Annual Reports 2008

Highlights Annual Report 2008 80

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Bt endotoxin (Bt) + Plant extract (E)

CIMAP The green path to better health and life

Central Institute of Medicinal and Aromatic Plants (CSIR), Lucknow

© copyright 2008 CIMAP, Lucknow, India

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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 pods Back Cover : Albizia lebbeck tree Albezzia lebbeck flowers and pods are important source of a synergistic formulation for controlling lepidopteran insects in combination with Bacillus thuringiensis δ -endotoxin (CIMAP invention).

Central Institute of Medicinal and Aromatic Plants

BIO-VILLAGE MISSION Artemisia annua Artemisia annua was successfully cultivated for the first time in south Indian agroclimatic conditions at CIMAP Resource Centre, Bangalore and good herb yield (15-20 t/ha) and artemisinin content (0.7-1.0%) were obtained. Artemisia biovillage programme was successfully extended to state of Tamil Nadu where 7 acre land at a village near Salem was put under Artemisia cultivation with excellent economic yield and artemisinin content (0.89%) Vetiver Under the Vetiver (Khus) Biovillage programme three varieties of Khus namely CIMAP/ KS-I, Gulabi and Dharini slips were distributed among 45 selected farmers of Sitapur, Barabanki and Raebareli districts of Uttar Pradesh. The feedback from 25 farmers for root and oil production indicated that average production of root was in the range of 15-20 q/ ha with oil recovery of 1.0 -1 .5% on tank basis. Digging of roots of Khus is the main hindrance and costly operation for its cultivation by the farmers. Field demonstration of root digging by J.C.B. machine (catterpillar) for large area and modified tractor operated Khus digger for medium area was done successfully and was found to several fold cheaper than manual digging. Withania Based on the agrotechnology and seeds provided by the CIMAP Resource Centre, Hyderabad a group of farmers started cultivation of aswagandha on pilot scale in Guntakal Kottala village in Guntakal region in 2003. During 2005-06, the cultivation was taken up in relatively large area and an association of medicinal plants growers was formed in Gooty, Aananthapur consisting mainly of small and marginal farmers (range of land holding 2 -12 ha). This has facilitated marketing of the root. Aswagandha is now cultivated by 79 farmers in 26 villages belonging to 13 revenue mandals in 495.5 ha replacing the traditional groundnut, redgram and Bengalgram in some villages. The largest area has been in Guntakal Kottala village (CIMAP's model biovillage) and total area was highest in Uravakonda revenue mandal. During 2006-07, CIMAP's agrotechnology was demonstrated for cultivation of Withania variety Poshitha. One hundred and seventy six farmers were trained at Guntakal - the heart of Aswagandha belt in Ananthapur district. Patchouli CIMAP Resource Centre, Bangalore launched a bio-village programme in this area in September 2003. Initially 10 farmers were selected and given planting material of a superior variety (Johore) of patchouli acceptable to the industry and other inputs for cultivation. Training programmes were conducted by CIMAP, RC, Bangalore to educate the farmers of the region in cultivation of patchouli. Now more than 350 farmers have taken up cultivation of patchouli covering an area of more than 300 acres. One of the farmers, who was trained by CIMAP on distillation, has established distillation unit and also entered into a buyback agreement with the farmers of the area. Over a period of 3 years, the returns from the patchouli cultivation have improved the rural livelihoods of the farmers in the area through increased income from their lands. Thus an end to end mission for patchouli has been successfully established at Siddapur in North Kanara District in Karnataka.

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Annual Report 2008 : Highlights

Aroma Biovillage

Central Institute of Medicinal and Aromatic Plants

GENETIC IMPROVEMENT & BREEDING EFFORTS

Aroma Biovillage jointly sponsored by Department of Science and Technology and Council of Scientific and th Industrial Research, New Delhi has been launched at CIMAP on 4 August, 2007 by Shri Kapil Sibal, Minister of Science and Technology and Earth Sciences, Government of India. In this programme about 100 beneficiary farmers (50 ha) of Sultanpur and Raebareli have been adopted for cultivation of mint in normal cultivated lands and Basil, Palmarosa, Lemongrass and Khus in salt affected soils.

