Menthol tolerant clones of Mentha arvensis: approach for in vitro ... Key words: Mentha arvensis L., menthol tolerance, mint oil, new variety, RAPD, somaclones, ...
Plant Cell, Tissue and Organ Culture 75: 87–94, 2003. 2003 Kluwer Academic Publishers. Printed in the Netherlands.
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Menthol tolerant clones of Mentha arvensis: approach for in vitro selection of menthol rich genotypes** Sunita Dhawan 1 , Ajit K. Shasany 1 , Ali Arif Naqvi 1 , Sushil Kumar 2 & Suman P.S. 1, Khanuja * 1
2
Central Institute of Medicinal and Aromatic Plants ( CIMAP), Lucknow 226015, India; National Center for Plant Genomic Research, JNU Campus, New Delhi, India ( * requests for offprints; Fax: ⫹91 -0522 -342 666; E-mail: khanujazy@ yahoo.com) Received 2 August 2002; accepted in revised form 27 February 2003
Key words: Mentha arvensis L., menthol tolerance, mint oil, new variety, RAPD, somaclones, yield
Abstract In vitro raised shoots of Mentha arvensis L. were screened for menthol tolerance level by growing them in media containing 0–100 g ml ⫺1 menthol. A total of 2850 regenerated shoots were step wise screened for menthol tolerance at the concentrations of 50 g ml ⫺1 followed by 60 and 70 g ml ⫺1 . In this screening, only 30 individual regenerated shoots were able to survive. The clones from the primary screen were inoculated into rooting medium and, after rooting, transferred to pots in the greenhouse. Ultimately, these 30 menthol tolerant clones were multiplied and grown in the field in replicated plots of 2.5⫻2.5 m sizes. Twigs of 30 clones from the replicated trials were rechecked for tolerant phenotypes at a concentration of 70 g ml ⫺1 menthol wherein, these survived even after 7 days (secondary screening). These clones were checked for oil and menthol content and were found to be better than the control plants. Out of these 30 plants, five tolerated 80 g ml ⫺1 menthol (tertiary level screening) and were found to contain the highest amount of menthol per g leaf biomass. Molecular analysis through RAPD showed distinct variation in the profiles of these five plants, in comparison to the control. Using this method the relationship between the primer OPT 04, menthol tolerance and high menthol content character of the genotype was established. Further, a cultivar ‘Saksham’ was released from the selections by CIMAP for superior performance. Abbreviations: RAPD – randomly amplified polymorphic DNA; RFLP – restriction fragment length polymorphism; BA – benzyl aminopurine; NAA – 1-naphthalene acetic acid; 2,4-D – 2,4-dichlorophenoxy acetic acid; 2iP – 6-(␥,␥-dimethylallyl amino) purine; 2aP – 6-(␥,␥-dimethylallyl amino) purine
Introduction Mints are high value crops containing monoterpenes with uses as pharmaceuticals, cosmetic ingredients and as flavouring agents in foods, beverages and tobacco products (Rech and Pires, 1986). Since the mints are of considerable interest to the industrial world, selection programmes for the isolation of desirable clones with improved terpene accumulation and suitable agronomic traits are being pursued in **US Patent no. 6423541.
several laboratories. However, due to mint’s sterility problem, conventional breeding programmes are severely hampered (Van Eck and Kitto, 1990; Caissard et al., 1996). Selection of mutant clones obtained by mutagenesis and somaclonal variation hold promise for the development of new plant types, by alternative methods like natural variant clone selection (Larkin and Scowcroft, 1981; Khanuja et al., 1998). Three new rice somaclonal variants were derived from rice protoplasts. The development of these commercial varieties following successful progeny testing indicated the importance of somaclonal variation (Kimura
88 et al., 1992). The comparison of DNA variation detected by RFLP analysis with phenotypic changes suggested that occurrence of mutations can be correlated with the degree of DNA variations. Some somaclonal variants derived from a landrace rice variety, Indrayani, were shown to be high yielding and resistant to multiple diseases. These were also found to have DNA changes that were detected through microsatellite fingerprinting (Chowdari et al., 1998). High pigment, reduced blossom end scar size and disease resistant tomato varieties were also developed from somaclones (Morrison and Evans, 1996). Secor et al. (2000) reported a method for in vitro selection of potato clones which were resistant to blackspot and bruising. We have earlier reported high efficiency protocols for regenerating internodes of Mentha arvensis to obtain somaclonal variants (Khanuja et al., 1998), as well as a method to limit somaclonal variation for micropropagation purposes (Shasany et al., 1998). In the present study, we have successfully attempted to develop and utilize a holistic method of screening the somaclones using menthol in the medium to produce high menthol yielding clones which could be transferred to the field and checked for their stability.
