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Annual Report2008
Central Institute of Medicinal and Aromatic Plants
CENTRAL INSTITUTE OF MEDICINAL AND AROMATIC PLANTS(Council of Scientific Industrial Research)P.O. CIMAP, LUCKNOW – 226 015, INDIA
Front Cover : Albizia lebbeck inflorescence and podsBack Cover : Albizia lebbeck treeAlbezzia lebbeck flowers and pods are important source of a synergistic formulation for controlling
lepidopteran insects in combination with Bacillus thuringiensis δ-endotoxin (CIMAP invention).
were used as soil drench on three week old seedlings which were planted in root-knot nematode infested soil.
Maximum reduction in root-knot severity and nematode population occurred with 4-chlorosalycylic acid, O-
acetylsalicylic acid and 2-chloronicotinic acid. The result envisages that these chemical activators may be used
in integrated management system for protection against root-knot nematodes.
The experiment conducted on integrated pest-management in mints indicated that initial adult population of
white fly was found building up from March and was maximum in the month of May. Among genotypes
evaluated for this resistance potential to whitefly population, significantly low number of flies observed in
variety - Kushal. Out of four samplings in field from March to May, the Begomovirus infection was also found
minimum in variety - Kushal as compared to others. The maximum infestation of Begomovirus was found in
cultivar Shivalik. The genotypes; MAS-35, MAS-216 and cultivar Shivalik were found moderately resistant to
gall forming root - knot nematode (Meloidogyne incognita). However, none of the varieties/genotypes was
found to be highly resistant or susceptible with respect to outbreak of M. incognita.
Highest root biomass (15.20 q/ha) and oil yield (40.10 kg/ha) was recorded in vetiver when harvested at age of
10-12 month followed by 9 month aged crop. The vetiver crop as annual crop successfully be grown under the
sub-tropical climate of Uttar Pradesh for better economic returns.
Two distinct leaf blight diseases of kalmegh have been investigated. One type is caused by Colletotrichum sp.
and another by Rhizoctonia sp. Rhizoctonia was found more destructive than the Colletotrichum sp. However,
both caused premature defoliation of leaves. In Senna the disease was found to be caused by Colletotrichum
Integrated management of foliar diseases of sarpagandha in field
Chemical activators: A novel and sustainable approach for management of Meloidogyne incognita in
Chamomilla recutita
Integrated pest management in mints
Effect of crop age on root and oil yield of vetiver
Leaf blight disease of Kalmegh (Andrographis paniculata), Senna (Cassia senna) and Aloe vera
Separation and quantification of lignans in Phyllanthus species by a simple chiral densitometric method
Influence of sources and doses of N and K on herbage, oil yield and nutrient uptake of patchouli in semi-arid
tropical climate
Influence of integrated nutrient management on yields, oil quality and soil fertility of rosemary
Qualitative performance of Java citronella (Cymbopogon winterianus) cultivars in Uttarakhand hills
Influence of different VAM fungi on the root biomass yield and reserpine content of Sarpgandha (Rauvolfia
serpentina) in unsterile soil
Effect of date of planting on the yield and disease incidence of Aloe vera
Influence of bioinoculants on the growth, yield and black spot disease of Aloe vera
A sensitive, selective, and robust high-performance TLC (HPTLC) method using chiral TLC plates for qualitative
and quantitative analysis of phyllanthin, hypophyllanthin, niranthin, and nirtetralin, the active lignans of
Phyllanthus species, was developed and validated.
Field experiments were conducted at CIMAP RC, Bangalore to study the influence of sources and doses of N and
K on herbage, oil yield, nutrient uptake, nitrogen use efficiency (NUE) and oil quality of patchouli and soil fertility -1 -1status. The results revealed that application of 200 kg N ha and 41.5 kg K ha produced significantly higher
patchouli herbage and oil yields compared with control.
Results revealed that all the treatment combinations significantly influenced the herb and essential oil yield over
control. Herb yield increased by 39-85% over control with application of different combinations of inorganic
fertilizers and organic manure (vermicompost). The corresponding increase with full nutrient supply through
the organic manure and full nutrient supply through inorganic fertilizers was 39 and 85%, respectively. Similarly,
oil yield increased by 37-84% over control with different combinations.
Hydrodistilled essential oils of Java citronella (Cymbopogon winterianus) cultivars grown in Uttarakhand hills
were analysed during summer, rainy and winter seasons. Cultivars Manjusha, Mandakini, Jalpallavi and Bio-13
recorded highest oil in winter (1.5 –1.82%), while cultivar Medini (1.1%) in summer season. In cultivar Manjusha,
Mandakini and Bio-13, citronellal content was higher in summer followed by rainy season, while cultivar
Jalpallavi recorded maximum citronellal content during rainy season. Cultivar Medini was geraniol rich, and
geraniol was found to be higher during summer (45.74%) followed by winter (43.71%).
The sole treatment of G. mosseae, G. fasciculatum, G. margarita and G. aggrigatum increased the fresh root
biomass yield of sarpgandha over untreated control by 203, 157, 135 and 103%, respectively.
Maximum herb yield/ plant (3.5kg) of Aloe vera was recorded in March to May planting with 15-17 months
duration. Yield was recorded to be more than 3kg/plant in 12-14 month- old plants. Thus, these plants can be
very safely cultivated as annual without any significant effect on herb yield.