IMPROVED VARIETIES & AGROTECHNOLOGIES CIM- Hariyali: High essential oil yielding variety of Rosemarinus officinalis Rosemarinus officinalis commonly known as 'Rosemary', a member of family Lamiaceae is an aromatic plant and its essential oil is used in high-grade perfumery, cosmetic and soaps. The variety CIM –Hariyali has 1.03% oil with a yield potential of nearly 350 litres of oil/ ha accounting for more than Rs. 3.0 lakhs per hectare after three years of planting. CIM-Shakti: High yielding variety of Satawar (Asparagus racemosus) with high root yield and high saponin content Variety CIM-Shakti consistently showed high saponin content in all field evaluation yield trials. The average dry root yield is about 148q/ha and saponin content 21%. The new variety is characteristically having numerous fingers and profuse seed formation. Vetiver as annual crop Vetiver is grown as a perennial crop and its harvesting is recommended 18-24 months after planting. The long gestation period of vetiver crop is thought to be one of the reasons for its low popularity among growers. So far, no agrotechnology on the productivity of vetiver as annual crop is available. Keeping these in view CIMAP has developed agrotechnology to grow vetiver as annual crop for which a period of 10-12 months has been found to be optimum for production of economic oil yield with good quality oil. It can suitably be intercropped with some important crops like Mentha arvensis, M. piperita and Kalmegh which could generate additional revenue. New dimensions in organic agriculture CIMAP has now been accredited in the areas of crop production, processing and trading of medicinal and aromatic plants by ECOCERT in accordance with requirements of National Organic Programme (NOP) US, National Programme for Organic Production (NPOP) India, and the Commission Regulation (EEC) 2092/91 and Regulation (EC) 1788/2001. CIMAP has also taken an initiative on Development of Good Agricultural Practices (GAPs) in MAPs. The work has been initiated with a move towards 'Green Agriculture' for developing practices requiring minimum essential synthetic agri-chemicals for achieving higher yields, safer products with a clear objective of sustainability. Initial studies conducted with Ocimum sanctum clearly indicated that chemical fertilizers can be reduced to an extent of 70% when used along vermicompost. In this context, it has been established that perhaps, integrated nutrient management is the most viable proposition.

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Evaluation of performance of a linalool-rich variant of Ocimum basilicum Four chemovariants of Ocimum basilicum clones rich in linalool were identified from a population raised from open-pollinated seeds. The contents of linalool in these variants varied from 49-73%. The variant with highest content of linalool i.e., 73% was multiplied and its seeds were produced in isolation through intermating of plants raised from stem cuttings. The resulting two generations were evaluated during two years at four plant growth stages. Herb yield, oil content and linalool content increased from pre-emergence to seed-set stage. Further, no deterioration in linalool content was noticed over two generations of evaluation when seeds were produced through open-pollination in isolation. Characterization of Ocimum germplasm lines The Ocimum germplasm bank at CRC, Hyderabad contains 39 germplasm lines comprising of 4 species i.e. O. basilicum, O. tenuiflorum, O. gratissimum and O. kilimandscharicum. Studies for morphological, chemical and molecular characterization were initiated at CRC, Hyderabad during 2007-08 in order to assign descriptive characters to the genotypes for precise identification. Morpho-agronomical characterization (52 traits) of the 39 Ocimum genotypes has been done. Screening and evaluation of Claviceps purpurea for improving productivity of ergot and ergot alkaloids Field study on yield and quality evaluation of ergot was conducted. Maximum calculated yield was more than 150kg/ha in five accessions while in remaining accessions (except two where it was less than 100kg /ha ),yield was more than 100kg/ha.This yield performance was only upto mid November sown crops. Beyond that sclerotial yield was dastically reduced upto12kg/ha in one of the accessions. A high yielding genotype of lemongrass (Cymbopogon khasianus) 'NS-1' suitable for marginal lands Two superior genotypes of lemongrass were identified for vigour, high herb and oil yield with high citral content. Genotype NS-1 was found to be highly promising for very fast growth, high herb and oil yield of better quality. Morphological tag developed in low latex line of poppy (Papaver sommiferum) for seed crop A recombinant genotype of opium poppy has been developed in near zero latex containing variety Sujata background to distinguish its plants from other latex producing varieties of poppy. The new recombinant has the distinguished marker in form of deep fringed leaves. Identification of a high yielding strain CIMAP–SR 3-3 of Stevia (Stevia rebaudiana) for northern Indian plains Two superior genotypes SR- 2-6 and SR 3-3 were identified where one genetic stock, namely SR 3-3 was found to be highly promising for high fresh and dry leaf yield with high steveoside, rebaudioside with low dulcoside - A content (%) in the leaf. Development of a high yielding dark purple pigmented strain STAS-3 of Shyam tulsi (Ocimum sanctum) Under genetic improvement programme of tulsi (Ocimum sanctum), the diverse genetic stocks of Shyam tulsi (the purple pigmented tulsi plant) were collected from Uttar Pradesh, Bihar, Madhya Pradesh, Orissa, Uttarakhand, Gujarat, Maharashtra, Jammu & Kashmir, West Bengal states of India. The strain STAS - 3 consistently showed high oil content in BST and PST.

