- LavandulioLatour dora Men 154
Yield (x 106) 11.11±1.41 9.78±1.03 8.88±0.62 10.18±0.51 ±3.79 89.45± 1.71 81.7 ±4.32 82.3 ± 1.59 Viability (%) 81 25.75±5 N.D Diameter (IJ.m) 29.97±5.27 N.D
was observed, so protoplast purification only consisted of two rinses in the washing solution. The protoplast yield ranged from 8.88 ± 1.03 X 106 per gram of fresh tissue for clone Bergamote-Latour (MCBL) to 11.11 ± 1.41 X 106 for the clone Mitcham-Milly (MPV), while protoplast viability exceeded 80 % (Table 3). They were relatively homogeneous in diameter, which was rather small, about 28/lm for leaf protoplasts (Table 3).
Protoplast culture First divisions occurred on the third or fourth day of culture in AE2, AE3 and AE4 media for both clones of M X piperita and on the fourth or fifth day for the clone M X citrata, Bergamote-Latour (MCBL). Concerning the clone Lavanduliodora Men 154 (MCL), protoplast suspension turned progressively brown and formed budding-like structures whatever the medium. No division was observed and finally protoplasts died. As shown in Fig. 1, relatively high percentages of division were obtained in all three tested media for both clones of M X piperita but especially for the clone 40
30
•
AD
[!j AEJ
EJ
'-<"' Q
AE4
20
Hongrie (MPS), for which ADF reached 31.3 ± 1.74 % in AE2 medium. No difference in ADF was noted between the AE2 and AE4 media for these clones. On the other hand, an increased spermidine concentration (AE3) produced very low division frequency at day 6 (Fig. 1). However, this result was due to a delay in protoplast development because ADF in medium AE3 reached that already mentioned a few days later. Since ADF in the AE2 medium was similar to that of the AE4 medium for all three clones, further experiments were performed using AE2 as initial protoplast culture medium. When the dividing cells were transferred to the AE 5 medium at week 3, most of them gave rise to microcalli, for all three clones. Reduction of both the medium osmolality and 2,4-D concentration allowed an intense development of microcalli. Two to three weeks later, it was possible to plate them directly onto the regeneration media.
Shoot regeneration Protoplast-derived calli of the three clones (MPV, MPS, MCBL) started to increase in size whatever the regeneration medium and turned green when they were exposed to a 16 h photoperiod (60/lmol.m- 2 .s- l PAR).
Shoot regeneration ofM.
X
piperita clones
Callus growth of the clones Mitcham-Milly (MPV) and Hongrie (MPS) was very weak (l-2mm in diameter) on regeneration media supplemented with 9.1 /lmol/L TDZ (R3) or with 4.4 /lmollL BA (R4). It was more intensive (2-6 mm in diameter) when media contained a lower concentration of TDZ (1.8/lmoI/L, Rl or 4.5/lmollL, R2) or a higher concentration of BA (8.9/lmoIlL, R5 or 13.2/lmoIlL, R6). In these conditions, protoplast-derived calli of the clone MPV exhibited a multi-nodular structure and gave rise to buds. The first regenerated shoots on Rl, R2, R5, R6 media were observed 3 months after protoplast isolation. The best results were obtained with media R2 and R6, but in all cases, the frequency of regenerating calli was low with a maximum of 3.8 % (Table 4) . Growth regulators affected the number of shoots per callus as well as the shoot morphology. When TDZ was used, the number of shoots per callus was variable and increased with TDZ concentration: with 4.5 /lmol/L TDZ it could reach 10 to 20. Shoots were vitreous and exhibited an abnormal morTable 4: Frequency of regenerating protoplast-derived calli of M x piperita, clone Mitcham-Milly (MPV) in different regeneration me-
10
dia.
MPS
MCBL
MPV
MCL
Clones
Fig. 1: Protoplast absolute division frequency (ADF) at day 6 of M. X piperita, clones Hongrie (MPS) and Mitcham-Milly (MPV), and of M X citrata, clones Bergamote-Latour (MCBL) and Lavanduliodora Men 154 (MCL), in AE2, AE3 and AE4 media. Means and SE were calculated on the base of 3 independent experiments.
Medi- Growth reurn gulators (J.l.M)
N° of protoplast- N° of calli Regeneration derived calli with shoots frequency (%)
Rl
165 210 210 210 210 210
R2
R3 R4 R5 R6
1.8TDZ 4.5TDZ 9.1 TDZ 4.4 BA 8.9BA 13.2 BA
3 8 0 0 2 7
1.8 3.8 0 0 0.9 3.3
485
Regeneration of plants from mint protoplasts; monoterpene composition
Table 5: Frequency of protoplast-derived calli of M x citrata, clone Bergamote-Latour (MCBL), producing shoots in different regeneration medium sequences. The number of regenerating calli estimated after a total of 2 months of culture on the medium sequence. Regeneration medium sequence
Growth regulators (f.1M) Medium I'
Growth regulators (f.1M) Medium 2b
RSI RS2 RS3 RS4 RS5 RS6 RS7 RSB
I.B I.B I.B I.B I.B I.B I.B I.B
3.6TDZ 3.6BA 3.6 BA (2 weeks) 3.6 BA (2 weeks) I.B BA (2 weeks) 3.7 Zeatin 4.6 Zeatin 4.6 Zeatin (2 weeks)
TDZ TDZ TDZ TDZ TDZ TDZ TDZ TDZ
(1 (1 (1 (1 (1 (1 (1 (1
week) week) week) week) week) week) week) week)
Growth regulators (f.1M) Medium 3
4.4 BA B.B BA 4.4 BA 2.3 Zeatin
N° of protoplast derived calli
Wofcalli with shoots
Regeneration frequency
164 135 75 75 55 75 61 135
3 2 B 6 6 0 0 0
I.B 1.5 10.7 B 10.9 0 0 0
(%)
': Calli are cultivated for 1 week on medium 1 and then transferred onto medium 2. b: Unless otherwise stated, calli are maintained on medium 2.
phology. The reduction of TDZ concentration promoted shoot elongation. On the contrary, few shoots were produced when BA was used (2-3 shoots per callus) but they appeared normal. When shoots had sufficiently elongated they were transferred onto a hormone-free medium where they rooted spontaneously and the abnormal morphology observed on the TDZ-containing medium progressively disappeared.
Shoot regeneration ofthe M x citrata clone Since in a preliminary experiment, protoplast-derived calli of clone Bergamote-Latour (MCBL) did not form shoots when using previous regeneration treatments (transfer onto RI, R2, R5, R6 media) another strategy was established that consisted of a regeneration medium sequence as shown in Table 1: all protoplast-derived calli were first placed onto medium RI (1.8 ~mol/L TDZ) for 1 week, then a few of them were transferred onto another medium supplemented with various concentrations of TDZ or BA or Zeatin. In some cases, a third medium was used 2 weeks later (Table 1). In these conditions, the clone Bergmaote-Latour (MCBL) produced compact and green calli. When a Zeatin-containing medium was used, a sharp increase in callus size was noted and calli seemed to spread on the surface of the medium but no shoots were recovered (Table 5). On the contrary, the other regeneration medium sequences allowed plant regeneration. The highest frequencies, around 10 %, were obtained when BA was used in the second and third medium of the sequence (RS 3, 4, 5). First shoots were observed 3-4 months after protoplast isolation. When calli grew in the presence of TDZ (RS 1, 2), they produced numerous but abnormal shoots similar to those already described with the clone Mitcham-Milly with the same growth regulator. In the presence of BA, shoots had a normal morphology and on a few occasions shoots rooted on the BA-containing medium.