Four selected bioinoculants namely, Bacillus subtilis, Glomus aggregatum, G. intraradices and Trichoderma
harzianum were used alone and in different combinations to evaluate thier influence on plant height, number of
INTEGRATED NUTRIENT AND PEST MANAGEMENT
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Central Institute of Medicinal and Aromatic Plants Annual Report 2008 : Highlights
grow in presence of 25 ppm of Cr in soil, became stunted and died with higher accumulation of Cr in root (989
ppm) and leaves (415ppm) as compared to that grown in normal soil taken as control.
The herb and oil yield of M. piperita significantly increased with the application of heavy metals to soils up to
certain level. The relatively high concentration of Cr, Cd, Pb and Ni in root tissues than in the shoot tissues
indicate that the M. piperita restricted the translocation of heavy metal from root to shoot. These results
indicate that the M. piperita could be grown on moderately heavy metal contaminated soils.
The study indicated that vermicompost made up of distillation waste of aromatic grasses besides other
beneficial effect on soil physical, chemical and biological properties, also helps in regulating the availability of
trace elements in soil.
Active constituents curcumin, demethoxycurcumin and bisdemethoxycurcumin were isolated from the
rhizomes of C. longa. The chloroform extract showed significant antimycobacterial activity. On isolating the
pure molecules based on activity guided fractionation, demethoxycurcumin was found to be the active
constituent. Some of its derivatives were also synthesized for structure and activity relationship studies.
Antimicrobial and drug resistance modifying activity of the essential oil from Rosmarinus officinalis was
evaluated against human pathogenic bacterial strains using disc diffusion and micro broth dilution assay. The
oil was found to be more active against the gram positive human pathogenic bacteria compared to gram
negative bacteria used in this study. The present finding suggests that characterization and isolation of the
active compound(s) from the rosemary oil may be useful in the treatment of bacterial including drug resistant
infections.
As a part of efforts to discover plant based biologically active molecules/compounds through bioprospection,
362 extracts and essential oils from MAPs were evaluated for their antimicrobial activity. Out of 311 extracts,
146 were found active against bacteria, 82 against fungi and 37 against both (bacteria and fungi). Out of 51
essential oils, 37 were found active against bacteria, 50 against fungi and 33 against both.
Effect of seasonal variation on biological activity of O. biennis root extract was studied against Staphylococcus
aureus and S. epidermidis. The roots were collected between April 2006 to July 2006 at 15 days interval and
extracts prepared were subjected to activity evaluation both qualitatively and quantitatively. The maximum
activity was observed in the samples collected in the month of June - July.
Effects of heavy metals (Chromium, Cadmium, Lead and Nickel) on the growth and yield of Mentha piperita
Vermicompost as a carrier as well as organic chelate for regulating Fe and Zn availability in soil and growth
and yield of geranium (Pelargonium graveolens)
Demethoxycurcumin: An antimycobacterial principle from turmeric (Curcuma longa)
Rosemary oil useful against drug resistant bacteria
Bioprospecting for antimicrobials from plant sources
Effect of seasonal variation on antibacterial activity of Oenothera biennis roots
BIO ACTIVITY AND DRUG MOLECULE PROSPECTION
sp.(new species), which leads to premature defoliation of the leaves. This is one of the serious diseases of Aloe
vera, caused by Colletotrichum sp.; the disease greatly reduced the yield of gel. The disease have been
successfully managed by three application of Tilt (Propiconazole) @ 0.1% at 15 days interval.
The germination percentage in T. minuta accessions ranged from 17.3% to 28.6% depending upon their
genotypic differences. Similar trend in result was recorded for germination energy, the parameter to measure ththe speed of germination. The germination period was mostly 10 day in all the accessions.
The results of an experiment on intercropping of Mentha arvensis and vetiver indicated that 80 kg oil of M. 2
arvensis was obtained in one harvest from an area of 6000 m . The digging of vetiver root was done in the month
of the Jan 2007. Approximately 12 .0 kg oil of vetiver was obtained from the same area. Thus both the crops
planted in the month of March and Mentha arvensis harvested in the month of May produced 135 kg oil /ha,
and 20 kg oil /ha of vetiver. This intercropping of M. arvensis with vetiver, generates additional revenue and
helps in better utilization of resources and additional profits to farmers.
With a view to identify MAPs suitable for understorey cropping with Phal van (fruit orchard), four experiments
have been established: under artificial shade, under Anola orchard, under Eucalyptus citrodora and Bananna
plantation and under natural shade of mango orchard at Central Institute for Sub-tropical Horticulture,
Lucknow. The experiments indicate that many crops like Kalmegh, Asparagus Patchouli etc. are moderate to
highly shade tolerant.
The herb and oil yield of Japanese mint significantly increased with the application of all the heavy metals to
soils to a certain level. The increase in the yield was 20.9, 35.2, and 34.2 % in herb and 27.5, 13.7, and 6.8 % in oil
with the application of 15, 30 and 45 mg Cr / kg soil, respectively. The menthol content in the essential oil was
not significantly affected by the application of heavy metals to the soil.
An experiment was conducted to study the effect of urease, amylase, dehydrogenase, acid phosphatase and
alkaline phosphatase on geranium, rosemary and Artemisia in rhizosphere. The different distances from the
mesh surface (0-15, 15-30, 30-45 cm) were analyzed for activities of different enzymes involved in C, N, P and S
cycling. The enzyme activities showed a clear gradient up to 0-15 cm depth for the root surface, while no such
gradient were observed in the unplanted soil. Enzyme activities were higher in the geranium and rosemary
rhizosphere, whereas in Artemisia rhizosphere the activity was found to be low.