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Annual Report 2008 : Highlights

PROCESS TECHNOLOGIES, VALUE ADDITION AND UPSCALLING Upscaling of improved processing technology for isolation of hepatoprotective CLIV 92 from Cleome viscosa A novel processing technology was developed for isolation of Cliv-92 from the seeds of Cleome viscosa. The technology has now been upscaled to a level of 50 kg / batch size on the pilot plant. The novel processing technology is based on the use of solid matrix adsorption method followed by partitioning with different organic solvents. Upscaling and improvement of extraction technology for isolation of calliterpenone from Callicarpa macrophylla The process technology developed for the isolation of the molecules has now been upscaled to a level of 30 kg / batch. Optimisation of the process parameters like extracting solvent, temperature of extraction, and downstream techniques have been optimized, resulting in many fold increase in the yield of the product. One of the major improvements in the upscaled technology is the application of membrane filtration technique for the concentration of the extract and removal of water. Improved process for the preparation of antimalarial drug artelinate (AL)

Central Institute of Medicinal and Aromatic Plants

Post harvest storage effect on essential content and composition of Salvia sclarea grown in Kumaon region of western Himalaya Storage of clarysage inflorescence under shade for 96 hours prior to distillation significantly reduced the essential oil recovery (from 0.30 –0.13%). Linalool was found to increase on storage (28.86% to 37.47%), while linalyl acetate initially increased (from 39.68% to 43.30%) otherwise it decreased on storage.

NEW ANALYTICAL METHODS High-performance thin-layer chromatographic method for quantitative analysis in Clerodendrum inerme A sensitive, selective, precise, and robust high-performance thin-layer chromatographic method has been developed and validated for analysis of two new sterols, 4-methyl-24 ß -ethyl-5-cholesta-14,25-dien-3 ß -ol and 24 ß -ethylcholesta-5,9(11),22E-trien-3 ß -ol, and a triterpene, betulinic acid, in Clerodendrum inerme extract. BRP-HPLC method for the quantitation of glabridin in “Yashti-madhu” (Glycyrrhiza glabra) A reverse phase high performance liquid chromatographic method has been developed for the quantitative estimation of glabridin in Glycyrrhiza glabra, using C18 column with acetonitrile-water containing 2% AcOH (70:30) as an eluent.

Two improved processes for the preparation of artelinate have been developed. In the first process reduction, alkylation and hydrolysis steps were carried out in one pot followed by work up and recrytallization to produce pure AL with 81-88% yield in 10-12 hours. In the improved (second) process, removal of unwanted, undissolved reaction produced after completion of reduction and alkylation steps improved the hydroysis step and produce much cleaner product (AL) which upon direct recrystallization, discarding the use of column chromatography, produced pure AL with 95-98% yield.

