In vitro rooting of Decalepis hamiltonii Wight & Arn., an endangered ...

RESEARCH COMMUNICATIONS
The observed mineral assemblages – phenocryst + matrix
+ volatile phases + ocelli – in the lamprophyres from the
nunataks of cDML suggest that these dikes belong to calcalkaline
lamprophyre clan. The presence of Type 2 ocelli,
though points more towards the alkaline nature of the
magma 4 , the preliminary whole-rock geochemical data
available for these dikes confirm the calc-alkaline nature
of the dikes. The absence of any carbonate phase –
primary or secondary (except in one sample, No. 7/2-B8,
wherein carbonate phase forms the part of the
pseudomorph assemblage of olivine), suggests CO 2 -poor
nature of the magma. The absence of zoning in any of the
phenocrysts and lack of corona around olivine, indicate
rapid crystallization of the phenocryst phases in the
volatile-rich magma and hence do not represent the
liquidus phase. It is interesting to note that a positive
correlation exists between the incidence of the dominant
volatile phase – apatite and ocelli. The lamprophyres from
Starheimtind nunatak contain no ocelli and apatite is
almost absent. The absence of the volatile phase suggests
a relatively high viscosity for the magma, which in turn
may have been a factor influencing the solidification of
lamprophyric magma before the immiscibility field was
encountered 3 and hence the absence of ocelli. On the
other hand, the presence of ocelli with sharp contacts with
the matrix and the volatile phases in the lamprophyres
from three other nunataks from cDML suggest their
evolution by slower cooling vis-à-vis solidification along
the composition–temperature slope of silicate–silicate
immiscibility field. Although difference of opinion exists
as to the origin of different types (Types 1, 2 and 3) 4 of
ocelli, the Type 2 ocelli in the lamprophyres from the
nunataks of cDML, by nature of their composition and
shape, suggest that they are globules of immiscible felsic
liquid in the mafic alkaline magma and the feldspathic
nature of the ocelli is indeed a case of the silicate–silicate
liquid immiscibility, as suggested by Philpotts 5 .
The deformed nature (foliation and elongated ocelli) of
the lamprophyres of Sonstebynuten may be due to the
slight early emplacement of the dikes compared to the
dikes in other nunataks, when the deformation regime of
the Pan-African event was in the waning stage.
A few dikes from Sonstebynuten and Baalsrudfjellet
chemically analysed by earlier workers 2 suggest that they
have shoshonitic affinity. Both calc-alkaline and alkaline
lamprophyres have been reported from nearby Schirmacher
Oasis and have been shown to be derived from
same source region – in a continental within-plate tectonic
setting during Pan-African collision event of west and east
Gondwana and consequent generation of alkaline magma 1 .
Dayal and Hussain 6 have obtained Rb–Sr whole-rock/
mineral isochron ages of 455 ± 12 Ma and 458 ± 6 Ma for
two lamprophyre dikes from Schirmacher Oasis. The
spatial and temporal (based on field relations) association
of the calc-alkaline lamprophyres in the nunataks with
those of Schirmacher Oasis 1 , thus suggests that the
tectonic model proposed for the Schirmacher Oasis may
hold good for the whole of cDML as well.
1. D’Souza, M. J. and Chakraborty, S. K., J. Geol. Soc. India, 56,
593–604.
2. Singh, R. K., Mukherji, S., Jayaram, S., Srivastava, D. and Kaul,
M. K., Dep. Ocean Dev. Tech. Publ., 1988, 5, 109–119.
3. Philpotts, A. R., Principles of Igneous and Metamorphic Petrology,
Prentice Hall of India Publ., 1994, p. 498.
4. Rock, N. M. S., Lamprophyres, Blacky Publ., London, 1991,
p. 285.
5. Philpotts, A. R., Am. J. Sci., 1976, 276, 1147–1177.
6. Dayal, A. M. and Hussain, S. M., J. Geol. Soc. India, 1997, 50,
457–460.
ACKNOWLEDGEMENTS. We thank the Director-General, GSI for
permission to participate in the XIX Indian Scientific Expedition to
Antarctica and to publish this note. Mr R. Ravindra and Mr M. K.