The results of the experiment to assess the influence of chromium on growth and yield of kalmegh and the
ability of organic matter to mitigate the toxic influence of chromium has revealed that Kalmegh was unable to
Comparative germination behaviour of marigold (Tagetes spp)
Intercropping of Mentha arvensis with vetiver for higher returns
Exploring companion cropping of MAPs in agroforestry and fruit orchards by shade tolerance evaluation
Japanese mint as potent crop for the effective utilization of heavy metal polluted soils
Variability of soil enzyme activities in the rhizosphere of geranium, rosemary, Artemisia and unplanted crop
Organic matter as amendment to mitigate the toxic influence of chromium on kalmegh
PHYTOREMEDIATION AND SOIL QUALITY MANAGEMENT
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Central Institute of Medicinal and Aromatic Plants Annual Report 2008 : Highlights
Antimicrobial potential of Glycyrrhiza glabra roots
Synergism between extract of Phyllanthus amarus and silymarin for liver protection
Isolation of bioactive phytomolecules from Bidens pilosa
Bioactive phytochemical leads as mosquitocidal agents
Gallic acid based steroidal chalcones as anticancer agents
Anticancer and hepatoprotective agent from Withania somnifera and biological evaluation of its ester
derivatives
Cytotoxic agents from Terminalia arjuna
Antimycobacterial activity of Glycyrrhiza glabra was found at 500 µg/mL concentration. Bioactivity guided
phytochemical analysis identified glabridin as potentially active against both Mycobacterium tuberculosis H Ra 37
and H Rv strains at 29.16 µg/mL concentration. It also exhibited antimicrobial activity against both Gram-37
positive and Gram-negative bacteria. These results indicate potential use of licorice as antitubercular agent.
In search of the effective and standardized hepatoprotective combination therapy, silymarin and standardized
extract of Phyllanthus amarus were evaluated against CCl induced hepatotoxicity in rats. The combination of 4
silymarin and Phyllanthus amarus exhibited synergistic effect for hepatoprotection and silymarin with
ethanolic extract of P. amarus showed better activity due to the higher concentration of phyllanthin in ethanolic
extract in comparison to aqueous extract of P. amarus as estimated by HPLC.
The phytochemical investigation of hexane extract of Bidens pilosa leaf gave a bright orange compound ß-
carotene, phytyl alkanoate, friedelin, stigmasterol, 1-phenyl heptatriyne and a carotenoid derivative.
Compound 4'-O-â-D-(4”,6”-diacetyl)-glucopyranoside showed significant antioxidant activity and was found to
be non toxic to erythrocytes in in vitro bioassays.
Essential oil of blue stem grass, Bothriocola bladhii with sirodecenone and camphene as the major constituents
was observed to exhibit bioactivities against mosquito species, Aedes aegypti. Essential oil was evaluated for
ovicidal, larvicidal, growth disruption and adulticidal activities. Out of various bioactivities evaluated, the oil
was found effective only as larvicidal.
Estradiol based steroidal chalcones were synthesized. Using MTT assay, the anticancer activity of all these
synthesized chalcones was evaluated against various human cancer cell lines. Several steroidal chalcones
showed higher levels of cytotoxicity.
Detailed chemical investigation of Withania somnifera roots resulted in the isolation and identification of a
cytotoxic and hepatoprotective agent, palmitic acid, which was converted to eight semi-synthetic ester
derivatives. 2,9 t-Butyl palmitate and amyl palmitate were 4-6 times more active than against adherent and
suspension colon cancer cell lines. Interestingly, palmitic acid and its ester derivatives also showed
hepatoprotective activity which is being reported for the first time.
Four novel cytotoxic agents arjunic acid, arjungenin, arjunetin and arjunoglucoside-I were isolated from the
bark of T. arjuna. Out of the four compounds, arjunic acid was significantly active against the human oral (KB),
ovarian (PA 1) and liver (HepG-2 & WRL-68) cancer cell lines. Further, the most active compound arjunic acid
was converted into seven semi-synthetic ester derivatives 5 - 11. 2-O-Palmitoyl arjunic acid showed two times
more activity, while 2, 3-di-O-acetyl-, 2-O-p-anisoyl-, 2, 3-di-O-benzoyl- and 2, 3-di-O-p-nitrobenzoyl arjunic
acid showed 1.7 - 2.3 times less activity than the cytotoxic drug vinblastine against the liver cancer cell lines
HepG-2 and WRL-68, respectively.
The methanol extract of the fruit rinds of Garcinia indica showed potent cytotoxic activity against three human
cancer cell lines- colon (COLO-320-DM), breast (MCF-7) and liver (WRL-68) as determined by the MTT assay.
Through in-vitro bioassay, 160 plant extracts from CIMAP repository were evaluated against human; 4 plant
extracts showed the cell line dependent cytotoxicity in terms of IC-50 and IC-90 values against all the human
cancer cell line used. Further of interest, these extracts did not exhibit activity against the mice hepatocytes up
to concentration of 100 µg/ml.