To avoid unnecessary time delays and at the same time to achieve better sensitivity, DEPT-Q type experiment was standardized and brought into routine use. A typical DEPT-Q spectrum reveals all four types of carbon resonances with primary and tertiary on one phase and rest on the other phase.

Improved process technology for dihydroartemisinin

Quality-Marker development in Gloriosa superba and Coleus forskohlii grown in South India

An improved process technology and know-how for the conversion of artemisinin to DHA has been developed. It reduces process steps of distillation of solvents, extraction, recrystallization, thus making the process economic, greener, less time consuming and having higher overall yield of DHA, 95% w/w, with 97 % purity.

Extraction of the marker compounds from the herb Coleus forskohlii was done at 2 kg level. The marker compound, Forskolin, was isolated in pure form (>95% purity by HPLC) from Coleus forskohlii roots by column chromatography and used as reference standard. Structure confirmation of the compound was done by NMR spectral studies. HPLC Quality control method was developed and standardized for the analysis of forskolin.

Aloe vera processing technology CIMAP has developed an improved and economical technology with better process efficiency and superior quality for the processing of Aloe vera for aloe juice, filleted leaf sap, Aloe vera gel and Aloe vera spray dried powder. Post harvest storage method for rose-scented geranium (Pelargonium graveolens) herb under hill conditions of Uttarakhand

NMR method for small molecule structural studies

Adulterant of commercial 'Guggul' identified Gum-oleo-resin of guggul is usually adulterated with the allied species C. agallocha, which is medicinally not of much importance. Both the species and the commercial samples of guggul were pharmacognostically evaluated and diagnostic characters were identified in the present study to distinguish the authentic sample from its adulterant. Method for identification and quantification of polyisoprenylated benzophenones of Garcinia cambogia

Geranium biomass can be stored in polythene bags up to 48 hrs without losing the essential oil. It was observed that esters (formate, acetate, butyrate and tiglate of citronellol and geraniol), neral and 10-epi--eudesmol improved and ketones (menthone, isomenthone) and alcohols (linalool, nerol, geraniol) decreased on post harvest storage. It is also noted that citronellol to geraniol ratio (C: G) improved on storage in the polythene bags.

A sensitive liquid chromatography/electrospray ionization tandem mass spectrometrical (LC/ESI-MS/MS) method was developed for simultaneous identification and quantification of two polyisoprenylated benzophenones, isoxanthochymol and camboginol in the extracts of the fruit rinds, stem bark,seed and leaves of Garcinia indica and in the fruit rinds of Garcinia cambogia.

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Annual Report 2008 : Highlights

Separation and quantification of lignans in Phyllanthus species by a simple chiral densitometric method 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.

INTEGRATED NUTRIENT AND PEST MANAGEMENT

Central Institute of Medicinal and Aromatic Plants

plantlet production and herb yield and incidence of black spot disease on Aloe vera.Maximum plant height attained was 56 cm in Glomus intraradices treated plants followed by 53cm in Bacillus subtilis and B. subtilis + Glomus aggregatum treatments. Maximum plantlets production (14) was recorded in the treatments of B. subtilis + Trichoderma harzianum + G. aggregatum followed by 12 in others. Maximum herb yield obtained was 3.65 kg/plant in G. aggregatum treated pots, followed by 3.35 kg/plant in G. aggregatum + B. subtilis and Glomus intraradices treated pots. Integrated management of foliar diseases of sarpagandha in field

Influence of sources and doses of N and K on herbage, oil yield and nutrient uptake of patchouli in semi-arid tropical climate 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 status. The results revealed that application of 200 kg N ha-1 and 41.5 kg K ha-1 produced significantly higher patchouli herbage and oil yields compared with control. Influence of integrated nutrient management on yields, oil quality and soil fertility of rosemary 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. Qualitative performance of Java citronella (Cymbopogon winterianus) cultivars in Uttarakhand hills 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%). Influence of different VAM fungi on the root biomass yield and reserpine content of Sarpgandha (Rauvolfia serpentina) in unsterile soil 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. Effect of date of planting on the yield and disease incidence of Aloe vera 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. Influence of bioinoculants on the growth, yield and black spot disease of Aloe vera

