RESEARCH COMMUNICATIONS

density intrusive material was deposited in the upper crust 16. Ghosh, D. B., Geol. Surv. India Misc. Publ., 1976, 34, 119–132.
to the north of the Narmada. Upper crustal deposition did 17. Venkata Rao, K., Srirama, B. V. and Rama Sastry, P., Geol. Surv.
India Spl. Publ., 1990, 28, 99–117.
not take place to the south of Narmada. 18. Shivaji, C. H. and Agarwal, B. N. P., Mem. Geol. Soc. India,
During phase II, activity along the south fault started 1995, 31, 495–518.
during the Jurassic–Cretaceous period. The mafic intru- 19. Singh, A. P. and Meissner, R., J. Geodyn., 1995, 20, 111–127.
sion in the upper crust caused due to this activity was 20. Acharya, S. K., Kayal, J. R., Roy, A. and Chaturvedi, R. K., J.
limited to the region south of Narmada and east of Geol. Soc. India, 1998, 51, 295–304.
Barwani–Sukta fault. ACKNOWLEDGEMENTS. We thank the Director, NGRI for permis-
Phase III coincided with the passage of India over the sion to publish this work. Our thanks are due to Department of Science
reunion plume during late Cretaceous. The plume head at and Technology for funding the project for reinterpretation of the seis-
mic data across Narmada region. We also thank Sri M. Shankarayya
that time was situated to the south of Narmada region. and Sri B. P. S. Rana for drafting the figures.
Due to tectonic disturbances close to the west coast, mate-
Received 16 September 2000; revised accepted 4 December 2000
rial from the plume head got emplaced at the base of the
crust and extended to east to a large distance. This caused
underplating in large parts of the crust. Subsequent extru-
sion from the plume led to the Deccan Trap activity and
its deposition. Around the same time some eruption might
also have taken place from the upper crustal mafic body Photoregulation of adventitious and
to the south of Narmada zone. axillary shoot proliferation in menthol
The reinterpreted results of the seismic data across mint, Mentha arvensis
NSL, lead to the following conclusions. (1) The Barwani–
Sukta fault divides the Narmada zone into two parts.
Savithri Bhat†, S. K. Gupta†, R. Tuli*, S. P. S. Khanuja†,
While the SW part is a graben, the NE part is a basement
S. Sharma†, G. D. Bagchi†, Anil Kumar‡ and
uplift. (2) The upper crust east of the Barwani–Sukta fault
Sushil Kumar†,#
shows a horst feature between the Narmada north and †
Central Institute of Medicinal and Aromatic Plants, P.O. CIMAP,
south faults, indicating that the Narmada zone is a ridge Lucknow 226 015, India
between two pockets of mafic intrusion of different ages. *National Botanical Research Institute, Rana Pratap Marg,
(3) Upwarp of the Moho and intra crustal layers in the Nar- Lucknow 226 001, India
‡
School of Biotechnology, Devi Ahilya Vishwavidyalaya,
mada zone suggest that the two faults involve deep-seated Indore 452 001, India
tectonics. (4) On the basis of present-day crustal structure,
at least three major phases of tectonic activity during the A direct and indirect methodology for efficient shoot
proliferation and regeneration from various explants
Archaean–Proterozoic, Jurassic–Cretaceous and late Cre-
of Mentha arvensis has been developed by studying the
taceous can be identified in the Narmada region. interactive effect of plant growth regulator and light
colour. Nodal explants cultured on Murashige and
1. Auden, J. B., Natl. Inst. Sci. India, 1949, 15, 315–340. Skoog’s (MS) medium supplemented with 10 µM
2. Kaila, K. L., Gaur, V. K. and Narain, K., Bull. Seismol. Soc. Am., thidiazuron and incubated under red light, prolife-
1972, 62, 1119. rated an average of 50 shoots after 30 days, whereas
3. West, W. D., Curr. Sci., 1962, 31, 143–144. internodal explants cultured in modified MS medium
4. Jain, S. C., Nair, K. K. K. and Yedekar, D. B., Geol. Surv. India supplemented with 40 µM 6-benzylaminopurine and
Spl Publ., 1995, 10, 1–154. 0.5 µM α-naphthaleneacetic acid and exposed to red
5. Kaila, K. L. and Krishna, V. G., Curr. Sci., 1992, 62, 117–153.
light, regenerated an average of 200 shoots after 60
6. Cerveny, V. and Psencik, I., Research Report, Institute of Geo-
physics, Charles University, Prague, 1981.
days. Among the six cultivars tested, internodal
7. Prakash Kumar, Tewari, H. C. and G. Khandekar, Geophys. J. Int. explants of cv. Gomti regenerated about 90 shoots
2000, 142, 95–107. after 20 days of culture. Under the above conditions,
8. Sridhar, A. R. and Tewari, H. C., J. Geodyn., 2000, 31, 19–31. leaf explants were observed to regenerate an average
9. Murty, A. S. N., Mall, D. M., Murty, P. R. K. and Reddy, P. R., of 60 shoots after 60 days of culture, which was pre-
Pure Appl. Geophys., 1998, 152, 247–266. ceded by callus development. The protocol standar-
10. Rao, C. K., Gokaran, S. G. and Singh, B. P., Geomagn. Geoelect., dized for high efficiency shoot proliferation and
1995, 47, 411–420. regeneration in M. arvensis from nodal, internodal and
11. Mishra, D. C., Tectonophysics, 1992, 212, 153–161.
leaf explants is suitable for micropropagation, genetic
12. Gokaran, S. G., Rao, C. K., Gupta, Gautam and Singh, B. P.,
Newsletter, 1999, 9, 2–12.
transformation and for obtaining somaclonal variants
13. Mall, D. M., Kaila, K. L. and Rao, V. K., in First International in this essential oil-yielding crop plant.
Seminar and Exhibition on Exploration Geophysics in Nineteen
MENTHA ARVENSIS, a member of Lamiaceae, is a source of
Nineties, 1991, pp. 298–303.
14. Verma, R. K. and Banerjee, P., Tectonophysics, 1992, 202, 375– several single monoterpenes, including menthol and their
397. mixtures that are extensively used in flavour, fragrance,
15. Agarwal, B. N. P., Das, L. K., Chakravorthy, K. and Sivaji, C. H.,
#
Mem. Geol. Soc. India, 1995, 31, 469–493. For correspondence. (e-mail: [email protected])