Under Control and Supervision of Experimentation Animals (CPCSEA) all institutions carrying animal
experimentation have to constitute Institutional Animal Ethics Committee (IAEC) and animal experiments have
to be approved by the CPCSEA. The protocols have been categorized into infecting disease, metabolic
disorders, and pharmacological and toxicity studies. We have tested a total 259 smaples out of which 73 were
found positive.
O. vulgare collected from the high altitude of Himachal Pradesh (Kothi-2500m) was found to be a new
chemotype rich in thymol (62%), p-cymene (9%) and 3-octanone (8%). Highest thymol content of 49% has so
far been reported in the oil obtained from plants of Mediterranean region .
The essential oil obtained from the inflorescence of Artemisia capillaris was analysed by GC and GC-MS. Forty
two compounds representing 93.82% of the oil were identified. The main constituents were capillene,
terpinene, eugenol, limonene, p-cymene, β-myrcene and -pinene.
Volatile fractions from flowers of four geranium chemotypes viz., Bourbon, CIM-Pawan, Kelkar and Algerian
were analyzed by capillary GC. Thirty compounds comprising 91.67%of the Bourbon, 29 compounds
representing 93.07% of the CIM-Pawan, 23 compounds representing 85.37% of the Kelkar and 26 compounds
accounting for 87.48% of Algerian flower oils have been characterized. Bourbon and CIM-Pawan were found to
be rich in citronellol (25.64% and 28.44%), geraniol (25.21% and 23.85%) and citronellyl formate (10.11% and
11.18%). Cultivar Kelkar had citronellol (62.48%) and isomenthone (12.67%) as principle components.
However, Algerian type was found to be rich in citronellol (29.92%), citronellyl formate (23.22 and %) and
geraniol (7.21%).
Cytotoxic activity of Gracinia indica
Cancer cell line dependent cytotoxic activity of essential oil as compared to pure compound
Pre-clinical study at “Jeevanika” (Animal house)
A new chemotype of Origanum vulgare identified
Essential oil constituents of Artemisia capillaris from Uttarakhand hills
Essential oil composition of flowers of geranium (Pelargonium graveolens) chemotypes from Uttarakhand
hill
PHYTOCHEMICAL PROSPECTION
β
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Central Institute of Medicinal and Aromatic Plants Annual Report 2008 : Highlights
Cloning of norcoclaurine synthase (ncs) gene involved in isoquinoline alkaloid biosynthesis in poppy
(Papaver somniferum)
An alpha-1 antitrypsin genetic variant identified in human liver biopsy sample
Polymorphism study in different chemotypes of ashwagandha (Withania somnifera) through AFLP
Semi-quantitative RT-PCR analysis of leaf-specific gene dat in Catharanthus roseus cv. “Dhawal”
Cloning of genes involved in isoquinoline alkaloid biosynthesis in poppy
Novel cytochrome P450 monooxygenase genes (cyp) from Artemisia annua
Semi-quantitative RT-PCR analysis of seasonal variation in transcript abundance of leaf specific/prominent
genes dat and sgd in Catharanthus roseus cv. “Dhawal”
The gene for this early pathway step in morphinan alkaloid biosynthesis has been isolated, cloned and
sequence-characterized from Papaver somniferum. This will prove beneficial for further metabolic engineering
of the morphinan alkaloid biosynthetic pathway in heterologous systems.
The study was carried out as an initial step for characterizing the AAT gene in an Indian sample with the ultimate
aim of developing a heterologous alpha-1 antitrypsin production system. The AAT gene was isolated and cloned
from a liver biopsy sample through RT-PCR and the full-length gene was sequenced.
AFLP analysis of 20 different W. somnifera germplasm accessions representing various chemotypes (for
withanolide content), present in the National Gene Bank of Medicinal and Aromatic Plants (NGBMAP) at
CIMAP, was carried out. A total of eight primer pairs (EcoRI/MseI) were used for the analysis, which yielded a
total of 4821 bands of which 4701 were polymorphic and 336 were unique thereby indicating 95.13 %
polymorphism among the chemotypes. This study will help in classifying the Withania germplasm for targeted
genomics studies in future.
Semi-quantitative RT-PCR analysis was carried out to measure the transcript abundance of leaf-specific gene
dat in the aerial and underground parts of the C. roseus (cv “Dhawal”) plant at three different plant
developmental stages (6 days, 6 weeks and 6 months). This gene is significant since its protein product catalyzes
the last step of vindoline biosynthesis, which then commits the metabolic flux into bisindole alkaloid
biosynthesis. Transcript abundance of the dat gene was found to decrease with the plant age.
Four new genes have been cloned from opium poppy cv “Sampada”. These genes are S-adenosyl-L-
methionine:norcoclaurine 6-O-methyltransferase (6omt) and S-adenosyl-L-methionine:3'-hydroxy-N-
methylcoclaurine 4'-O-methyltransferase (4'omt) in the pre-reticuline pathway and salutaridinol 7-O-
acetyltransferase (salAT) codeinone reductase (cor) in the post-reticuline pathway.
A bioinformatics-based approach has been used to hunt for novel cyp genes from Artemisia annua, whereby
the information from the conserved domains of cytochrome P450 genes have been utilized for cyp gene
amplification. Using primers based on these conserved domains, partial sequences of 22 novel cyp genes were
obtained, of which, 2 (cyp73 and cyp83) have been isolated full length using 5'-RACE.