Six applications of mancozeb @ 0.3% as foliar spray were found to be most effective giving 187.8 g fresh root/plant and 78.8% protection against foliar blight/spot (A. alternata, C. rauvolfia, C. serpentina, C. gloeosporioides, C. cassiicola, C. lunata, M. phaseolina and R. solani) diseases. Application of two doses of neemcake @ 50 g/plant at 30 days interval plus two sprays of B. subtilis in September and October along with three foliar spray of mancozeb at 15 days interval plus two foliar sprays of carbendazim @ 0.15% at 21 days interval provided, 201.5 g fresh root/plant, and 75.5% protection against foliar blight pathogens. Chemical activators: A novel and sustainable approach for management of Meloidogyne incognita in Chamomilla recutita Nine chemical activators namely, isonicotinamide, 2-chloronicotinic acid, 5-nitrosalicylic acid, 4-chlorosalcylic acid, DL-2 aminobutyric acid, 2-aminobutyric acid, O-acetylsalicylic acid, 4-aminosalicylic acid and salicylic acid 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, Oacetylsalicylic 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. Integrated pest management in mints 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. Effect of crop age on root and oil yield of vetiver 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. Leaf blight disease of Kalmegh (Andrographis paniculata), Senna (Cassia senna) and Aloe vera

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

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

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Annual Report 2008 : Highlights

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. Comparative germination behaviour of marigold (Tagetes spp) 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 the speed of germination. The germination period was mostly 10th day in all the accessions. Intercropping of Mentha arvensis with vetiver for higher returns 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.

Central Institute of Medicinal and Aromatic Plants

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. Effects of heavy metals (Chromium, Cadmium, Lead and Nickel) on the growth and yield of Mentha piperita 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. 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) 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.

BIO ACTIVITY AND DRUG MOLECULE PROSPECTION

Exploring companion cropping of MAPs in agroforestry and fruit orchards by shade tolerance evaluation 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.

PHYTOREMEDIATION AND SOIL QUALITY MANAGEMENT

Demethoxycurcumin: An antimycobacterial principle from turmeric (Curcuma longa) 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. Rosemary oil useful against drug resistant bacteria

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.

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.

Variability of soil enzyme activities in the rhizosphere of geranium, rosemary, Artemisia and unplanted crop

Bioprospecting for antimicrobials from plant sources

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.

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.

Japanese mint as potent crop for the effective utilization of heavy metal polluted soils

Organic matter as amendment to mitigate the toxic influence of chromium on kalmegh 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

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Effect of seasonal variation on antibacterial activity of Oenothera biennis roots 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.

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Annual Report 2008 : Highlights

Antimicrobial potential of Glycyrrhiza glabra roots 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 H37Ra and H37Rv strains at 29.16 µg/mL concentration. It also exhibited antimicrobial activity against both Grampositive and Gram-negative bacteria. These results indicate potential use of licorice as antitubercular agent. Synergism between extract of Phyllanthus amarus and silymarin for liver protection

Central Institute of Medicinal and Aromatic Plants

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. Cytotoxic activity of Gracinia indica 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.

In search of the effective and standardized hepatoprotective combination therapy, silymarin and standardized extract of Phyllanthus amarus were evaluated against CCl4 induced hepatotoxicity in rats. The combination of 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.

Cancer cell line dependent cytotoxic activity of essential oil as compared to pure compound

Isolation of bioactive phytomolecules from Bidens pilosa

Pre-clinical study at “Jeevanika” (Animal house)

The phytochemical investigation of hexane extract of Bidens pilosa leaf gave a bright orange compound ßcarotene, 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.

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.

Bioactive phytochemical leads as mosquitocidal agents 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. Gallic acid based steroidal chalcones as anticancer agents 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. Anticancer and hepatoprotective agent from Withania somnifera and biological evaluation of its ester derivatives 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.

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.