878 CURRENT SCIENCE, VOL. 80, NO. 7, 10 APRIL 2001

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cosmetics, pharmaceutical and agrochemical industries1,2. was more efficient in regenerating shoots than NAA
Since Mentha species are largely cultivated by the use of alone, either at lower or higher concentrations (data not
vegetatively produced root suckers as planting material3, included). A comparative study of the effect of red and
successive use of the same material carries with it any white light on shoot regeneration response was carried out
associated bacterial, fungal and/or viral infections to the by incubating the internodal (10 mm) and leaf (5 × 5 mm)
next crop. Rapid and convenient regeneration and micro- explants cultured in MB medium containing 0.5 µM NAA
propagation procedures are required for cleaning of plant- and 10, 20, 40, 60 and 80 µM BAP under respective light
ing material free of infection and for transformation of conditions. To compare the intervarietal differences in
genome with homologous or heterologous genes for the response to shoot regeneration, internodal segments of 6
incorporation of desired characters such as disease and cultivars of M. arvensis were cultured in MB medium
pest resistance and modification of monoterpene pathway supplemented with 40 µM BAP and 0.5 µM NAA and
towards more economic production of essential oil of exposed to red light. In all the experiments, cultures were
better quality4. In recent years, several protocols have incubated under respective light conditions at 25 ± 2°C
been described for micropropagation via shoot prolifera- with 16 h photoperiod. The regenerated and proliferated
tion and regeneration from stem5–7 and leaf8,9 explants in in vitro shoots were carefully separated and transferred to
Mentha species. However, these have certain disadvan- PGR-free MS medium to permit elongation and rooting.
tages such as callus intervention and/or low-level shoot After 15 days, rooted shoots were transferred to distilled
regeneration efficiency to varying extent. The present water for 7 days for acclimatization and were transferred
study describes experiments specifically designed for to pots containing 1 : 1 (v/v) mixture of soil and compost
standardization of efficient protocols for regeneration and kept under green-house conditions. The experiments
and proliferation of shoots at high efficiency from nodal, were arranged in randomized complete blocks and repli-
internodal and leaf explants of M. arvensis. cated 30 times. Analysis of variance was done by F-test
The nodal segments excised from potted plants of six and the differences between means tested using CD (criti-
cultivars (Gomti, Shivalik, MAS-1, Himalaya, MAH-3 cal difference) at 1%. For histological observations, the
and Kalka) of M. arvensis were sterilized by dipping into regenerating and proliferating explants were fixed in
Teepol detergent solution (2% v/v, Sigma Chemicals, St. 18 : 1 : 1 mixture of 50% (v/v) ethanol, glacialacetic acid
Louis, USA) for 5 min followed by thorough washing and formaline, dehydrated in ethanol and ethanol–xylene
under running water for 3–4 h. Surface disinfestation was series before embedding in mixture of paraffin : bee wax
carried out by treating the nodes for 10 min in 0.5% (8 : 1). Twenty µm thick sections were cut on a rotary
sodium hypochlorite (4% w/v commercial bleach, Qua- microtome (Spencer make) and the sections were stained
ligens Fine Chemicals, India) followed by rinsing with with saffranin and fast green.
sterile distilled water. The explants were then prepared by The effects of differentially coloured visible light on
trimming at the edges prior to inoculation in Murashige axillary bud proliferation were examined with and without
and Skoog’s (MS) medium10 supplemented with 10 µM BAP or TDZ supplementation. It was observed that while
6-benzylaminopurine (BAP) and solidified with 0.6% the incubation of explants under light passed through blue
agar, pH had been adjusted to 5.8 prior to autoclaving for or green filter lowered the rate of shoot proliferation,