Kaul, Directors, Antarctica Division (present and former, respectively)
are acknowledged for their encouragement and guidance. Mr M. P.
Gaur and Mr M. J. D’Souza, are acknowledged for useful discussions.
Suggestions of an anonymous reviewer are duly acknowledged. The
National Center for Antarctic and Ocean Research (NCAOR) under
whose aegis the Indian expeditions to Antarctica are launched is duly
acknowledged.
Received 4 January 2001; revised accepted 29 August 2001
In vitro rooting of Decalepis hamiltonii
Wight & Arn., an endangered shrub,
by auxins and root-promoting agents
B. Obul Reddy, P. Giridhar and G. A. Ravishankar*
Plant Cell Biotechnology Department, Central Food Technological
Research Institute, Mysore 570 013, India
In the present study micropropagation of an endangered
shrub, Decalepis hamiltonii Wight & Arn.,
optimization and standardization of efficient methods
for rooting of in vitro-derived micro-shoots by using
phloroglucinol (PG), activated charcoal and CoCl 2
have been achieved. Transfer of in vitro-derived shoots
to MS medium supplemented with 8.8 µM indole-3-
butyric acid (IBA) and 1.43 µM indole-3-acetic acid
resulted in root induction. In another treatment, IBA
was found to be a good root-promoting agent, wherein
dipping of explants in 4.4 µM IBA for 30 min and
subsequent inoculation on MS basal medium was also
beneficial for root induction. Successful field transfer
was accomplished in rooted plantlets.
DECALEPIS hamiltonii Wight & Arn. (Swallow root) is
a monogeneric climbing shrub endemic to the Deccan
peninsula, which is used as a culinary spice due to its
*For correspondence. (e-mail: pcbt@cscftri.ren.nic.in)
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highly aromatic roots. It is useful as an appetizer, blood
purifier, preservative and as a source of bioinsecticide for
stored food grains 1,2 .
Over-exploitation of these plants by destructive harvesting
for the aromatic roots, almost endangered this
plant in its natural habitat. Measures to develop micropropagation
protocols for this endangered shrub with high
field survival are essential. George et al. 3 reported the
plantlet regeneration from leaf callus of D. hamiltonii.
Bais et al. 4 reported the influence of silver nitrate on
in vitro rooting of tissue culture-derived shoots of this
plant. In this communication we report the use of cobalt
chloride, an inhibitor of ethylene biosynthesis, phloroglucinol
(PG) and activated charcoal in combination with
auxins on the rooting of in vitro-grown shoots of D.
hamiltonii.
Healthy plants of D. hamiltonii Wight & Arn. were
collected from the Gumballi forest range located in B.R.
Hills, 80 km from Mysore. Axillary buds were excised
and washed under running tap water to remove soil and
other superficial contamination. The bud explants were
sterilized using 0.15% (w/v) mercuric chloride for five
minutes. Explants having one axillary bud were inoculated
on Murashige and Skoog medium 5 supplemented with
8.8 µM BAP and 2.85 µM IAA for shoot proliferation.
Shoots measuring 2–3 cm were transferred individually to
media containing various levels of α-napthaleneacetic
acid (NAA), indole-3-butyric acid (IBA) and indole-3-
acetic acid (IAA) for root induction. All the explants
were maintained at 25 ± 2°C with a light intensity of
45 µmol m –2 s –1 for 16 h photoperiod using fluorescent
lights (Philips India Ltd) and 60–70% relative humidity.
Experiments were performed with a minimum of five
replicates and repeated four times. PG (Sigma, USA) was
filter-sterilized using 0.22 µM filters (Sartorius Ltd,
Germany) and was incorporated in a range of 17–69 µM;
cobalt chloride was added at 5–10 µM range in MS
medium with activated charcoal at a concentration of
a
b
Figure 1. Effect of root-promoting agents on in vitro rooting of tissue
culture-derived shoots of D. hamiltonii. a, Activated charcoal 0.25%
(explants soaked in 4.4 µM IBA for 30 min); b, Activated charcoal
0.25% + 69 µM PG + 4.4 µM IBA.