The dat transcript levels in the mature leaf of 6 months old-field grown plant were found to be higher in winter
(December) than in summer (June) season. The sgd transcript levels in the mature leaf of 6 months old-field
grown plant did not vary much with the seasonal change but were found to be slightly higher in summer (June)
than in winter (December) season.
Variation in the chemical composition and yield of essential oil of rose scented geranium (Pelargonium
species) by foliar application of metallic salts
New thaiocarbamates from Moringa oleifera
Composition of North Indian pyrethrum (Chrysanthemum cinerariaefolium) flower oil
Effect of altitude on the essential oil constituents of a new variety of Artemisia (A. roxburghiana var.
purpurascens)
Heterologous expression of menthol biosynthetic pathway gene in bacteria
Cloning of cyp71AV1 gene from Artemisia annua (variety “CIM Arogya”)
The concentration of linalool, geraniol and 10-epi- gamma eudesmol in the essential oil of geranium
significantly increased by the application of metallic salt, whereas the concentration of isomenthone,
citronellol and citronellyl formate was not significantly affected as compared with control plants. The oil yield
of geranium significantly increased by the foliar application of metallic salts as compared with control plants.
The increase in the oil yield was 41.9, 23.4, 29.6, 29.6, 41.9 and 41.9 % over control by the application of sodium
chloride, calcium chloride, iron sulphate, zinc sulphate, copper sulphate and nickel sulphate, respectively.
From the pods of Moringa two new thiocarbamates, O-n-butyl 4-[(á-L-rhamnopyranosyloxy) benzyl]
thiocarbamate (E) and O-ethyl-4-[(á-L-rhamnopyranosyloxy)-3hydroxybenzyl] thiocarbamate (E) along with
niazirin and 5,5-dimethyloxazlidine-2-thione were isolated.
Twenty two compounds representing 85.27% of the total oil were identified. Trans-chrysanthemumic acid
and á-eudesmol (7.07%) were the major compounds. The oil of Indian species is uniquely different from the oil
obtained from pyrethrum of Kenya.
In-vitro testing of dried ethanolic extracts of the plant showed promising insulin secretagogue activity. Plants
collected from different altitudes (850m, 1218m and 2205m) of Garhwal Himalayas showed considerable
variation in yield and quality of oil. The oil yield was lowest (0.2%) in the plants collected from higher altitudes
and it was rich in borneol (21.2%) followed by linalyl acetate (7.4%) and á-humulene (6.7%). While, the plants
collected from the lower altitudes yielded higher percentage of oils (0.8-0.85%) which were dominated by â-
caryophylene (16.3-18.4%) followed by eugenol (16.2%)or á-thujone (12.0%).
The menthol biosynthetic pathway gene pulegone reductase (pr) cloned and sequenced earlier at CIMAP
(submitted to NCBI under Accession Number EF426467) from Mentha arvensis was cloned in pET100 vector
and expressed in bacterial E. coli strain BL21 (DE3) background.
The gene 1467bp for cyp 71AV1, which catalyzes the three step oxidation of amorpha-4,11-diene to artemisinic
acid in the artemisinin biosynthetic pathway of A. annua has been isolated, cloned and sequence-
characterized.
PLANT GENOMICS & MOLECULAR BIOLOGY
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Central Institute of Medicinal and Aromatic Plants Annual Report 2008 : Highlights
14in shoot cultures was corroborated with radiolabel incorporation studies using [2- C] acetate as precursor.
Production of withaferin A was also found in the in vitro shoot cultures.
Method for hairy root induction was developed in Solanum nigrum. In vitro raised plant's leaf and stem were
used as explant source for the Agrobacterium mediated genetic transformation of Solanum nigrum. Infected
plant tissues after 2-3 weeks of incubation showed the hairy root induction.
CIMAP has developed an Agrobacterium rhizogenes-mediated hairy root line (CR-N2) of C. roseus (Var.Nirmal)
that exhibits unique capability to regenerate transgenic plants via adventive shoot bud organogenesis on a
modified medium.
The multiple shoot cultures of H. perforatum entered the exponential phase of in vitro growth between 15-25 th
days of the culture cycle (GI= 2379.29) followed by a gradual incremental biomass gain up-to 40 day (3173.69) th th
and a second rapid growth phase between 40 to 45 day of incubation (GI= 4328.07). Hypericine production thin these shoots occurred after 25 day of growth and reached the peak between 35-45 days of the culture cycle
that coincides with the onset of stationary phase of growth after the two exponential growth peaks.
Using young offset as explants, a rapid in vitro micro-cloning method has been developed to produce disease-
free stocks of Aloe vera (Var. CIM-Sheetal). A multiplication rate of one:eight was achieved within two weeks
when explants were cultured on a cytokinin containing medium.
Tissue culture studies in Chlorophytum borivilianum were undertaken for standardizing multiplication
protocols employing somatic embryogenesis aiming towards its high frequency rapid in vitro multiplication.
Growth kinetic studies were carried out in R. serpentina to find out a co-relation between growth period of the
root clones and higher biomass as well as secondary metabolite production. On the basis of behavior of the five
root clones it may be inferred that, alkaloid production in transformed roots in R. serpentina is associated with
growth and reserpine content was found to be highest at the end of active growth period i.e. 10 weeks of
culture duration.