Materials and methods Plant variety, explants and culture conditions The plant variety used was cv. Himalaya (Kumar et al., 1997) of Mentha arvensis L. (2n⫽96). The suckers of M. arvensis L. cv. Himalaya used for multiplication of the plant material and experimentation were obtained from CIMAP’s gene bank. Explant material was collected from field grown plants and surface sterilized by washing sequentially with 2% detergent (Labolene, Qualigen Chemicals) for 10 min, distilled water containing a few drops of Savlon (Johnson and Johnson, India) for 1 h, 0.1% acidified mercuric chloride for 1 min and four times in autoclaved distilled water before inoculation (5 min, each washing). About 1 cm long internode pieces were inoculated in the MS solid medium (Murashige and Skoog, 1962) containing 2.68 M NAA and 22.19 M of BA (Shasany et al., 1998). The cultures were grown at 25⫾2 ⬚C and 54–81 mole m ⫺2 s ⫺1 light intensity with 16-h photoperiod. The regenerated shoots were separated 12 weeks after the explant inoculation to determine the critical menthol concentration for
screening the somaclones. Similarly, 1 cm long internode pieces were inoculated in the MS medium containing 0.9 M of 2,4-D and 34.44 M of 2iP or 2aP (Khanuja et al., 1998) to obtain callus derived somaclonal variants tolerant to the critical level of menthol in the medium (Figure 1a). Screening for menthol resistant somaclones Regenerated shoots (3–4 cm long, containing the apical bud and 2–3 nodes) from the NAA / BA medium (Shasany et al., 1998), were inoculated into MS basal medium, with or without menthol. The menthol used had been crystallized and purified from the hydrodistilled essential oil of Mentha arvensis cv. Himalaya. In this experiment menthol, 0–100 g ml ⫺1 was used, where the concentration was stepped up by 10 g in each treatment. Five independent regenerated shoots of each variety were inoculated per flask; four flasks were inoculated per replication and each treatment was replicated five times, making the number of flasks to 20 and number of explants to 100 for each concentration of menthol. Control flasks contained only MS basal medium and no menthol. The experiment was laid out in a completely randomized design (CRD). Cultures were incubated and maintained at 25⫾2 ⬚C and 54–81 mole m ⫺2 s ⫺1 light intensity with 16-h photoperiod. The nature of initial responses in explants was recorded every 24 h over a period of 1 week (Shasany et al., 2000). Large scale screening of clones was carried out in MS basal medium in various batches by raising the somaclones raised through callus mediated regeneration (Khanuja et al., 1998) in 2,4-D / 2iP medium. A total of 2850 shoots were transferred to the screening medium containing 50 g ml ⫺1 menthol and incubated for 1 week. The surviving shoots from this medium were step-wise transferred to media containing higher concentrations of menthol (60 and 70 g ml ⫺1 ). Finally, tolerant clones surviving at 70 g ml ⫺1 menthol concentration were transferred individually to MS basal medium containing vitamins for rooting. The rooted plantlets were subsequently multiplied using the medium as described by Shasany et al. (1998), hardened and transferred to pots in the glass house to raise sufficient planting material (sucker) for field experiments. Transfer of plants to the field The acclimatized selected plantlets from the glass-
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Figure 1. (a) Regenerated shoots 12 weeks after the internodal explant inoculation in 0.2 g ml ⫺1 2,4-D and 7 g ml ⫺1 2iP. (b) Shoots inoculated in the NAA and BA medium containing menthol 70 g ml ⫺1 (left control flask shows browning and death after 24 h). (c) Selected five clones are growing in menthol (80 g ml ⫺1 ) containing medium, after 7 days (left flask is control). (d) Uniformly growing clone 27 in the field. (e) Uniformly growing clone 30 in the field.