PHYTOCHEMICAL PROSPECTION A new chemotype of Origanum vulgare identified 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 . Essential oil constituents of Artemisia capillaris from Uttarakhand hills 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 β -pinene. Essential oil composition of flowers of geranium (Pelargonium graveolens) chemotypes from Uttarakhand hill

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

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%).

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Central Institute of Medicinal and Aromatic Plants

Variation in the chemical composition and yield of essential oil of rose scented geranium (Pelargonium species) by foliar application of metallic salts

Cloning of norcoclaurine synthase (ncs) gene involved in isoquinoline alkaloid biosynthesis in poppy (Papaver somniferum)

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.

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.

New thaiocarbamates from Moringa oleifera 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. Composition of North Indian pyrethrum (Chrysanthemum cinerariaefolium) flower oil Twenty two compounds representing 85.27% of the total oil were identified. Trans-chrysanthemumic acid (19.61%), decanoic acid (7.63%), nerolidol (9.63%), spathulenol (4.58%), guaiol (5.93%), â-eudesmol (9.81%) 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. Effect of altitude on the essential oil constituents of a new variety of Artemisia (A. roxburghiana var. purpurascens) 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 âcaryophylene (16.3-18.4%) followed by eugenol (16.2%)or á-thujone (12.0%).

PLANT GENOMICS & MOLECULAR BIOLOGY Heterologous expression of menthol biosynthetic pathway gene in bacteria 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. Cloning of cyp71AV1 gene from Artemisia annua (variety “CIM Arogya”)

An alpha-1 antitrypsin genetic variant identified in human liver biopsy sample 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. Polymorphism study in different chemotypes of ashwagandha (Withania somnifera) through AFLP 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 of leaf-specific gene dat in Catharanthus roseus cv. “Dhawal” 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. Cloning of genes involved in isoquinoline alkaloid biosynthesis in poppy Four new genes have been cloned from opium poppy cv “Sampada”. These genes are S-adenosyl-Lmethionine:norcoclaurine 6-O-methyltransferase (6omt) and S-adenosyl-L-methionine:3'-hydroxy-Nmethylcoclaurine 4'-O-methyltransferase (4'omt) in the pre-reticuline pathway and salutaridinol 7-Oacetyltransferase (salAT) codeinone reductase (cor) in the post-reticuline pathway. Novel cytochrome P450 monooxygenase genes (cyp) from Artemisia annua 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. 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 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 sequencecharacterized.

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.

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Studies on drug resistance development and mechanism in Staphylococcus aureus through molecular genetics approach

in 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.

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.

Establishment of hairy root cultures in Solanum nigrum

Role of efflux pump modulator in altering the activity of ATPase in Mycobacterium smegmatis 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. Bioconversion of a monoterpene into food flavoring agent vanillin 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). Standardization of cerebral malaria model in Mus musculus using markers of chemokine pathway 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. Modulation of inflammatory cytokines by Ocimum gratissimum under in-vitro conditions 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. Toxicity profile of Ocimum sanctum leaf at acute and sub-acute level in rat 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.

PLANT TISSUE CULTURE TECHNOLOGY Withanolide-A biosynthesis in in vitro shoot and root cultures of ashwagandha

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. Plantlet regeneration from hairy roots of Catharanthus roseus 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. Biogenesis of hypericine in multiple shoots cultures of Hypericum perforatum as a function of culture age and precursor feeding 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 th in 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. Micro-cloning of Aloe vera Using young offset as explants, a rapid in vitro micro-cloning method has been developed to produce diseasefree 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. Induction and development of somatic embryos of Chlorophytum borivilianum in liquid culture 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 kinetics studies in Agrobacterium rhizogenes mediated genetically transformed roots in Rauvolfia serpentina 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. Establishment and chemical profiling of hairy root culture of Rauvolfia serpentina

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

Agrobacterium rhizogenes (strain A4) mediated hairy root cultures have been induced in leaf explants of Rauvolfia serpentina at 81.25% relative transformation frequency. Most of the emerging roots of the present study tend to form calli in the half-strength, hormone-free, liquid basal MS medium during further subculturing.