Agrobacterium rhizogenes ( mediated hairy root cultures have been induced in leaf explants of
Rauvolfia serpentina at M
Establishment of hairy root cultures in Solanum nigrum
Plantlet regeneration from hairy roots of Catharanthus roseus
Biogenesis of hypericine in multiple shoots cultures of Hypericum perforatum as a function of culture age
and precursor feeding
Micro-cloning of Aloe vera
Induction and development of somatic embryos of Chlorophytum borivilianum in liquid culture medium
Growth kinetics studies in Agrobacterium rhizogenes mediated genetically transformed roots in Rauvolfia
serpentina
Establishment and chemical profiling of hairy root culture of Rauvolfia serpentina
strain A )4
81.25% relative transformation frequency. ost of the emerging roots of the present
study tend to form calli in the half-strength, hormone-free, liquid basal MS medium during further sub-
culturing.
Studies on drug resistance development and mechanism in Staphylococcus aureus through molecular
genetics approach
Role of efflux pump modulator in altering the activity of ATPase in Mycobacterium smegmatis
Bioconversion of a monoterpene into food flavoring agent vanillin
Standardization of cerebral malaria model in Mus musculus using markers of chemokine pathway
Modulation of inflammatory cytokines by Ocimum gratissimum under in-vitro conditions
Toxicity profile of Ocimum sanctum leaf at acute and sub-acute level in rat
Withanolide-A biosynthesis in in vitro shoot and root cultures of ashwagandha
In the present study, the sensitive Staphylococcus aureus (SA) and different drug-resistant mutants of SA
including MRSA were tested for their cross resistance to different antibiotics representing various structural and
functional groups. The ATPase activity of drug-resistant mutants were found to be decreased compared to
sensitive strain indicating less transport of drug thereby helps in acquiring resistance.
In presence of ciprofloxacin there was significant decrease in ATPase activity of drug-resistant mutant, when
compared with control suggesting effluxing of the drug in multi drug resistant mutant MDR-Q. However, in
presence of ciprofloxacin and plant molecule citral together, ATPase activity was increased.
The present work was done with the objective of the conversion of abundantly available phytomolecules
eugenol into vanillin a compound of industrial importance using microorganisms Aspergillus flavus, A. niger and
Pseudomonas aeruginosa. These microbes were found to be capable of converting eugenol to industrially
important cost-effective products, vanillin (used as flavouring agent).
In an effort to bioprospect medicinal plants for cerebral malaria a model was standardized for evaluating leads
against malaria using Plasmodium bergheii (Chloroquine sensitive) and P. yoelii nigeriensis (chloroquine
resistant) strains where in as a conventional parameter, parasitaemia and mean survival time (MST) are is being
monitored.
It was observed that O. gratissimum @ 1 and 10 mg/ml, exhibited the anti-inflammatory activity through
inhibition of pro-inflammatory cytokines (IL-1â and IL-6).No significant change were observed in IL-4
expression.
The in-vivo acute and sub-acute toxicity study of Ocimum sanctum (Aqueous extract) was conducted as per the
approved protocol by Institute's Animal Ethical Committee and confirmed to national guidelines on the care and
use of laboratory animals with an intention to study the safety profile of the plant (leaf) which is one of the most
commonly used medicinal plants in Ayurvedic system of medicine. It may be concluded that chemically
characterized aqueous extract of Ocimum sanctum leaf is well tolerated by Charles Foster rat at acute and sub
acute level.
Multiple shoot cultures of the experimental lines of Withania somnifera plants established using nodal segment
as ex-plant under specific hormonal combinations of benzyl adenine and kinetin resulted in significantly higher
level of biogeneration of withanolide A in the in vitro shoots. The enhanced de novo biogenesis of withanolide A
PLANT TISSUE CULTURE TECHNOLOGY
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Central Institute of Medicinal and Aromatic Plants Annual Report 2008 : Highlights
inhibitors involved and post-translational modification to its cloned commentary. Through user friendly GUI
based database one can find out all the relevant information about menthol and biosynthetic pathways.
DbAlbioPath is an alkaloid biosynthetic pathway database of medicinal and aromatic plants (MAPs). The
present database has graphical user interface (GUI). Through this user friendly GUI based database one can
find out relevant information about alkaloids and their pathways. The database contains information of the
alkaloids metabolic pathways and related genes. It comprises of chemical structures with name, formulae,
physical properties, SMILES generation, industrial application and map viewer of medicinal plants.
In this study, an in silico analysis was performed in which SSRs were mined from ESTs corresponding to alkaloid,
phenylpropanoid and terpenoid biosynthesis reported in a large number of important plants species .The
abundance and distribution of various type of SSRs were also analysed. Comparative genomic study was
performed for finding the corresponding genes to these SSR- ESTs related to alkaloids, phenylpropanoid and
terpenoid biosynthesis, using Arabidopsis thaliana as a model. Results revealed that the SSRs are distributed
throughout the complete datasets of EST, almost 941 trinucleotide, 213 dinucleotide, 157 tetranucleotide and
A rapid and efficient method was developed to initiate multiple shoots from internodal and leaf explants.
Excised tissues (leaf blade and internode) were transferred in half strength MS medium supplemented with BAP
and TDZ. Multiple shoots were initiated within three-four weeks. Internodes / leaf blades with regenerated
multiple shoots were transferred to fresh half strength MS medium without supplements for elongation and
rooting.