house were transferred to the field for sucker production in the month of September. In the month of January, of the following year the suckers developed from each plant were field planted with five replicates using randomized block design. The plants grown with standard agronomic practices (Kumar et al., 1997) were first harvested 110 days after planting and subsequently 70 days after the first harvest. Oil extraction and GLC analysis Oil samples from the field grown clones were extracted by hydrodistillation using Clevenger’s apparatus and weighed to record the yield. Over ground shoot samples were collected from the whole plant selected randomly from the middle of the row of each replicated plots (2.5⫻2.5-m sizes), 110 days after planting and 70 days after first harvest. Shoots col-
lected from individual plants were bulked for each treatment and essential oil was distilled from all the replicates taking 500 g of bulked shoots containing leaves. The final analysis of all the essential oil samples was accomplished on Varian CX-3400 using a 30 m⫻0.25 mm (0.25 ) Supelcowax-10 column. The injector and detector temperature were maintained at 200 and 225 ⬚C, respectively, with oven temperature programmed from 60 to 200 ⬚C at the rate of 7 ⬚C min ⫺1 increase, with initial and final holds of 2 and 5 min, respectively. Hydrogen gas was used as carrier at the rate of 1 ml min ⫺1 and 0.1 l of sample was injected with a split ratio of 1:50. Data were processed in the electronic integrator Varian 4400 and the identification was based on retention time of authentic samples of 1-menthol (Takasago, Japan) and retention indices calculations (Jennings and Shibamoto, 1980).
90 DNA isolation and PCR amplification reactions DNA was isolated from leaf tissue essentially according to the protocol described previously (Khanuja et al., 1999) and pooled DNA (equal amount from 20 individual plants of a genotype in a field) constituted the samples for polymerase chain reactions (PCRs) which were carried out in 25 l volume. A reaction tube contained 25 ng of DNA, 0.2 unit of Taq DNA polymerase, 100 M each of dNTPs, 1.5 mM MgCl 2 and 5 pmol of decanucleotide primers. The amplifications were carried out using the DNA Engine thermal cycler (MJ Research, USA) following the protocol of Khanuja et al. (2000). The amplified products were loaded in 1.2% agarose gel containing 0.5 g ml ⫺1 of ethidium bromide and photographed by Polaroid system. Forty decamer primers procured from Operon Technologies, USA (OPJ and OPT) were used to detect polymorphism in the selected clones.
Results and discussion Menthol tolerance of regenerated shoots Monoterpenes are known to be cytotoxic to plant tissues, inhibiting respiration and photosynthesis by drastically affecting the mitochondria, golgi bodies etc. and decreasing cell membrane permeability (Brown et al., 1987). Monoterpenes are either sequestered in the plants in specialized structures like glandular hairs in Pelargonium (Brown and Charlwood, 1986), trichomes in Mentha or stored in the form of non-toxic glycoside derivatives in vacuoles, e.g. Rosa spp. Effects of exogenous monoterpenes on cellular viability in suspension cultures of Pelargonium fragrans have been studied (Brown et al., 1987). The end product toxicity in such cases may be related to feed-back inhibition of mechanism(s) that regulates the extent of monoterpenes accumulation. This natural regulation mechanism, thus, may also offer a handle to researchers interested in selection of high monoterpene yielding genotypes. In Mentha arvensis, specific glands known as trichomes are formed which store the oil containing toxic monoterpenoid metabolic products (Spencer et al., 1993). The essential oil produced by Mentha arvensis mainly contains the monoterpene menthol whose content determines oil quality. Lethal concentration of menthol on in vitro raised shoots was determined by comparing the percent