Figure 3. In vitro raised plants of D. hamiltonii transferred to pots
for hardening.
a b c d
Figure 2. In vitro rooting of tissue culture-derived shoots of D.
hamiltonii. a, 39 µM PG + 4.4 µM IBA; b, 69 µM PG + 4.4 µM IBA;
c, 5 µM CoCl 2 + 4.4 µM IBA; d, 10 µM CoCl 2 + 4.4 µM IBA.
Figure 4.
Micropropagated hardened plants of D. hamiltonii.
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0.25%. In an another experiment, basal portions of the
in vitro-derived shoot explants were dipped in 4.4 µM
IBA for 30 min and then placed on MS basal medium
as well as on MS basal medium supplemented with
4.4 µM IBA. These experiments were repeated thrice
with five replicates, each replicate containing five explants.
Rooting efficiency was calculated as the percentage of
shoots producing roots after four weeks of culture in all
the treatments. The number of roots produced, length of
roots and the mean values were recorded. Rooted plantlets
were removed from culture medium, freed of agar by
washing in running tap water and were planted in a
sand : compost mixture (1 : 2) under the polythene hood
in a greenhouse. The plantlets were hardened for 20 days
and then were transplanted to the field.
Nodal explants having one axillary bud gave rise to
multiple shoots on MS medium with 8.8 µM BAP and
2.85 µM IAA, which were subsequently used for the
rooting experiments.
Of all the auxins tried for rooting on in vitro-derived
shoots, the best result was obtained in IBA (4.4 µM) with
100% of explants producing roots and exhibiting maximum
field survival of 90%, followed by NAA (1.32 µM)
and IAA (1.42 µM) with 83% and 50% of field survival,
respectively.
Influence of PG and activated charcoal on rooting of
in vitro-derived shoots was studied individually and in
combination. When basal regions of shoot explants were
dipped in IBA (4.4 µM) solution for 30 min and inoculated
on MS basal medium with 0.25% activated charcoal
(Figure 1 a), this treatment gave 100% rooting response
with 66.6% field survival of plantlets. Shoots pretreated
with 4.4 µM IBA solution for 30 min and transferred to
PG (69 µM)-containing medium also resulted in 100%
rooting, but with survival of 60% of the plants transferred
to field. Whereas, shoots which were not pretreated with
IBA solution but cultured on PG (69 µM) with 4.4 µM IBAcontaining
medium, resulted in 100% rooting (Figure 2 b).
Moreover, field survival was high (73.3%). Shoot explants
transferred onto MS medium containing 4.4 µM IBA
along with PG (69 µM) and activated charcoal 0.25%
(w/v) resulted in 80% rooting response, with 75% field
survival. Cobalt chloride at 5 µM with 4.4 µM IBA was
most effective in eliciting 100% rooting response and
80% survival of plants upon field transplantation (Figure
2 c). Compared with 2.85 µM IAA used in our earlier
study with 40 µM AgNO 3 (ref. 4), it was observed that
4.4 µM of IBA was found to be very effective in promoting
healthy rooting even at lower concentration
of silver nitrate (10 µM), with 60–80% field survival of
plantlets.
PG (1,3,5-trihydroxy benzene), one of the degradation
products of phloridzin is well known for
its growth-promoting, and root-inducing property 6–11 ,
especially in several woody species 12–14 , and in in vitro
shoot tip cultures 15,16 . Its effect was pronounced in
combination with an auxin. A similar observation was
also made in the present study. It is highly probable that
the beneficial effect of PG is due to auxin–phenol
synergism resulting in suppression of the peroxidase
activity in the cultured shoot tips, thereby protecting
the endogenous auxin from peroxidase-catalysed oxidation
16,17 . Activated charcoal might stimulate rooting
by adsorbing growth regulators and/or inhibitors 18–23 .
We have earlier reported use of silver nitrate, a potent
inhibitor of ethylene activity in promoting in vitro rooting
in this woody climber 4 . The root stimulating effect of
cobalt chloride may be due to its inhibition of ethylene
biosynthesis 24–28 .
Thus the present study showed that cobalt chloride,
charcoal and PG along with IBA treatment had beneficial
effect on rooting of in vitro-derived shoots (Figures 3, 4),
which was useful for hardening and survival of in
vitro-derived plants of D. hamiltonii upon transfer to
field.