An in silico analysis of metal binding proteins was performed for identifying putative metal binding proteins for
Cd, As, Hg, Co, Zn, Hg, As, Mo, Mn, Mg and Ni ions in medicinal plants. Related metal binding protein sequences
based on gene name were retrieved using UniProtKB/Swiss-Prot database. The conserved/consensus patterns
was obtained through MSA performed by ClustalX. Motifs were designed and validated through
UniProtKB/Swiss-Prot and UniProtKB/TrEMBL database search which yielded true positives showing the high
specificity of each motif.
Promoters with known transcriptional start sites for Mycobacterium tuberculosis genes in whole genome
(4,411,529 bp) was predicted, compiled and analyzed. Different nucleotide bases were detected as
transcription start site in different promoter sequences. In total of 1970 promoter sequences, adenine residue
was observed to be base for transcription initiation in 347 sequences, guanine in 608 sequences, thymine in 411
sequences and cytosine in 604 sequences. Thus the present study showed guanine was most frequently acted
as start site for the transcription. All the promoters were aligned to determine the consensus elements (-35
hexamer and -10 hexamer).
MBPdb database provides information of various enzymes participating in the pathway under different heads
e.g. EC number, pathway involved (related to KEGG database), reaction type, natural substrate, co-factors,
BIOINFORMATICS/ IN SILICO BIOLOGY
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Central Institute of Medicinal and Aromatic Plants Annual Report 2008 : Highlights
also known as plastid-pathway was very well conceived in all the above experiments conducted on
monoterpenes whereas the cytosol pathway oriented from MVA was found in the experiments conducted on
compounds of sesquiterpenoidal nature.
Artificial polyploids have been developed in a seed forming clone of vetiver. The polyploids thus developed
evince not only high gametic and seed sterilty but are also accompanied by enhanced biological potential for
the characters associated with increased cell size and physico-physiological efficiency. This has far reaching
implications in utilizing a given ideotype for efficient application of vetiver ecotechnology for its multifarious
environmental and industrial applications.
Analysis of alkaloid content showed that treatment duration of 12 hrs of Cr at 10 & 20mM and combination of
Cr- (10 & 20mM), Cd- (10 & 20 mM), and Mn- (10 & 20) with SA- (1 & 5mM) reduced alkaloid content possibly by
influencing biosynthetic steps. Treatments particularly Cd at (10 & 20 mM), Mn at (10 & 20mM) and YE at (1 & 5
g/L) and combination of Mn at 20 mM and 1 mM SA and YE at 5 g/L with SA at 5 mM showed enhancement in
total alkaloid content possibly by influencing biosynthetic pathway. These treatments can thus enhance
accumulation of total alkaloid in Catharanthus.
Results revealed that 0.00 Fe mg/l have the nil vinblastine and vincristine percentage with 0.93 % and 0.89 % of
total alkaloids in Dhawal-Ga treated and etheral treated plants. Vinblastine and vincristine are higher in Dhawal
etheral treted one then the Ga. Furthermore, the vincristine is totally absent in all the alkaloid of the 0.00 to
11.2 cultured plant of Catharanthus.
The virus was efficiently sap transmitted from naturally infected periwinkle plants to healthy plants and to a
variety of plant species mainly from Chenopodiaceae, Cucurbitaceae and Solanaceae. Total RNA was extracted
using Qiagen kit. The isolated RNA was used for cDNA synthesis using reverse transcriptase for PCR
amplification. The PCR reaction (25ìl) using primers specific to CMV coat protein (F:5'-
GCATTCTAGATGGACAAATCTGAATC-3'/ R:5'GCATGGTACCTCAAACTGGGAGCAC-3') was performed in an
automated thermal cycler. A part of the PCR amplification product was electrophoresed in 1.2% agarose gel in
1X TAE that exhibited an amplified DNA fragment of expected size (~650 bp) only from virus infected samples,
not from healthy ones.
About 179 accessions have been added to seed gene bank raising the present tally to accession of 2465 species.
In vitro bank has been enriched by 17 accessions and now comprises of 265 accessions. The DNA bank now
comprises of 1345 accessions with the addition of 23 accessions.
Towards development of non-invasive vetiver – Vetiveria zizaniodes
Elicitation studies in post-harvested leaves of Catharanthus roseus
Effect of gibberellin and ethereal and Fe nutrition in Catharanthus roseus tissue concentration and chemical
composition
Natural infection on periwinkle (Catharanthus roseus) by cucumber mosaic virus
GENE BANK STATUS/UPDATES
BASIC SCIENCE
Effect of zinc and paclobutrazol on growth and pyrethrin quantity and quality of pyrethrum
Effect of catechol treatment on post-harvested alkaloid content in leaves of Catharanthus
Changes in enzymes activities, proline, protein and IAA contents in relation to flower development in Rosa
damascena
Withanolide metabolism in ashwagandha (Withania somnifera)
Standardization of germination parameters in medicinal and aromatic plants (MAPs)
Mevalonate and non-mevalonate pathways for isoprenoid compounds in MAPs
Application of zinc enhanced pyrethric acid ester (Cinerin-2, pyrethrin-2 and jasmolin-2). Cinerin-2 -1
concentration increased significantly, and the increase was 53% and 40% over control at 5 and 10 kg ha of Zn
respectively, while pyrethrin-2 increased by 24% and 31%, respectively. Similarly, an increase of 10% and 42% in -1jasmolin-2 concentration was obtained by 5 and 10 kg ha zinc application, respectively.