survival of shoots at different concentrations of menthol added to the medium. Shoots regenerated from the internodal explants were inoculated into media containing a series of step-wise increasing concentrations (0–100 g ml ⫺1 ) of 1-menthol. Shoots were scored for browning and death of shoot tissues. As reported earlier by Shasany et al. (2000), lethality for cv. Himalaya was observed at concentrations of 50 g ml ⫺1 of menthol or above, within 24 h and in case of 40 g ml ⫺1 concentration, 7 days after inoculation. Below these concentrations, though some lower leaves were desiccated, the shoots still survived in culture. At a concentration of 70 g ml ⫺1 shoot survival was about 45% within 24 h of inoculation but ultimately leading to death of the shoots (almost 99%) within 7 days. Figure 1b shows browning and death of shoots (left flask) at a menthol concentration of 70 g ml ⫺1 while the other two flasks shows the survival of some tolerant clones. No shoots were able to survive at 80 g ml ⫺1 and above of menthol by day 7 (Shasany et al., 2000). Therefore, a concentration of 70 g ml ⫺1 menthol was taken as the critical concentration for large scale screening of tolerant clone(s) in step wise fashion of selection. Menthol, the major component of the essential oil is considered to be cytotoxic to the plant. Thus the level of menthol accumulated may be linked to the tolerance level of the tissue(s) and uncoupling of these two factors may deregulate the rate-limiting step in the production of menthol. It was assumed that through certain alternate cellular mechanism(s), it may escape toxicity and damaging effect of higher menthol in the tissues. Such a condition may lead to more menthol accumulation in the trichomes of the regenerated menthol tolerant plants by circumventing feed back inhibition. Utilizing the advantage of variation through callus mediated regeneration (Khanuja et al., 1998), a total of 2850 regenerated shoots (which show variation at RAPD level also) were taken in the step wise screening for menthol tolerance starting with a concentration of 50 g ml ⫺1 followed by 60 and ultimately 70 g ml ⫺1 for the surviving clones. The obvious symptom of menthol toxicity, observed at 70 g ml ⫺1 was immediate chlorophyll loss and irreversible wilting of the shoots within 24 h. Treated shoots were nonrescuable even by culturing in normal medium without menthol. Lowest survival was detected at a concentration of 70 g ml ⫺1 , where only 1% shoots survived. Since at 50 g ml ⫺1 concentration of menthol, lethality was observed to be irreversible, a
91 stepwise selection pressure of menthol concentration in the medium was employed in the large scale screening experiment. This is for the enrichment of the potentially tolerant shoots which presumably resort to an adaptive response by tolerating a lower level of menthol through the induction of genes required for the tolerance, before transfering to the higher level. In this three tier selection approach, 1263 shoots survived the 50 g ml ⫺1 concentration of menthol. These surviving shoots were subsequently transferred to the medium containing 60 g ml ⫺1 menthol. At this concentration 942 shoots became brown and died within 7 days. The remaining 321 shoots, which survived 60 g ml ⫺1 menthol, were inoculated into the selective medium containing 70 g ml ⫺1 menthol. At this tertiary level selection, only 30 shoots survived (Table 1). These clones were inoculated into the rooting medium and, after rooting, transferred to pots in the greenhouse. Ultimately, these 30 menthol tolerant clones were multiplied, transferred and grown in the field in replicated plots of 2.5⫻2.5-m sizes. Shoots of 30 field grown clones were rechecked for the menthol tolerant phenotype against a concentration of 70 g ml ⫺1 menthol, showed survival even after 7 days. These 30 clones were then tested for survival at one step higher concentration of menthol (80 g ml ⫺1 ). At this concentration of menthol only five clones (clone no 3, 14, 24, 27 and 30) survived (Figure 1c). These were further analyzed for oil and menthol production under field conditions and compared with other clones. Figure 1d,e shows the growth of the clones 27 and 30, respectively, in the field.