Oil composition Callus-derived plants and controls of M X piperita vulgaris were harvested in early September, after 4 months of growth in field conditions. The amounts of men thone, menthol and carvone in the leaves were determined by gas chromatography.
<{? [>
Callus - derived plants Control plants
Menthol (mglg.d.w.) 2.97
2.44
1.90
3.74 2.74
Carvone (mglg.d.w.)
Menthone (mglg.d.w.) 0.024 0.72
Fig.2: Distribution of callus-derived plants (40 plants: 10 calli x 2 shoots per callus x 2 replicates) and control plants (12 replicates of Mentha x piperita vulgaris), according to menthone, menthol and carvone amounts.
Table 6: Mean of the control (12 replicates) and experimental error (root MSE*) for menthone, menthol and carvone amounts (mg/ g.d.w.).
Control mean Root MSE*
Menthone
Menthol
Carvone
2.Bl
2.44 0.273
0.15 0.062
0.401
* Square root of the Mean Square for Error from analysis of variance = Estimation of the standard deviation of replications.
Among the three compounds analysed, menthone and menthol were the most abundant in all the plants studied, carvone being much less represented (Fig. 2). For example, control plants contained an average of 2.81 and 2.44 mg per g.d.w. of menthone and menthol, respectively, and only 0.15 mg carvone (Table 6). However, variations in the content of the three compounds studied were observed when comparing the population of callus-derived plants with that
486
MAruE-HELENE CHAPUT, HAN SAN, LOUIS DE HyS, EMMANUEL GRENIER, HELENE DAVID, and ALAIN DAVID
Table 7: Differences between the mean of the 2 replicates of each
callus-derived plant (Mentha x piperita vulgaris) and the mean of the control expressed in root MSE for menthone, menthol and carvone amounts. Al and A2 represent two plants derived from callus A, B1 and B2 from callus B ... (see materials and methods). Plant
Menthone
Menthol
Carvone
Al A2 B1 B2 C1 C2 D1 02 E1 E2 F1 F2 G1 G2 HI H2 II 12 J1 J2
0.3 0.6 -1.7 -3.2* -3.5* -3.2* -3.7* -3.8* -3.5* -4.8* -4.5* -3.0* -4.0* -4.1* -3.3* -3.2* -1.3 -2.9* -3.2* -1.5
1.1 1.4 -0.5 -2.2 -1.1 -0.4
3.5* 2.4 4.0* 2.4 2.2 2.6* 1.7 2.1 1.1 0.7 -0.1 0.5 -0.1 1.0 1.5 0.9 0.8 0.1 -1.7 0.2
-1.3
-1.0 -2.6* -2.5* -3.1* -0.7 -1.0 -2.8* -3.2* -1.7 -1.6 -3.6* -1.6 -3.3*
* significant at the 0.05 level according to Dunnett's test.
of the control. The shift resulted in a decrease in menthone and menthol amounts and, on the contrary, a carvone increase in the leaves of the in vitro produced plants (Fig. 2). The validity of these observations was confirmed using Dunnett's test in which the mean value of the two replicates of a callus-derived plant for each of the three components was compared with those of the control. Significant differences (5 % level) were always negative for menthone and menthol and positive for carvone. For example, (Table 7) plant E2 produced significantly less menthone than the control. The difference between the mean values of menthone for the two replicates of the in vitro plants and that of the control corresponded to a 4.8-fold experimental error (Table 7). On the contrary, essential oil of the plant B 1 contained significantly more carvone than the control, the difference between the two mean values corresponding to a 4-fold experimental error (Table 7). In order to determine the origin of the variations mentioned above, the amount of menthone, menthol and carvone was compared in plants produced on the same callus. The difference is not significant (analysis of variance; p = 0.36, p=0.06 and p=0.44, respectively). The procedure from the protoplast to the plant in the field can be divided into two steps, from the protoplast to the callus and from the callus to the plant in natural conditions. Since there is no significant difference between plants on the same callus, the conclusion is that the changes in the amount of the three terpenes versus the control result from physiological events occurring during the first step, i.e. from the protoplast to the callus.
Discussion
Since the possibility of producing secondary metabolites in mint tissue cultures has been demonstrated (Bricout and Paupardin, 1975), attempts have been made to modifY the essential oil content of plants using somaclonal variation. In this context, two strategies for adventitious budding are available: organ culture (Bricout et al., 1978; Kukreja et al., 1991) and protoplast culture (Sato et al., 1993). The present paper reports on the use of this second strategy. Plants were regenerated from leaf protoplasts of two micro propagated mint species, M. X piperita and M x citrata . In M. X piperita, the amounts of three components of the essential oil, menthone, menthol and carvone, were compared with that of the control plants after transfer to field conditions. In the given experimental conditions, the first protoplast divisions of the M X piperita clones occurred on the third or fourth day of culture in liquid media, but they were only observed after 6 days of culture in media solidified with gelrite (Sato et al., 1993). It has been demonstrated recently that flax protoplast divisions were delayed in culture media solidified with agarose, while plating efficiency was promoted (unpublished laboratory data). Bud differentiation was highly influenced by the type of cytokinins and by the medium sequence used. In addition to BA-containing media also successfully employed by Sato et al. (1993), shoot regeneration was observed in the presence of TDZ for both M. X piperita and M X citrata (Tables 4 and 5); however, the two species exhibited different responses. M. X piperita could withstand TDZ for a long period of time. On the contrary a short induction with TDZ followed by a weaker cytokinin was more effective for M. X citrata. TDZ, a phenyl urea type cytokinin, has been found to stimulate shoot organogenesis (Cambecedes et al., 1991; Bates et al., 1992) and somatic embryogenesis (Bates et al., 1992). TDZ was also effective for the development of poplar protoplast derived calli and shoot formation (Chupeau et al., 1993). The beneficial effect of TDZ could be the result of the stimulation of the endogenous cytokinin synthesis or the limitation of their degradation by cytokinin oxidase (Hare and Van Staden, 1994). Concerning the essential oil composition, protoplast- derived plants were analysed 120 days after transfer to field conditions. Court et al. (1993) studied the effects of harvest date on the terpene content of peppermint plants in field trials. They observed that essential oil yield increased throughout the season, achieving a maximum from late August to early September. During plant development, the concentration of menthone decreased in favour of menthol, one of the major components of the essential oil of peppermint. At harvest time (early September), menthol represented 50.1 % against 11 % for menthone (Court et al., 1993). Based on the results mentioned above, plants used in the present study were harvested at this period of the year. However, contrary to the observations of Court et al. (1993), the amount of menthone remained high in the plants, e.g. in the controls, concentrations of menthone and menthol were 2.81 and 2.44 mg per g.d.w., respectively. However, these differences may result from quick changes in the composition of the oil, which occur as the maximum yield is ap-