The effects of catechol as elicitor for enhancement of alkaloid accumulation in post-harvested leaves of
Catharanthus roseus were investigated. The effect of catechol was dependent of dose and duration.
Treatments of catechol at 1.0mM at 12h resulted in elevation in content of total leaf alkaloid content.
•Pre-licensing evaluation agreement with JK Agri-Genetics for complete technology package for novel plant
growth promoter “Calliterpenone” from Callicarpa macrophylla and Albizia lebbeck extract effective
against Bt-Cofton.
•Agreement with Biogentek BG (I) Pvt. Ltd. for exploring the possibilities of collaboration in the area of hairy
root culture/cell culture.
•Confidentiality agreement between CIMAP and Laila Nutraceuticals, Vijayawada for pre-licensing
evaluation of nutraceutical formulations and other leads of CIMAP.
•Confidentiality agreement signed with TVC SKY Shop, Mumbai for pre-licensing evaluation of CIMAP
herbal formulations.
•Memorandum of Understanding (MoU) for joint research project between CIMAP and University of
Mysore.
Technology licensing
Pre – licensing agreements
Collaborations
•Memorandum of Understanding (MoU) for joint research project between CIMAP and University of
Lucknow for studies on insect bioassay of plant extracts.
•Memorandum of Understanding (MoU) for joint research project with Ocimum Biosolutions, Hyderabad
on development of cost effective Microarray chip with periwinkle as a model.
•Agreement with Avitech Animal Health Pvt. Ltd., New Delhi for exploring the possibilities of collaboration
in the area of developing hepato-protective and immunostimulant product/feed for poultry.
•Designing, fabrication and development of plant/ herb distillation and processing units/ plants/
accessories for MAPs clients on demand at Narayan Mahavidyalaya, Goreakothi, Siwan (Bihar).
•Preparation of feasibility report for M/s SV Farms (P) Ltd., Hyderabad.
•Designing, fabrication and installation of two state- of- art distillation units for distillation of citronella and
lemongrass crops cultivated by the tribal farmers and supported by Integrated Tribal Development Agency
(ITDA), Govt. of Andhra Pradesh.
•Designing, fabrication and installation/commissioning of 1.0 ton capacity improved field distillation unit
on turn key basis to Mahamaya College of Agricultural Engineering and Technology, Akbarpur, Ambedkar
Nagar.
•Design/ Drawings for CIMAP improved FDU to Mr Vijayan, Vishakhapatnam.
•Survey of ash dyke area and preparation of a pre-feasibility report for NTPC, Unchahar, Raibareli.
•Survey of land and preparation of a pre-feasibility report for Vaid Organics, Hardoi.
•Crop consultancy in patchouli to Caps Ayur Krishi, Bangalore.
•Four CIMAP scientists visited foreign research establishments/institutions.
•Eighteen CIMAP scientists bagged reputed national awards/recognitions. CIMAP scientists were also
nominated to different academies, elected fellows and members of the high level expert/advisory
committees.
•Five Ph.D. degrees were awarded to the staff and students of CIMAP.
•More than 112 persons were trained at CIMAP, which included foreign scholars, students, industry
representatives and State and Central Government officials.
•First JNU-CIMAP Ph.D. Programme started.
nd •100 day CIMAP Certificate Course on Medicinal and Aromatic Plants (100 d CCC) held during 22 February thto 4 Junue, 2007.
CNP projects undertaken
HUMAN RESOURCE DEVELOPMENT
20 21
Central Institute of Medicinal and Aromatic Plants Annual Report 2008 : Highlights
•CIMAP Summer Training (CST-2007) on Techniques and Tools of Biotechnology and Bioinformatics for th th
Graduate and Master Students held during 11 June-10 July, 2007.
•CIMAP Summer School (CSS-2007) on "Molecular Techniques in Bioprospection and Biodiversity th
Analysis" held during June 15th - July 30 , 2007.
•CIMAP Training School on Advance Instrumentation and Analytical Techniques for Natural Products (AIAT-rd th
2007) held during 3 -16 September, 2007.
th th•CSIR Programme for Youth for Leadership in Science (CPYLS) held during 15 -16 November, 2007.
•CIMAP Winter School (CWS-2007) on "Recent Techniques in Structural and Functional Genomics" st thheld during 1 -10 December, 2007.
•CIMAP Winter Training (CWT-2008) on “Techniques and Tools of Biotechnology and Bioinformatics for th thGraduate and Master Students” held during 20 January-20 February, 2008.
•A Unique "Know each other" meet of Scientists-Students and Teachers during CPYLS at CIMAP during 19th-
20th February, 2008.
th•National Technology Day (11 May 2007): BEE – MAPs 2007: Emphasis on creation of skilled human
resource: Farmers–industry–scientist converge at CIMAP; entrepreneurial training imparted to women
•'CIMAP Technology Fest-2007' Novel Community-Public-Private-Partnership model to show the way for th thrural prosperity (5 – 11 August, 2007) launched by Vice President, CSIR (Honorable Minister of Science
and Technology, Government of India, Shri Kapil Sibal). Aroma Biovillage Project jointly sponsored by DST
and CSIR, New Delhi was also launched.
th•CIMAP Day Function: MAPs show the way to adopt Integrative agriculture mode (Iam) (11 August, 2007)
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