20 min at 105 kPa and 121°C. The cultures were incu- exposure to light passed through yellow or red filter
bated under 16 h photoperiod (60 µmol m–2 s–1) at 25 significantly improved the shoot proliferation. On an
± 2°C for 30 days. Shoots proliferated from nodal seg- average, 23 and 52 shoots proliferated per nodal explant
ments were rooted in plant growth regulator (PGR)-free under light passed through red filter on MS medium
MS medium. The nodal, internodal and leaf explants pre- containing 10 µM BAP and TDZ, respectively (Table 1).
pared from such in vitro raised shoots were used to opti- Histological studies of sections of proliferating nodal
mize culture conditions for proliferation and regeneration explants revealed darkly-stained vascularized areas aris-
in M. arvensis cv. Gomti. The interactive effect of light ing directly from the original vascular bundle. The inter-
colour and cytokinin on axillary bud proliferation from nodal explants of M. arvensis cv. Gomti cultured on MB
nodal explants was evaluated on MS medium supple- medium containing 40 µM BAP and 0.5 µM NAA and
mented with or without thidiazuron (TDZ) or BAP at 0, 2, incubated under red light, regenerated about 90 shoots
10 or 20 µM concentrations; blue, green, yellow or red after 20 days and 200 shoots after 60 days of culture
light was obtained by combining respective filters with (Table 2). Microscopic examination of thin sections of
cool white fluorescent tubes (Philips, TL-40W/54, regenerating internodal explants revealed darkly-stained
6500 K). In the preliminary test for shoot regeneration vascularized areas in the cortical region and regeneration
from internodal explant, α-naphthaleneacetic acid (NAA) of shoot buds. This showed clear evidence of non-
at 0.2 to 1.0 µM concentration in combination with BAP involvement of any callus in the shoot bud initiation
at 10 to 80 µM concentration was supplemented in MB events in the internodal explant. Leaf explants cultured on
medium (MS salts + B5 vitamins11). It was observed that MB medium containing 60 µM BAP and 0.5 µM NAA
NAA at 0.5 µM in combination with BAP (10–80 µM) formed little callus at the cut ends and regenerated on an
CURRENT SCIENCE, VOL. 80, NO. 7, 10 APRIL 2001 879
RESEARCH COMMUNICATIONS

average 20 shoots per explant after 60 days of culture. The studies described above define a protocol for high-
Observations of sections of leaf explants showed that efficiency shoot proliferation from excised nodes, inter-
callus originated from palisade cells and regeneration of nodes and leaves of M. arvensis. Kukreja et al.6 described
shoots from the callus. Replacement of white light with a method for obtaining 30–40 shoots via callus formation
red light in the same regeneration medium significantly in 60 days, thus rendering the protocol unsuitable for
enhanced shoot regeneration, resulting in 60 shoots per maintaining genetic integrity, since it led to the develop-
explants after 60 days of culture (Table 2). The internodal ment of somaclones. On the other hand, the direct prolif-
explants from all the six cultivars of M. arvensis cultured eration procedure standardized by Rech and Pires5,
on MB medium supplemented with 40 µM BAP and produced only 2–4 shoots per explant over a period of 4–
0.5 µM NAA and incubated under red light regenerated 8 weeks. In the present work, the supplementation with
about 50–90 shoots after 20 days. Among the cultivars TDZ in medium was more effective in proliferating shoots
tested, Gomti and Himalaya were found to be superior in compared to an alternate cytokinin screened. It has been
regenerating shoots from internodal explants compared to demonstrated that addition of TDZ in place of BAP in MS
other cultivars (Table 3). The proliferated and regenerated medium increased shoot differentiation and proliferation
shoots were individually separated, rooted on MS medium in the cultures of certain medicinal plant species like
and transferred to greenhouse for acclimatization. Cinnamomum12 and Saussurea13. In the study of adventi-