Regeneration of plants from mint protoplasts; mono terpene composition proached by the end of the growing season (Court et aI.,
1993). It was noted that plant tissue culture conditions induced variations in the quantity of menthone, menthol and carvone in the protoplast-derived plants. Several papers have already reported on the influence of the in vitro treatment on secondary metabolite biosynthesis, such as terpenes (Bricout and Paupardin, 1975; Jain et al., 1991; Calvo and Sanchez-Gras, 1993) or alkaloids (Huizing et aI., 1983; Jung et al., 1992; Gontier et al., 1993). In the present study, a decrease of menthone and menthol and an increase of carvone were observed for all the protoplast-derived plants. Similar results have been described by Bricout and Paupardin (1975), when studying the essential oil content of plants obtained by adventitious budding on stem fragments of M x piperita cultivated on a BA-containing medium. A sharp decrease in menthol production was noticed. Kukreja et al. (1991) observed variations in four major constituents of the essential oil of M. arvensis, namely menthol, menthone, isomenthone and menthyl-acetate, in plants obtained by adventitious budding and grown for 110 days in the field. Out of the 80 somaclones analysed, only 8 had an amount of menthol slightly higher (88-94 %) than that of the control (86 %), the others having a lower or, in the best case, an equal concentration. The authors suggested that these modifications could be induced by somaclonal variation. Carvone is one of the major components of the essential oil of spearmint, M spicata (Hirate et al., 1990), while it is poorly represented in peppermint, M. x piperita. It was noticed in the evaluation trial that carvone had a tendency to increase in protoplast-derived plants, compared with the control plants. So in this study, the tissue culture treatment inversely affected menthone-menthol and carvone biosynthesis. Since the three of them, and terpenes in general, have a common precursor, Geranyl-PP, the inhibition of the biosynthetic pathway of menthone-menthol could result in the stimulation of the biosynthesis of carvone, but at this stage there is no evidence to sustain this hypothesis. Protoplasts were isolated from the leaves of plants micropropagated on a medium devoid of hormones. This step of the in vitro treatment should not induce long standing modifications. Moreover, no changes in the three monoterpenes analysed were observed between plants issuing from the same callus. Then it can be concluded that the variation between in vitro-produced plants and the control may result from events occurring before bud differentiation on the protoplast-derived calli, i.e. origin of the protoplast in the leaf, the response of a single cell faced with a stress condition andlor the first cycles of division leading to the callus formation. In summary, this paper reports on the conditions for the regeneration of Mentha X piperita L., Mitcham-Milly ssp. vulgaris (MPV) and Mentha x citrata Ehrh., Bergamote- Latour (MCBL) from leaf protoplasts of micropropagated shoots. It was observed that the procedure induces modifications in the three terpenes analyzed, namely a decrease of menthone and menthol and an increase of carvone. However, replications of oil analysis over several successive growing seasons are necessary before concluding that stable modifications can be induced by this procedure and before studying the modulation of the terpene metabolism pahtway by in vitro treatment.
487
Acknowledgements
We would like to thank the following ISAB colleagues: K. Chapelain for help with the statistical analysis and J. Gibson for reviewing the translation.
References BATES, S., J. E. PREECE, N. E. NAVARRETE, J. W. VAN SAMBEEK, and G. R. GAFFNEY: Thidiazuron stimulates shoot organogenesis and somatic embryogenesis in white ash (Fraxinus americana L.). Plant Cell Tissue and Organ Culture 31, 21-29 (1992). BRICOUT, J. and C. PAUPARDIN: Sur la composition de l'huile essentielle de Mentha piperita L. cultivee in vitro: influence de quelques facteurs sur sa synthese. C. R. Acad. Sc. 281, 383-386 (1975). BRICOUT, J., M. J. GARCIA-RoDRIDUEZ, C. PAUPARDIN, and R. SAUSSAY: Biosynthese de composes monoterpeniques par les tissus de quelques especes de Menthes cultivees in vitro. C. R. Acad. Sc. 287, 611-613 (1978). CALVO, M. C. and M. C. SANCHEZ-GRAS: Accumulation of monoterpenes in shoot-proliferation cultures of Lavandula latifolia Med. Plant Science 91, 207-212 (1993). CAMBECEDES, J., M. DURON, and L. DECOURTYE: Adventitious bud regeneration from leaf explants of the shrubby ornamental honeysuckle, Lonicera nitida Wils. cv. Maigriin: effects of thidiazuron and 2,3,5-triiodobenzoic acid. Plant Cell reports 10, 471474 (1991). CHUPEAU, M. c., M. LEMOINE, and Y. CHUPEAU: Requirement of thidiazuron for healthy protoplast development to efficient tree regeneration of a hybrid poplar (Populus tremula X p. alba). J. Plant Physiol. 141,601-609 (1993). COURT, w. A., R. C. Roy, and R. Pocs: Effect of harvest date on the yield and quality of the essential oil of peppermint. Can. J. Plant Sci. 73,815-824 (1993). FREARSON, E. M., J. B. POWER, and E. C. COCKING: The isolation, culture and regeneration of Petunia leaf protoplasts. Dev. bioI. 33, 130-137 (1973). GONTIER, E., M. A. FUNIAUX, J. N. BARBOTIN, and B. S. SANGWAN-NoRREEL: Tropane alkaloid levels in the leaves of micropropagated Datura innoxia plants. Planta Med. 59, 391-484 (1993). HARE, P. D. and J. VAN STADEN: Inhibitory effect of thidiazuron on the activity of cytokinin oxidase isolated from soybean callus. Plant Cell Physiol. 35, 1121-1125 (1994). HIRATA, T., S. MURAKAMI, K. OGIHARA, and T. SUGA: Volatile monoterpenoid constituents of the plantlets of Mentha spicata produced by shoot tip culture. Phytochemistry 29, 493-495 (1990). HUIZING, H. J., E. C. PFAUTH, TH. M. MAUNGRE, and J. H. SIETSMA: Regeneration of plants from tissue and cell suspension cultures of Symphytum officinale L. and effect of in vitro culture on pyrrolizidine alkaloid production. Plant Cell Tissue Organ Culture 2, 227-238 (1983). JAIN, M., R. BANER]I, S. K. NIGAM, J. J. C. SCHEFFER, and H. C. CHATURVEDI: In vitro production of essential oil from proliferating shoots of Rosmarinus officinalis. Planta Med. 57, 122-124 (1991). JUNG, K. H., S. S. KWAK. S. W. KIM, C. Y. CHOI, G. S. HEO, and J. R. LIU: Selection of protoclones for high yields of indole alkaloids from suspension cultures of Catharanthus roseus and the qualitative analysis of the compounds by LC-MS. Biotechnology Techniques 6, 305-308 (1992). KAo, K. N.: Chromosomal behaviour in somatic hybrids of soybean - Nicotianaglauca. Mol. Gen. Genet. 150,225-230 (1977).