1. George, J., Pereira, J., Divakar, S., Udaysankar, K. and Ravishankar,
G. A., A method for the preparation of active fraction
from the root of Decalepis hamiltonii, useful as bioinsecticide,
Indian Patent No. 1301/Del/98, 1998.
2. George, J., Pereira, J., Divakar, S., Udaysankar, K. and Ravishankar,
G. A., Curr. Sci., 1999, 77, 501–502.
3. George, J., Bais, H. P., Ravishankar, G. A. and Manilal, P., Hortic.
Sci., 2000, 35, 296–299.
4. Bais, H. P., Sudha, G., Suresh, B. and Ravishankar, G. A., Curr.
Sci., 2000, 79, 894–898.
5. Murashige, T. and Skoog, F., Physiol. Plant., 1962, 15, 473–497.
6. Jones, O. P., Nature, 1976, 262, 392.
7. Chang, D. C. N., Peng, K. H., Nichols, M. and Swain, D., Acta
Hortic., 1996, 415, 411–416.
8. Aklan, K., Cetiner, S., Aka-Kacar, Y., Yalcin-Mendi, Y., Kuden,
A. B. and Dennis, F. G. Jr, Acta Hortic., 1997, 441, 325–
327.
9. Goudarzi, R., Majedi, A., Talaie, A. R. and Mostafavi, M., Iran J.
Agric. Sci., 1997, 28, 133–143.
10. Hammatt, N. and Grant, N. J., Plant Cell Tiss. Org. Cult., 1997,
47, 103–110.
11. Zanol, G. C., Fortes, G. R., de, L., Campos, A. D., da Silva, J. B.,
Centellas, A. Q. and da Silva, J. B., Rev. Bras. Fisiol. Vegetal.,
1998, 10, 65–68.
12. James, D. J., Physiol. Plant, 1983, 57, 149–153.
13. James, D. J., Hortic. Sci., 1979, 54, 273–277.
14. Jones, O. P. and Hopgood, M. E., J. Hortic. Sci., 1979, 54,
63–66.
15. James, D. J. and Thurbon, I. J., J. Hortic. Sci., 1981, 56,
15–20.
16. Sarkar, Debabrata and Naik Prakash, S., Plant Cell Tiss. Org.
Cult., 2000, 60, 139–149.
17. De Klerk, G. J., Vander Krieken, W. and de Jong, J. C., In Vitro
Cell Dev. Biol. Plant, 1999, 35, 189–199.
18. Fridborg, G. and Eriksson, T., Physiol. Plant., 1975, 34, 306–
308.
19. Fridborg, G., Pederson, M., Landstorm, L.-E. and Eriksson, T.,
Physiol. Plant., 1978, 43, 104–106.
20. Eliasson, L., Physiol. Plant., 1981, 51, 23–26.
21. Dumas, E. and Monteuuis, O., Plant Cell Tiss. Org. Cult., 1995,
40, 231–235.
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22. Weatherhead, M. A., Burdon, J. and Henshaw, G. G.,
Z. Pflanzenphysiol., 1978, 89, 141–147.
23. Tyagi, A. K., Rashid, A. and Maheswari, S. C., Physiol. Plant.,
1980, 49, 296–298.
24. Auderset, G., Moncousin, C. and Rourke, J. O., Hortic. Sci., 1996,
31, 240–242.
25. Ma, J. H., Yao, J. L., Cohen, D. and Morris, B., Plant Cell Rep.,
1998, 15, 87–90.
26. Biondi, S., Diaz, T., Iglesias, I., Gamberin, G. and Bagri, N.,
Physiol. Plant., 1990, 78, 474–483.
27. Bollmark, M. and Eliasson, L., Physiol. Plant., 1990, 80, 534–
540.
28. Reid, D. M. and Bradford, K. J., Flooding and Plant Growth,
Academic Press, New York, 1984, pp. 195–219.
ACKNOWLEDGEMENTS. We thank the Department of Science and
Technology, Govt. of India, New Delhi for a research grant.
Received 21 April 2001; revised accepted 18 September 2001
In vitro induction and enlargement of
apical domes and formation of
multiple shoots in finger millet,
Eleusine coracana (L.) Gaertn and
crowfoot grass, Eleusine indica (L.)