monoterpene profiles. For comparative menthol productivity of these genotypes (Table 2) menthol yield per g shoot biomass was also estimated. In this comparative analysis, six of the 30 clones (GRB 6, 24, 26, 27, 29 and 30) yielded 1% or more oil in the first harvest. Three of these clones, namely GRB 24, 27 and 30, produced about or more than 2% oil. Compared to the parent cv Himalaya clones GRB 4, 5, 17, 18 and 30 produced more menthol in their oil. Menthol yield per unit shoot biomass showed that the majority (20 out of 30) clones produced more menthol per g biomass in the first harvest compared to the control. In the second harvest clones GRB 1, 2, 3, 4, 5, 6, 15, 19, 21, 25, 27, 28, 29 and 30 showed sustained superiority over cv. Himalaya for menthol. The total menthol yield per g shoot mass in both the harvest in all the selected clones was higher than the control cv. Himalaya. Higher menthol production per g shoot mass was observed in clones GRB 24, 27 and 30 (23.04, 24.91 and 25.05 mg per g shoot mass, respectively) compared to 10.58 mg g ⫺1 by the control cv. Himalaya. These genotypes thus represent promising material for developing improved varieties with high menthol production in Mentha arvensis. These clones were checked for their menthol tolerance and menthol yield for two subsequent years and observed to be similar to the presented data. Considering the stability of the multiplied clones for menthol tolerance and increased menthol production, it is assumed that the mother cells in vitro might have undergone some genetic changes to give rise to these variant (clones) and as a consequence of selection pressure of menthol, ultimately were selected as menthol tolerant genotypes with much higher tolerance level than the control Himalaya. We, in our earlier study, have observed a positive correlation between menthol content in oils of genotypes and the level of menthol tolerance by the shoots.
Oil and menthol yields of selected clones Plant shoot samples of the selected clones (500 g) collected were analyzed for oil yield (w / w) and
Table 1. Large scale screening and survival of regenerated clones at different concentrations of menthol Screening for menthol tolerance
Shoots* screened
Number
2850
Survival percentage
Survival in medium containing menthol ( g ml ⫺1 ) 50
60
70
1263
321
30
44.31
11.26
1.05
*Source of regenerants (callus derived on MS medium containing 2,4-D (0.9 M) and 2iP (34.44 M) according to Khanuja et al. (1998).
92 Table 2. Comparative oil and menthol yields of the menthol tolerant clones in field evaluation Clone
GRB 1 GRB 2 GRB 3 GRB 4 GRB 5 GRB 6 GRB 7 GRB 8 GRB 9 GRB 10 GRB 11 GRB 12 GRB 13 GRB 14 GRB 15 GRB 16 GRB 17 GRB 18 GRB 19 GRB 20 GRB 21 GRB 22 GRB 23 GRB 24 GRB 25 GRB 26 GRB 27 GRB 28 GRB 29 GRB 30 Control Himalaya LSD* at 5% 1%
Oil yield (%)
Menthol content of oil (%)
Menthol yield (mg per g biomass)
First harvest
Second harvest
First harvest
Second harvest
First harvest
Second harvest
Total
0.81 0.81 0.88 0.79 0.84 0.93 0.80 0.85 0.85 0.53 0.72 0.69 0.71 0.70 0.65 0.82 0.58 0.73 0.75 0.77 0.75 0.88 0.78 1.03 0.84 1.14 1.06 0.85 0.94 0.95 0.64 0.2056756 0.2735486
1.17 1.00 1.45 0.77 1.30 1.06 1.36 1.18 1.40 0.90 1.19 1.08 1.04 1.45 1.28 0.59 1.12 1.02 1.49 0.72 1.40 0.99 0.86 1.91 0.96 1.36 2.03 0.88 0.87 2.22 0.70 0.5922415 0.786813
69.9 75.1 76.1 77.7 77.6 76.9 76.8 75.6 75.0 75.1 74.6 74.8 70.5 73.4 75.4 76.3 77.5 79.3 75.6 76.8 74.2 74.1 75.6 76.7 73.9 74.0 74.5 76.0 64.7 77.7 77.1 6.133469 8.157515
80.1 79.2 80.3 83.0 79.6 81.6 78.0 78.4 76.5 76.0 78.8 75.6 71.9 77.8 79.1 74.3 77.9 77.6 79.6 71.1 80.8 72.6 75.6 76.2 83.6 77.7 80.9 79.1 80.6 80.7 78.0 5.647517 7.511198