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MARIE-HELENE CHAPUT, HAN SAN, LoUIS DE HyS, EMMANUEL GRENIER, HELENE DAVID, and ALAIN DAVID
KUKREJA, A. K., O. P. DHAWAN, A. K. MATHUR, P. S. AHUJA, and S. MANDAL: Screening and evaluation of agronomically useful somaclonal variations in japanese mint (Mentha arvensis L.). Euphytica 53, 183-191 (1991). MOREL, G. and R. H. WETMORE: Fern callus tissue culture. Amer. J. Bot. 38, 141-143 (1951). MURASHIGE, T. and F. SKOOG: A revised medium for rapid growth and bio assay with tobacco tissue cultures. Physiol. Plant. 15, 476-497 (1962).
SATO, H., S. ENOMOTO, S. OKA, K. HOSOMI, and Y. ITo: Plant regeneration from protoplasts of peppermint (Mentha piperita L.). Plant Cell Reports 12,546-550 (1993). SCHENK, R. U. and A. C. HILDEBRANT: Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can. J. Bot. 50, 199-204 (1972). WIDHOLM, J. M.: The use of fluorescein diacetate and phenosafranine for determining viability of cultured plant cells. Stain Tech. 47, 189-194 (1972).
149. pp. 481-488 (1996)
How Plant Regeneration from Mentha x piperita L. and Mentha x citrata Ehrh. Leaf Protoplasts Affects their Monoterpene Composition in Field Conditions 1 2 MARIE-HELE:NE CHAPUT , HAN SAN , LOUIS DE Hys2, EMMANUEL GRENIER3, 1 1 HELENE DAVID , and ALAIN DAVID 1
Laboratoire de Biotechnologie et Physiologie Vegetaies, Facuite des Sciences Universite de Picardie Jules Verne, 33 rue Saint-Leu, 80039 Amiens Cedex, France
2
InstitU( du Tabac de Bergerac, Domaine de la Tour, 24100 Bergerac, France
3
Departemenr Mamematiques et Systemes d'information, Institut Superieur Agricole de Beauvais, BP 313, 60026 Beauvais Cedex, France
Received Ocrober 23, 1995 . Accepted March 8, 1996
Summary
A procedure to regenerate plants from leaf protoplasts of two micropropagated hybrid species of mint, Mentha x piperita L. and Mentha x citrata Ehrh., has been developed in order to determine whether the in vitro treatment could influence the monoterpene composition. Purified protoplasts were first plated in liquid media containing 3.5 ~mol/L BA, 1.25 ~mol/L zeatin, 2.5 ~mol/L NAA and 2.25 or 5.5 ~mol/L 2,4-0 as growth regulators to induce initial divisions. In these conditions, high percentages of protoplast-derived cells divided, especially for M X piperita clones (ADF ranging from 17% to 31 % at day 6). Reduction of both medium osmolality and 2,4-0 concentration resulted in a sustained development of microcalli for both species. Calli were then transferred onto solidified regeneration media. The first regenerated shoots of M. X piperita were observed 3 months after protoplast isolation on media containing 8.9 ~mol/L and 13.2 ~mol/L BA or 1.8 ~mol/L and 4.5 ~mol/L TOZ. Regeneration frequency did not exceed 4 %. Regeneration medium sequences were required for shoot regeneration on M. X citrata calli. At first, they were cultured on a 1.8 ~mol/L TOZ containing-medium for 1 week. They were then transferred onto media supplemented with various concentrations ofTOZ or BA. The highest frequencies of shoot regeneration were around 10 %. Shoot morphology was affected by TOZ for both species, but not by BA. In a field trial, the amount of menthone, menthol and carvone in the regenerated plants of M x piperita vulgaris was compared with that of the control. Results showed a decrease in the amount of menthone and menthol and an increase of carbone levels in all protoplast-derived plants.
Key words: Mentha X piperita L., Mentha x citrata Ehrh., mint, protoplast, plant regeneration, essential oil monoterpenes. Abbreviations: ADF = absolute division frequency; BA = 6-benzylaminopurine; 2,4-0 = 2,4dichlorophenoxyacetic acid; IAA = indolacetic acid; NAA = 1-naphthaleneacetic acid; PAR = photosynthetically active radiation; TOZ = thidiazuron.
© 1996 by Gustav Fischer Verlag, Stuttgart
482
MARlE-HELENE CHAPUT, HAN SAN, loUIS DE HyS, EMMANUEL GRENIER, HELENE DAVID, and ALAIN DAVID
Introduction
Aromatic components are widely encountered in plants, such as those belonging to Apiaceae, Myrtaceae or Lamiaceae. They represent an important group of natural products with industrial interest. They are used as perfume (lavender), as flavouring agents in food (mint, basil, rosemary), as drugs (thyme, eucalyptus) and in cosmetics (mint, borage). Essential oils of mint are also of great use for the aromatic industry (tooth-paste and sweet aromas). Each mint species is characterized by its essential oil content (Bricout et al., 1978), which confers its medicinal or culinary qualities. Linalool and linolyl-acetate are the major terpenes in Mentha X citrata Ehrh. , carvone in Mentha spicata (spearmint) and menthol in Mentha X piperita L. (peppermint), which also contains high amounts of menthone and/or menthyl-acetate. Due to the sterility of many mint species, conventional breeding methods are often difficult. So in vitro techniques, such as protoplast culture or protoplast fusion, are expected to provide a new possibility for increasing genetic variability and also a means of transferring desirable traits to plants. However, the validity of this approach depends on the ability of the regenerated plants to produce high yields of essential oil of suitable quality. Bricout and Paupardin (1975) and Bricout et al. (1978) showed that tissue culture could modify the production and/or the quality of essential oils in Mentha X citrata and in Mentha X piperita. Recently, Sato et al. (1993) obtained shoot regeneration from peppermint protoplasts, but the terpene composition of regenerated plants was not studied. This paper reports on the successful culture and plant regeneration of leaf protoplasts of two hybrid species of mint, Mentha X pipenta L. and Mentha X citrata Ehrh. Regenerated plants of Mentha X piperita were also assessed in the field for oil composition. Analysis was carried out using some of the terpenoid components as markers of a putative somaclonal variation.