Table 1. Effect of differentially coloured visible light in the presence of different concentrations
and types of cytokinins supplemented in MS medium on the proliferation ability of
nodal explants in Mentha arvensis cv Gomti

Mean number of shoots formed per explant under differentially

Plant growth regulator coloured filter

Concentration
Compound (µM) Nil Blue Green Yellow Red Mean

Nil 0 1.2 1.5 0.8 1.4 1.5 1.3

BAP 2 3.7 1.7 1.7 5.5 5.4 3.6
BAP 10 6.5 4.5 5.9 12.4 23.0 10.4
BAP 20 3.1 3.1 3.1 5.1 5.4 4.0
TDZ 2 6.2 3.9 3.5 6.4 14.4 6.9
TDZ 10 10.0 7.1 3.4 17.7 52.1 18.1
TDZ 20 3.1 3.7 2.1 6.7 15.4 6.2
Mean 4.8 3.6 2.9 7.9 16.7 7.2

CD values at 1% between: Two main factors (filters) = 0.9; Two sub factors (PGR) = 1.1; Sub factors
at fixed level of main factors = 2.6; Main factors at fixed level of sub factors = 2.5.

Table 2. Regeneration and shoot proliferation responses of internodal and leaf explants of Mentha
arvensis cv Gomti, growing on MB medium (MS salts + B5 vitamins) containing 0.5 µM
NAA and different concentrations of BAP as affected by the red colour light

Concentration Internodal Mean number of Leaf explants Mean number of

of BAP in explants that shoots regener- that regenerated shoots regener-
Colour of the medium regenerated into ated per inter- into shoots ated per leaf
the filter (µM) shoots (%) nodal explant (%) explant

Nil 10 54.9 11.9 18.9 11.6

20 74.8 27.9 38.4 14.9
40 90.3 90.4 58.8 17.0
60 77.3 37.6 79.0 22.6
80 54.2 8.2 28.5 10.9
Red 10 75.9 30.9 19.5 28.4
20 83.9 45.4 40.6 46.0
40 99.1 198.6 62.1 52.1
60 77.0 47.3 85.5 62.0
80 53.1 62.8 30.8 36.0
Mean 66.6 38.0 46.2 30.1
CD 1% 4.8 6.9 3.4 3.6

880 CURRENT SCIENCE, VOL. 80, NO. 7, 10 APRIL 2001

 RESEARCH COMMUNICATIONS
Table 3. Relative shoot regeneration responses of the internodal explants of different cultivars of
Mentha arvensis cultured on MB medium (MS salts + B5 vitamins) containing 40 µM BAP
and 0.5 µM NAA and exposed to red light

M. arvensis variety Gomti Shivalik MAS-1 Kalka Himalaya MAH-3 Mean CD 1%

Percentage of explants 92.2 86.7 58.8 80.2 73.1 71.6 80.3 5.8
that regenerated shoots
Mean number of shoots 92.8 78.8 52.0 79.2 85.8 80.2 78.1 7.2
regenerated per explant