Gaertn
Satish Kumar, Kalpana Agarwal and S. L. Kothari*
Department of Botany, University of Rajasthan, Jaipur 302 004, India
In cereal crops, somatic embryogenesis is the most
common pathway of plant regeneration. We describe a
protocol for high frequency regeneration of Eleusine
coracana via formation of large apical domes from
seeds, mature embryos, immature inflorescences and
immature embryos. The induction of apical domes
occurred on MS medium supplemented with different
auxins [2,4-dichlorophenoxyacetic acid (2,4-D);
2,4,5-trichlorophenoxyacetic acid (2,4,5-T); and parachlorophenoxyacetic
acid (pCPA)] and cytokinins
[kinetin (Kn) and 6-benzylaminopurine (BAP)]. The
primary domes, after four weeks of subculturing on
MS + 2,4-D (0.1, 0.2 mg/l), proliferated rapidly and
gave rise to secondary and tertiary domes along with
green, nodular, compact callus. The domes on MS
medium containing GA 3 ; (1 mg/l) or 1-naphthaleneacetic
acid (NAA) (1 mg/l) gave rise to high-frequency
shoot bud differentiation. Multiple shoot buds arose
over the entire surface of the dome, exogenously.
Histological observations provided a clear evidence
that the shoot apices of the mature and immature
embryos transformed into enlarged apical domes and
the surface of the domes was occupied by highly
meristematic cells which differentiated into shoot
buds. De novo differentiation of meristematic giant
domes and shoot buds was also observed in the
repeatedly subcultured callus raised from mature
embryos as well as inflorescence. Similar structures
were also seen in cultures raised from immature
inflorescence of crowfoot grass (Eleusine indica).
*For correspondence. (e-mail: slkothari@lycos.com)
1482
CEREALS are the most important crops in the world in
terms of production, area under cultivation, and contribution
to the diet of man and livestock. Therefore,
they have received maximum attention in the past few
decades towards regeneration of plants from cultured
cells. Today, almost all major cereal crops can be regenerated
from tissue cultures 1–3 . Further, improvement of
in vitro regeneration and production of transgenics in this
group of plants is under intense investigation. Somatic
embryogenesis has been described as the most common
method of plant regeneration in tissue cultures of
cereals 1,2 . Organogenesis (or shoot morphogenesis) involving
the development of axillary buds or de novo
development of shoot meristems in callus cultures is also
not uncommon in cereals 4,5 . There are many examples
where both embryogenesis and shoot bud formation
occurred in the same cultures 6–8 . Regeneration of plants
via somatic embryogenesis is preferred to organogenesis
because of the single cell origin of embryoids, thereby
making such embryogenic cells suitable for genetic
manipulation. In this paper we have described the formation
of multiple shoots on the enlarged primary apical
domes (apical domes are present between the embryonal
leaf primordia and enlargement occurs due to culture
conditions) as well as on the de novo-formed meristematic
nodules (meristematic nodules are formed de novo in the
dedifferentiated callus) in callus cultures. Formation of
enlarged apical domes is rare. Histological evidence has
been provided for both, the formation of adventitious
buds on apical giant domes and meristematic nodules
formed in callus cultures. Wakizuka and Yamaguchi 9 have
previously described induction and multiplication of
enlarged apical domes as continuous cultures of apical
domes, without any formation of callus. De novo formation
of meristematic nodules described in this paper
has not been reported previously. Structures similar
to enlarged apical domes have also been reported in
Musa 10 , wheat 11 , sorghum 12 and Pennisetum 13 . Plant
regeneration previously interpreted as occurring from
somatic embryos 14,15 as well as shoot buds 16 , may well be
a case of multiple shoot formation from the enlarged
apical domes.
Seeds, mature embryos, immature inflorescences and
immature embryos of three cultivars of Eleusine coracana
(L.) Gaertn, viz. PR-202 (brown ragi), and L-216, GE-
4971 (white ragi) were used as source material for
induction of cultures. Seeds of all the three cultivars were
CURRENT SCIENCE, VOL. 81, NO. 11, 10 DECEMBER 2001