Materials and Methods
Plant material Two sets of clones were used [Mentha x piperita L.. MitchamMilly ssp. vulgaris (MPV) and Hongrie ssp. Sylvestris (MPS); Mentha x citrata Ehrh., Bergamote-Latour (MCBL) and Lavanduliodora Men 154 (MCL»), both provided by the National Institute of Medicinal, Aromatic and Industrial Plants (91490 Milly-La Foret. France). Stems of each clone were surface-sterilized by dipping in «Mercryl-lauryJe» solution for 2 min, followed by a 45 min treatment with a 15 % (v/v) solution of commercial hypochlorite (Domestos). After three rinses with sterile distilled water, stems were divided into single node cuttings of 1 cm in length. which were transferred in vitro Onto a MS basal medium (Murashige and Skoog, 1962) containing 2 % (w/v) glucose. 0.05 % (w/v) casein enzymatic hydrolysate. 0.01 % (w/v) L ascorbic acid (as an antioxidant agent) and solidified with 0.25 % (w/v) Phytagel (Sigma). The pH was adjusted to 5.S before autoclaving at 11S·C for 20 min. Cultures were placed in a growth chamber at 23·C, with 16 hI day illumination (60 Ilmol. m -z. s-1 PAR) . When axillary buds had elongated and produced a stem with 4 to 5 nodes, this stem was
used for a new generation of microcuttings. Cycles of micropropagation occurred at 4-week intervals on the same medium without ascorbic acid.
Protoplast isolation Lamina from 4-week-old plants were slightly scarified and placed onto a 6-mL filter-sterilized enzyme solution composed of cell and protoplast washing (CPW) salts (Frearson et aI., 1973). O.S % (w/v) cellulase RIO (Yakult, Tokyo. Japan). 0.05 % (w/v) pectolyase Y23 (Sheishin. Tokyo. Japan), 9.1 % (w/v) mannitol and 0.05% (w/v) 2(N-morpholino) ethane sulfonic acid (MES) buffer at pH 5.6. Lamina were then incubated overnight in the dark at 31 .c. At the end of the digestion period, the mixture was gently shaken (30 rpm) for 10-15 min and then passed through a nylon sieve (SO Ilm) to separate released protoplasts from cellular debris and undigested tissue. Protoplast suspension was washed twice by centrifugation at 62 gn for 6 min in a CPW solution containing 0.25 mollL mannitol and 0.125mo1/L NaCl (washing solution). Yield and viability of protoplasts were evaluated from 4 independent experiments. Yield represented the number of purified protoplasts per gram of fresh tissue and viability was assessed with fluorescein diacetate (Widholm, 1972) and expressed as the percentage of purified protoplasts.
Protoplast culture Purified protoplasts were initially cultured in the dark at 23 ·C and at a density of 40 x 103 protoplasts per mL. They were suspended in three different culture media: AE2. AE3. AE4. The basal medium contained 6.3 mmollL KN0 3 • 2.5 mmollL NH 4N0 3, 4.1 mmollL CaCI2> 0.75 mmollL MgS0 4, 0.6mmollL KH zP0 4 and MS micronutrients (Murashige and Skoog. 1962). 7.2 % (w/v) glucose, 0.025 % (w/v) casein enzymatic hydrolysate. 2.5 % (v/v) coconut water (Sigma), vitamins, sugars and organic acids as described by Kao (1977) and supplemented with 0.01 % (w/v) spermidine (AE2 and AE4 media) or 0.02 % spermidine (AE3 medium). Growth regulators were as follows: 3.5 IlmollL BA. 1.25 Ilmoi/L Zeatin. 2.51lmo1lL NAA with either 5.5 IlmollL 2,4-0 (AE2 and AE3 media) or 2.251lmo1lL 2,4-0 (AE4). The pH was adjusted to 5.7-5.S (KOH). Osmolality ranged from 4S0 to 510 mOsm (Kg H ZO)-I. Absolute Division Frequency (ADF) was defined as the number of divided protoplast-derived cells at day 6 per initial number of cultured protoplasts. It was estimated on the basis of three independent experiments. After 2 weeks of culture. microcalli were resuspended in AE5 medium, which was the same as AE4 but with the osmolality reduced to 300-330 mOsm (Kg H ZO)-1 decreasing the glucose concentration.
Shoot regeneration Two to three weeks later. when their development was sufficient in AE5 medium. calli were transferred onto solidified regeneration media. and exposed to a 16h photoperiod (60llmol.m-z.s-1 PAR). All regeneration media contained SH mineral salts (Schenk and Hildebrandt. 1972). vitamins (Morel and Wetmore. 1951). 2 % (w/v) sucrose. 0.61lmo1lL IAA and 0.25 % (w/v) phytagel (Sigma) . For the clones Mitcham-Milly (MPV) and Hongrie (MPS). the media were supplemented with 1.S. 4.5. 9.1 Ilmoi/L TDZ or 4.4. S.9. 13.2IlmoI/L BA (Table 4). For the clone Bergamote-Latour (MCBL), calli were first transferred onto a regeneration medium containing 1.Sllmol/L TDZ for 1 week. They were then submitted to different sequences of growth regulators as shown in Table 1.
Regeneration of plants from mint protoplasts; mono terpene composition
Table 1: Growth regulator composition of regeneration medium sequences tested for the culture of protoplast-derived calli of M. x citrata, clone Bergamote-Latour (MCBL). Regeneration Growth remedium gulators (~M) Medium 1a sequence
Growth regulators (~M) Medium 2~
RS 1 RS 2 RS 3 RS4 RS 5 RS 6 RS 7 RS8
3.6TDZ 3.6BA 3.6 BA (2 weeks) 3.6 BA (2 weeks) 1.8 BA (2 weeks) 3.7 Zeatin 4.6 Zeatin 4.6 Zeatin (2 weeks)
a
b
1.8TDZ 1.8 TDZ 1.8 TDZ 1.8 TDZ 1.8 TDZ 1.8 TDZ 1.8TDZ 1.8 TDZ
Growth regulators (~M) Medium 3
4.4 BA 8.8 BA
4.4 BA 2.3 Zeatin
Calli are cultivated for 1 week on medium 1 and then transferred onto medium 2. Unless otherwise stated, calli are maintained on medium 2.
The percentage of regenerating calli (number of calli bearing at least one shoot/number of transferred calli) was estimated after 2 months of culture on the regeneration media. Regenerated shoots were propagated in vitro as mentioned above, acclimatized in a greenhouse and finally transferred to the field. The chronology of the successive culture steps is shown in Table 2.
Field trial Shoots of Mentha x piperita vulgaris were randomly isolated from ten calli designated from A to] (two shoots per callus, e.g. AI , A2; Bl, B2; .. .), rooted, and the plants transferred to greenhouse conditions. They were cultivated from April to May 1994 in pots containing 0.2 L ofTriohum Tray substrate (Klasmann-France). Plants were watered daily. Two blocks were established in the field on May 17th (SW France). Both of them contained one replicate of each callus-derived plant, obtained by vegetative propagation (twenty plants), and six control plants originating from the same M. x piperita vulgaris genotype grown in natural conditions. Plants were randomly distributed within each block (interval between plants = 2 m) and fertilised with ammonium nitrate (60 kg/ha) . Plants were harvested 120 days after transfer to the field.