tious shoot regeneration, callus formation was avoided by 5. Rech, E. L. and Pires, M. J. P., Plant Cell Rep., 1986, 5, 17–18.
increasing the proportion of BAP : NAA through use of 6. Kukreja, A. K., Dhawan, O. P., Mathur, A. K., Ahuja, P. S. and
Mandal, S., Euphytica, 1991, 53, 183–191.
higher concentration of BAP and lower concentration of 7. Shasany, A. K., Khanuja, S. P. S., Dhawan, S., Yadav, U.,
NAA. Earlier, BAP and NAA have been effectively used Sharma, S. and Kumar, S., J. Biosci., 1998, 5, 641–646.
to culture internodal14,15 and leaf16–18 segments of several 8. Van Eck, J. M. and Kitto, S. L., Plant Cell Tissue Org. Cult.,
medicinal plants. It has been reported that plants sense 1992, 30, 41–49.
many aspects of light, including its wavelength, duration, 9. Faure, O., Diemer, F., Moja, S. and Jullien, F., Plant Cell Tissue
Org. Cult., 1998, 52, 209–212.
intensity and direction19. Irradiation of plants with light at 10. Murashige, T. and Skoog, F., Physiol. Plant, 1962, 57, 407–497.
various wavelengths is known to markedly affect expres- 11. Gamborg, O. L., Miller, R. A. and Ojima, K., Exp. Cell Res.,
sion of genes encoding proteins related to photosynthesis, 1968, 50, 151–158.
morphogenesis and organogenesis20. In the present study, 12. Huang, Li, Huang, B. L. and Murashige, T., In Vitro Cell Dev.
Biol., 1998, 34, 141–146.
incubation of cultured explants under red light almost
13. Johnson, D. S., Narayan, S. B. and Narayana, D. B. A., In Vitro
doubled the shoot proliferation and regeneration response Cell Dev. Biol., 1997, 33, 128–130.
within the same period of culture. As observed in the pre- 14. Nikam, T. D., Plant Cell Tissue Org. Cult., 1997, 51, 225–228.
sent study, the potentiating effects of red light on axillary 15. Anand, A., Srinivasa, Rao, Latha, R., Josekutty, P. L. and
shoot proliferation has been previously demonstrated in Balakrishna, P., In Vitro Cell Dev. Biol., 1998, 34, 136–140.
16. Paniego, M. B., Maligne, A. E. and Giulietti, A. M., Biotech in
several dicotyledonous plant species21–23. The enhance-
Agriculture and Forestry, Medicinal and Aromatic Plants V (ed.
ment effect of red light in proliferating shoots has been Bajaj, Y. P. S.), 1993, vol. 24, pp. 64–78.
attributed to phytochrome action in Pelargonium cul- 17. Augustine, A. C. and Souza, L. D., In Vitro Cell Dev. Biol., 1997,
tures24 or to enhanced protein synthesis and cell division 33, 111–113.
in Crepis cultures25. A direct relationship between phyto- 18. Dronne, S., Colson, M., Moja, S. and Faure, O., Plant Cell Tissue
Org. Cult., 1999, 55, 193–198.
chrome and cell division in the in vitro cultures has been
19. Chory, J., Plant Cell, 1997, 9, 1225–1234.
earlier postulated and evidenced26. The adventitious bud 20. Batschauer, A., Gilmastin, P. M., Nagy, F. and Schafer, E., in
formation and improvement in shoot regeneration res- Photomorphogenesis in Plants (eds Kendrick, R. E. and Kronen-
ponse on exposure to red light during culture period in berg, G. H. M.), Kluwer Academic Publishers, Dordrecht, The
several plants have been reported earlier24,27. Netherlands, 1994, 2nd edn.
21. Chee, R., Plant Cell Tissue Org. Cult., 1986, 7, 121–134.
22. Chee, R. and Pool, R. M., J. Am. Soc. Horti. Sci., 1989, 114, 350–
1. Osol, A., Pratt, R. and Altschule, M. D., JB Lippincott Philadel- 354.
phia, 1960, 1, 277. 23. Moreira da Silva, M. H. and Debergh, P. C., Plant Cell Tissue
2. Tyler, V. E., Brady, L. R. and Robbers, J. E., Pharmacognosym, Org. Cult., 1997, 51, 187–193.
Lea and Febiger, Philadelphia, 1976, 7th edn. 24. Wand, H. B. and Vance, B. D., J. Exp. Biol., 1968, 19, 119–121.
3. Cellarova, E., Repcakova, K. and Honcariv, R., Proceedings of 25. Husemann, W. and Reinert, J., Protoplasma, 1976, 90, 353.
International Symposium on Plant Tissue and Cell Culture: Appli- 26. Davidson, A. W. and Yeoman, M. M., Ann. Bot., 1974, 38, 545.
cation to Crop Improvement, Czechoslovak Academy of Sciences, 27. Kadkade, P. G. and Seibert, M., Nature, 1977, 49, 270.
Prague, 1984, pp. 515–516.
4. Roberts, D. D., Plant Dis., 1981, 65, 322–324. Received 24 July 2000; revised accepted 16 January 2001

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