5.87 6.11 6.71 6.09 6.55 7.15 6.16 6.44 6.38 4.00 5.42 5.17 5.20 5.38 5.79 6.86 5.16 6.05 5.93 5.63 5.69 5.98 6.32 8.60 6.09 6.91 8.24 6.96 6.13 7.12 5.12 1.37 1.82
9.33 7.92 11.68 6.35 10.33 8.67 10.62 9.24 10.75 6.88 9.44 8.11 7.67 11.33 10.1 4.40 8.69 7.89 11.91 4.99 11.38 7.39 6.43 14.44 7.74 10.66 16.67 6.98 7.05 17.9 5.46 4.778079 6.354845
15.2 14.03 18.39 12.44 16.88 15.82 16.78 15.68 17.13 10.88 14.86 13.28 12.87 16.71 15.93 11.26 13.85 13.94 17.84 10.62 17.07 13.37 12.75 23.04 13.83 17.57 24.91 13.94 13.18 25.05 10.58 5.111729 6.900825
*LSD⫽Least significant difference.
The use of this relationship as a selectable biochemical marker opens the practical applicability in largescale in vitro screening of the germplasm, clones and breeders material for selection of elite genotypes (Shasany et al., 2000). The selected clones with significantly high menthol production per unit biomass represent the superior genotypes with the capability to be developed into improved varieties of Mentha arvensis upon large scale yield trials.
Molecular differentiation of the genotypes Among the variants selected for higher tolerance level against menthol, five clones (GRB 3, 14 24, 27 and
30) were found to be consistent in their phenotypes surviving even under 80 g ml ⫺1 menthol. Since these were the result of independent selection experiments, the variation at molecular level was assessed through RAPD analysis. Polymorphic bands differentiating these genotypes could be detected using 40 random primers establishing their distinctiveness (Table 3, Figure 2). Randomly Amplified Polymorphic DNA (RAPD) can serve as the rapid method for determining variation. We have earlier reported the rapid screening of somaclones in Mentha arvensis (Khanuja et al., 1998) through RAPD. These five clones showed distinct profiles among them as well as in comparison to the parent plant Himalaya indicating a change in the genotype of these plants. All the
93 Table 3. Polymorphic bands observed in the RAPD profiles of the clones with different primers compared to cv. Himalaya Clones
Primers showing polymorphic bands
GRB 3 GRB 14 GRB 24 GRB 27 GRB 30
OPJ 09, 17 and OPT 04, 05, 12, 20, OPJ 05, 14, 15, 16, 17 and OPT 04, 05, 13, 15, 16 OPT 04, 08 OPJ 08 and OPT 04, 13, 17 OPJ 01, 03, 09, 16, 19, 20 and OPT 01, 04, 05, 08, 12, 16, 20
primers tested were not able to differentiate all the selected plants from the parent Himalaya when used separately, but in combination this was possible. OPT 04 (Figure 2) which differentiated the RAPD profiles of all the five selected plants compared to the parent plant Himalaya indicated a relationship between the primer OPT 04 and menthol tolerance character. Further the selected clones maintained the polymorphic profiles after two generations of field evaluation with the primers described in Table 3, indicating stable inheritance of the genetic change acquired during the stages of menthol screening. In conclusion, it is possible to screen, identify and develop menthol tolerant plants / genotypes or clones rich in menthol, through tissue culture utilizing generation of somaclones. Using this method a cultivar ‘Saksham’ of Mentha arvensis from the clonal screening has already been released by CIMAP (Khanuja et al., 2001) for superior performance.
Figure 2. RAPD profiles of the selected plants at 80 g ml ⫺1 menthol compared to the parent plant Himalaya (H), and Hind III digest (M) with OPT 04.
Acknowledgements We thankfully acknowledge the Department of Biotechnology (DBT) and Council of Scientific and Industrial Research (CSIR), Government of India, for the financial support.
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