483
Oil composition analysis Leaves of each plant of M x piperita vulgaris were frozen at -18 ·C, then Iyophilised and ground into powder (particle size 0.5 mm) . Two grams of leaf powder were extracted with 25 mL of 95 % ethanol. The extraction was carried out on an orbital shaker for 2 hours at 20·C. Then the suspension was filtered and the filtrate analysed immediately. Two microliters of filtrate (ethanolic solution) were analysed by gas chromatography in a glass column, external diameter 7 mm, internal diameter 2 mm, length 2 m packed with SE 30 5 % on chromosorb. The chromarographic conditions were: gas pressure (nitrogen) 2.5 bars; injector temperature 200 ·C, column temperature 50·C (isotherm), FlO detector temperature 200 .c. The retention time of the three compounds analysed (Aldrich products) were: (-)-menthone 4 min 0 s; DL-menthol 4 min 15 s; (- )-carvone 5 min 30 s. Calibration was effected with solutions containing the three compounds (menthone, menthol and carvone) and anethol (retention time 6 min 36 s) as the internal standard. The areas of the peaks were calculated by an integrator SP 4290. The quantity of each compound is expressed in mg per g.d.w.
Statistical analysis Data was subjected to analysis of variance. Comparison between the mean of each callus-derived plant and the mean of the control were performed using Dunnett's two-tailed test. The effect of blocks was removed from the error component. When testing the difference between plants derived from the same callus, the variation within calli was distinguished, leading to a partially hierarchical model. The use of a logarithmic scale was necessary to normalize the distribution of residuals for menthone and menthol amounts. However, as the transformation did not affect the conclusions. results are presented from the original data.
Results
Protoplast isolation For each clone, numerous green protoplasts were easily released when lamina were treated with an enzyme solution containing 0.8 % cellulase RIO and 0.05 % pectolyase Y23. At the end of the digestion period, very little cellular debris
Table 2: Chronology of the successive steps from protoplast to plants transferred in greenhouse conditions. Time
Culture Step
day 0
Isolation and culture of protoplasts in media AE2. AE3, AE4 (darkness. 23 ·C. density = 40x 103 p/mL; osmolality 480-510 mOsm (KgH 2 0)-I)
=
day 6 week 3 week 5/6 week 13/14 week 21
Estimation of ADF for each clone Transfer of protoplast-derived colonies into AE5 medium (osmolality = 300/330 mOsm (KgH 20)-I) Transfer of calli onto the regeneration media (16h photoperiod. 60 ~mol . m- 2 . s- 1 PAR. 23 ·C) Estimation of the percentage of regenerating calli Micropropagation of regenerated plants Transfer to the greenhouse
Growth Regulators and polyamines in culture medium AE2: AE3: AE4:
BA 3.5 3.5 3.5
AE5:
BA 3.5
Zea 1.25 1.25 1.25
NM 2.5 2.5 2.5
NM Zea 2.5 1.25 see Tables 1 and 4
~M)
2,4-0 5.5 5.5 2.25
Sper 39 78 39
2,4-0 2.25
Sper 39
484
MARIE-HEd:NE CHAPUT, HAN SAN, LOUIS DE HyS, EMMANUEL GRENIER, HELENE DAVID, and ALAIN DAVID
Table 3: Yield, viability and diameter of leaf protplasts. Means and SE were calculated from 4 independent experiments. Yield: Number of purified protoplasts per gram of fresh tissue; viability: percentage of fluorescent protoplasts; N.D: not determined.
Mentha x citrata
Mentha x piperita
Species clone
MitchamMilly
Hongrie
Bergamote- LavandulioLatour dora Men 154
Yield (x 106) 11.11±1.41 9.78±1.03 8.88±0.62 10.18±0.51 ±3.79 89.45± 1.71 81.7 ±4.32 82.3 ± 1.59 Viability (%) 81 25.75±5 N.D Diameter (IJ.m) 29.97±5.27 N.D
was observed, so protoplast purification only consisted of two rinses in the washing solution. The protoplast yield ranged from 8.88 ± 1.03 X 106 per gram of fresh tissue for clone Bergamote-Latour (MCBL) to 11.11 ± 1.41 X 106 for the clone Mitcham-Milly (MPV), while protoplast viability exceeded 80 % (Table 3). They were relatively homogeneous in diameter, which was rather small, about 28/lm for leaf protoplasts (Table 3).
Protoplast culture First divisions occurred on the third or fourth day of culture in AE2, AE3 and AE4 media for both clones of M X piperita and on the fourth or fifth day for the clone M X citrata, Bergamote-Latour (MCBL). Concerning the clone Lavanduliodora Men 154 (MCL), protoplast suspension turned progressively brown and formed budding-like structures whatever the medium. No division was observed and finally protoplasts died. As shown in Fig. 1, relatively high percentages of division were obtained in all three tested media for both clones of M X piperita but especially for the clone 40
30
•
AD
[!j AEJ
EJ
'-<"' Q
AE4
20
Hongrie (MPS), for which ADF reached 31.3 ± 1.74 % in AE2 medium. No difference in ADF was noted between the AE2 and AE4 media for these clones. On the other hand, an increased spermidine concentration (AE3) produced very low division frequency at day 6 (Fig. 1). However, this result was due to a delay in protoplast development because ADF in medium AE3 reached that already mentioned a few days later. Since ADF in the AE2 medium was similar to that of the AE4 medium for all three clones, further experiments were performed using AE2 as initial protoplast culture medium. When the dividing cells were transferred to the AE 5 medium at week 3, most of them gave rise to microcalli, for all three clones. Reduction of both the medium osmolality and 2,4-D concentration allowed an intense development of microcalli. Two to three weeks later, it was possible to plate them directly onto the regeneration media.
Shoot regeneration Protoplast-derived calli of the three clones (MPV, MPS, MCBL) started to increase in size whatever the regeneration medium and turned green when they were exposed to a 16 h photoperiod (60/lmol.m- 2 .s- l PAR).
Shoot regeneration ofM.
X
piperita clones
Callus growth of the clones Mitcham-Milly (MPV) and Hongrie (MPS) was very weak (l-2mm in diameter) on regeneration media supplemented with 9.1 /lmol/L TDZ (R3) or with 4.4 /lmollL BA (R4). It was more intensive (2-6 mm in diameter) when media contained a lower concentration of TDZ (1.8/lmoI/L, Rl or 4.5/lmollL, R2) or a higher concentration of BA (8.9/lmoIlL, R5 or 13.2/lmoIlL, R6). In these conditions, protoplast-derived calli of the clone MPV exhibited a multi-nodular structure and gave rise to buds. The first regenerated shoots on Rl, R2, R5, R6 media were observed 3 months after protoplast isolation. The best results were obtained with media R2 and R6, but in all cases, the frequency of regenerating calli was low with a maximum of 3.8 % (Table 4) . Growth regulators affected the number of shoots per callus as well as the shoot morphology. When TDZ was used, the number of shoots per callus was variable and increased with TDZ concentration: with 4.5 /lmol/L TDZ it could reach 10 to 20. Shoots were vitreous and exhibited an abnormal morTable 4: Frequency of regenerating protoplast-derived calli of M x piperita, clone Mitcham-Milly (MPV) in different regeneration me-
10
dia.
MPS
MCBL
MPV
MCL
Clones
Fig. 1: Protoplast absolute division frequency (ADF) at day 6 of M. X piperita, clones Hongrie (MPS) and Mitcham-Milly (MPV), and of M X citrata, clones Bergamote-Latour (MCBL) and Lavanduliodora Men 154 (MCL), in AE2, AE3 and AE4 media. Means and SE were calculated on the base of 3 independent experiments.
Medi- Growth reurn gulators (J.l.M)
N° of protoplast- N° of calli Regeneration derived calli with shoots frequency (%)
Rl
165 210 210 210 210 210
R2
R3 R4 R5 R6
1.8TDZ 4.5TDZ 9.1 TDZ 4.4 BA 8.9BA 13.2 BA
3 8 0 0 2 7
1.8 3.8 0 0 0.9 3.3
485
Regeneration of plants from mint protoplasts; monoterpene composition
Table 5: Frequency of protoplast-derived calli of M x citrata, clone Bergamote-Latour (MCBL), producing shoots in different regeneration medium sequences. The number of regenerating calli estimated after a total of 2 months of culture on the medium sequence. Regeneration medium sequence
Growth regulators (f.1M) Medium I'
Growth regulators (f.1M) Medium 2b
RSI RS2 RS3 RS4 RS5 RS6 RS7 RSB
I.B I.B I.B I.B I.B I.B I.B I.B
3.6TDZ 3.6BA 3.6 BA (2 weeks) 3.6 BA (2 weeks) I.B BA (2 weeks) 3.7 Zeatin 4.6 Zeatin 4.6 Zeatin (2 weeks)
TDZ TDZ TDZ TDZ TDZ TDZ TDZ TDZ
(1 (1 (1 (1 (1 (1 (1 (1
week) week) week) week) week) week) week) week)
Growth regulators (f.1M) Medium 3
4.4 BA B.B BA 4.4 BA 2.3 Zeatin
N° of protoplast derived calli
Wofcalli with shoots
Regeneration frequency
164 135 75 75 55 75 61 135
3 2 B 6 6 0 0 0
I.B 1.5 10.7 B 10.9 0 0 0
(%)
': Calli are cultivated for 1 week on medium 1 and then transferred onto medium 2. b: Unless otherwise stated, calli are maintained on medium 2.
phology. The reduction of TDZ concentration promoted shoot elongation. On the contrary, few shoots were produced when BA was used (2-3 shoots per callus) but they appeared normal. When shoots had sufficiently elongated they were transferred onto a hormone-free medium where they rooted spontaneously and the abnormal morphology observed on the TDZ-containing medium progressively disappeared.
Shoot regeneration ofthe M x citrata clone Since in a preliminary experiment, protoplast-derived calli of clone Bergamote-Latour (MCBL) did not form shoots when using previous regeneration treatments (transfer onto RI, R2, R5, R6 media) another strategy was established that consisted of a regeneration medium sequence as shown in Table 1: all protoplast-derived calli were first placed onto medium RI (1.8 ~mol/L TDZ) for 1 week, then a few of them were transferred onto another medium supplemented with various concentrations of TDZ or BA or Zeatin. In some cases, a third medium was used 2 weeks later (Table 1). In these conditions, the clone Bergmaote-Latour (MCBL) produced compact and green calli. When a Zeatin-containing medium was used, a sharp increase in callus size was noted and calli seemed to spread on the surface of the medium but no shoots were recovered (Table 5). On the contrary, the other regeneration medium sequences allowed plant regeneration. The highest frequencies, around 10 %, were obtained when BA was used in the second and third medium of the sequence (RS 3, 4, 5). First shoots were observed 3-4 months after protoplast isolation. When calli grew in the presence of TDZ (RS 1, 2), they produced numerous but abnormal shoots similar to those already described with the clone Mitcham-Milly with the same growth regulator. In the presence of BA, shoots had a normal morphology and on a few occasions shoots rooted on the BA-containing medium.
Oil composition Callus-derived plants and controls of M X piperita vulgaris were harvested in early September, after 4 months of growth in field conditions. The amounts of men thone, menthol and carvone in the leaves were determined by gas chromatography.
<{? [>
Callus - derived plants Control plants
Menthol (mglg.d.w.) 2.97
2.44
1.90
3.74 2.74
Carvone (mglg.d.w.)
Menthone (mglg.d.w.) 0.024 0.72
Fig.2: Distribution of callus-derived plants (40 plants: 10 calli x 2 shoots per callus x 2 replicates) and control plants (12 replicates of Mentha x piperita vulgaris), according to menthone, menthol and carvone amounts.
Table 6: Mean of the control (12 replicates) and experimental error (root MSE*) for menthone, menthol and carvone amounts (mg/ g.d.w.).
Control mean Root MSE*
Menthone
Menthol
Carvone
2.Bl
2.44 0.273
0.15 0.062
0.401
* Square root of the Mean Square for Error from analysis of variance = Estimation of the standard deviation of replications.
Among the three compounds analysed, menthone and menthol were the most abundant in all the plants studied, carvone being much less represented (Fig. 2). For example, control plants contained an average of 2.81 and 2.44 mg per g.d.w. of menthone and menthol, respectively, and only 0.15 mg carvone (Table 6). However, variations in the content of the three compounds studied were observed when comparing the population of callus-derived plants with that
486
MAruE-HELENE CHAPUT, HAN SAN, LOUIS DE HyS, EMMANUEL GRENIER, HELENE DAVID, and ALAIN DAVID
Table 7: Differences between the mean of the 2 replicates of each
callus-derived plant (Mentha x piperita vulgaris) and the mean of the control expressed in root MSE for menthone, menthol and carvone amounts. Al and A2 represent two plants derived from callus A, B1 and B2 from callus B ... (see materials and methods). Plant
Menthone
Menthol
Carvone
Al A2 B1 B2 C1 C2 D1 02 E1 E2 F1 F2 G1 G2 HI H2 II 12 J1 J2
0.3 0.6 -1.7 -3.2* -3.5* -3.2* -3.7* -3.8* -3.5* -4.8* -4.5* -3.0* -4.0* -4.1* -3.3* -3.2* -1.3 -2.9* -3.2* -1.5
1.1 1.4 -0.5 -2.2 -1.1 -0.4
3.5* 2.4 4.0* 2.4 2.2 2.6* 1.7 2.1 1.1 0.7 -0.1 0.5 -0.1 1.0 1.5 0.9 0.8 0.1 -1.7 0.2
-1.3
-1.0 -2.6* -2.5* -3.1* -0.7 -1.0 -2.8* -3.2* -1.7 -1.6 -3.6* -1.6 -3.3*
* significant at the 0.05 level according to Dunnett's test.
of the control. The shift resulted in a decrease in menthone and menthol amounts and, on the contrary, a carvone increase in the leaves of the in vitro produced plants (Fig. 2). The validity of these observations was confirmed using Dunnett's test in which the mean value of the two replicates of a callus-derived plant for each of the three components was compared with those of the control. Significant differences (5 % level) were always negative for menthone and menthol and positive for carvone. For example, (Table 7) plant E2 produced significantly less menthone than the control. The difference between the mean values of menthone for the two replicates of the in vitro plants and that of the control corresponded to a 4.8-fold experimental error (Table 7). On the contrary, essential oil of the plant B 1 contained significantly more carvone than the control, the difference between the two mean values corresponding to a 4-fold experimental error (Table 7). In order to determine the origin of the variations mentioned above, the amount of menthone, menthol and carvone was compared in plants produced on the same callus. The difference is not significant (analysis of variance; p = 0.36, p=0.06 and p=0.44, respectively). The procedure from the protoplast to the plant in the field can be divided into two steps, from the protoplast to the callus and from the callus to the plant in natural conditions. Since there is no significant difference between plants on the same callus, the conclusion is that the changes in the amount of the three terpenes versus the control result from physiological events occurring during the first step, i.e. from the protoplast to the callus.
Discussion
Since the possibility of producing secondary metabolites in mint tissue cultures has been demonstrated (Bricout and Paupardin, 1975), attempts have been made to modifY the essential oil content of plants using somaclonal variation. In this context, two strategies for adventitious budding are available: organ culture (Bricout et al., 1978; Kukreja et al., 1991) and protoplast culture (Sato et al., 1993). The present paper reports on the use of this second strategy. Plants were regenerated from leaf protoplasts of two micro propagated mint species, M. X piperita and M x citrata . In M. X piperita, the amounts of three components of the essential oil, menthone, menthol and carvone, were compared with that of the control plants after transfer to field conditions. In the given experimental conditions, the first protoplast divisions of the M X piperita clones occurred on the third or fourth day of culture in liquid media, but they were only observed after 6 days of culture in media solidified with gelrite (Sato et al., 1993). It has been demonstrated recently that flax protoplast divisions were delayed in culture media solidified with agarose, while plating efficiency was promoted (unpublished laboratory data). Bud differentiation was highly influenced by the type of cytokinins and by the medium sequence used. In addition to BA-containing media also successfully employed by Sato et al. (1993), shoot regeneration was observed in the presence of TDZ for both M. X piperita and M X citrata (Tables 4 and 5); however, the two species exhibited different responses. M. X piperita could withstand TDZ for a long period of time. On the contrary a short induction with TDZ followed by a weaker cytokinin was more effective for M. X citrata. TDZ, a phenyl urea type cytokinin, has been found to stimulate shoot organogenesis (Cambecedes et al., 1991; Bates et al., 1992) and somatic embryogenesis (Bates et al., 1992). TDZ was also effective for the development of poplar protoplast derived calli and shoot formation (Chupeau et al., 1993). The beneficial effect of TDZ could be the result of the stimulation of the endogenous cytokinin synthesis or the limitation of their degradation by cytokinin oxidase (Hare and Van Staden, 1994). Concerning the essential oil composition, protoplast- derived plants were analysed 120 days after transfer to field conditions. Court et al. (1993) studied the effects of harvest date on the terpene content of peppermint plants in field trials. They observed that essential oil yield increased throughout the season, achieving a maximum from late August to early September. During plant development, the concentration of menthone decreased in favour of menthol, one of the major components of the essential oil of peppermint. At harvest time (early September), menthol represented 50.1 % against 11 % for menthone (Court et al., 1993). Based on the results mentioned above, plants used in the present study were harvested at this period of the year. However, contrary to the observations of Court et al. (1993), the amount of menthone remained high in the plants, e.g. in the controls, concentrations of menthone and menthol were 2.81 and 2.44 mg per g.d.w., respectively. However, these differences may result from quick changes in the composition of the oil, which occur as the maximum yield is ap-
Regeneration of plants from mint protoplasts; mono terpene composition proached by the end of the growing season (Court et aI.,
1993). It was noted that plant tissue culture conditions induced variations in the quantity of menthone, menthol and carvone in the protoplast-derived plants. Several papers have already reported on the influence of the in vitro treatment on secondary metabolite biosynthesis, such as terpenes (Bricout and Paupardin, 1975; Jain et al., 1991; Calvo and Sanchez-Gras, 1993) or alkaloids (Huizing et aI., 1983; Jung et al., 1992; Gontier et al., 1993). In the present study, a decrease of menthone and menthol and an increase of carvone were observed for all the protoplast-derived plants. Similar results have been described by Bricout and Paupardin (1975), when studying the essential oil content of plants obtained by adventitious budding on stem fragments of M x piperita cultivated on a BA-containing medium. A sharp decrease in menthol production was noticed. Kukreja et al. (1991) observed variations in four major constituents of the essential oil of M. arvensis, namely menthol, menthone, isomenthone and menthyl-acetate, in plants obtained by adventitious budding and grown for 110 days in the field. Out of the 80 somaclones analysed, only 8 had an amount of menthol slightly higher (88-94 %) than that of the control (86 %), the others having a lower or, in the best case, an equal concentration. The authors suggested that these modifications could be induced by somaclonal variation. Carvone is one of the major components of the essential oil of spearmint, M spicata (Hirate et al., 1990), while it is poorly represented in peppermint, M. x piperita. It was noticed in the evaluation trial that carvone had a tendency to increase in protoplast-derived plants, compared with the control plants. So in this study, the tissue culture treatment inversely affected menthone-menthol and carvone biosynthesis. Since the three of them, and terpenes in general, have a common precursor, Geranyl-PP, the inhibition of the biosynthetic pathway of menthone-menthol could result in the stimulation of the biosynthesis of carvone, but at this stage there is no evidence to sustain this hypothesis. Protoplasts were isolated from the leaves of plants micropropagated on a medium devoid of hormones. This step of the in vitro treatment should not induce long standing modifications. Moreover, no changes in the three monoterpenes analysed were observed between plants issuing from the same callus. Then it can be concluded that the variation between in vitro-produced plants and the control may result from events occurring before bud differentiation on the protoplast-derived calli, i.e. origin of the protoplast in the leaf, the response of a single cell faced with a stress condition andlor the first cycles of division leading to the callus formation. In summary, this paper reports on the conditions for the regeneration of Mentha X piperita L., Mitcham-Milly ssp. vulgaris (MPV) and Mentha x citrata Ehrh., Bergamote- Latour (MCBL) from leaf protoplasts of micropropagated shoots. It was observed that the procedure induces modifications in the three terpenes analyzed, namely a decrease of menthone and menthol and an increase of carvone. However, replications of oil analysis over several successive growing seasons are necessary before concluding that stable modifications can be induced by this procedure and before studying the modulation of the terpene metabolism pahtway by in vitro treatment.
487
Acknowledgements
We would like to thank the following ISAB colleagues: K. Chapelain for help with the statistical analysis and J. Gibson for reviewing the translation.
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