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Chemical profiling of callus cultures of Taxus wallichiana

inhibitors involved and post-translational modification to its cloned commentary. Through user friendly GUI

Five C-14 oxygenated taxoids were characterized from the analysis of the callus cultures of the Taxus wallichiana which include the expression of three previously identified taxoids, namely—yunnanxane (1), 2á,5á,10â,14â-tetraacetoxy-4(20),11-taxadiene (2) and 2á,5á,10â-triacetoxy-14â-(2-methyl)butyryloxy-4(20),11-taxadiene (3) and two newly identified taxoids, i.e.2á,5á,10â-triacetoxy-14âpropionyloxy-4(20), 11-taxadiene(4) & 2á, 5á, 10â-triacetoxy-14â-isobutyryloxy-4(20),11-taxadiene (5). The confirmation of the structures of the identified taxoids was made through their accurate molecular formula determinations.

based database one can find out all the relevant information about menthol and biosynthetic pathways. Alkaloid biosynthetic pathway database (DbAlbioPath) 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,

Rapid and efficient organogenesis in Mentha piperita

physical properties, SMILES generation, industrial application and map viewer of medicinal plants.

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.

Bioinformatics based analysis of SSR's in ESTs corresponding to alkaloid, terpenoid and phenylpropanoid biosynthesis 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

BIOINFORMATICS/ IN SILICO BIOLOGY In silico insight into phytoremediation: Identification of metal binding motifs in medicinal plants 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. In silico identification of genes expressed in macrophage stage of Mycobacterium tuberculosis H37Rv strain 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). Menthol biosynthetic pathway database (MBPdb)

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 179 pentanucleotide reported in, Coptis japonica, Oryza sativa, Papaver somniferum, Catharanthus roseus, Phaseolus vulgaris, Capsicum annuum, Mentha piperita, Euphorbia tirucalli. Primers associated with the SSREST were also designed. Pattern analysis in CYP450 proteins of some medicinal plants Cytochrome p450 protein sequences were analyzed to find a conserved sequence in several medicinal plants (Mentha spicata, Ocimum basilicum, Stevia rebaudiana), and to find patterns involved in their active role. The pattern analysis reflects motifs, which were found to be associated with the function of heme-binding activity of Cytochrome p450 proteins, and were conserved in distant families of plant. The conserved motif sequences were found to be involved in binding pocket formation, which validate their role in functionality of Cytochrome p450. Terpene bio-synthetic pathway database (Db-Terpene) Information pertaining to the different aspects of terpenes viz; classification of terpenes (monoterpenes, diterpenes), physical properties of classified terpenes (for monoterpenes- linalol, nerol), categorization of

MBPdb database provides information of various enzymes participating in the pathway under different heads

plants, chemical substances (toxaphene, geniposide), functional genes involved (cypla, ESR2) can be accessed

e.g. EC number, pathway involved (related to KEGG database), reaction type, natural substrate, co-factors,

from this database.

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BASIC SCIENCE Effect of zinc and paclobutrazol on growth and pyrethrin quantity and quality of pyrethrum 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 jasmolin-2 concentration was obtained by 5 and 10 kg ha-1 zinc application, respectively. Effect of catechol treatment on post-harvested alkaloid content in leaves of Catharanthus 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. Changes in enzymes activities, proline, protein and IAA contents in relation to flower development in Rosa damascena Peroxidase, polyphenol oxidase, succinate dehydrogenase, amylase, catalase, proline content, protein content and IAA content were studied at different stages. The higest peroxidase activity was recorded during green bud stage and coloured bud stage coinciding with early flower development.

Central Institute of Medicinal and Aromatic Plants

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. Towards development of non-invasive vetiver – Vetiveria zizaniodes 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. Elicitation studies in post-harvested leaves of Catharanthus roseus 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.

Withanolide metabolism in ashwagandha (Withania somnifera)

Effect of gibberellin and ethereal and Fe nutrition in Catharanthus roseus tissue concentration and chemical composition

A high resolution DAD-ELSD-HPLC method of analysis of complex mixture of withanolides from leaves and roots of Withania somnifera has been developed and validated through application of various chromatogram quality parameters like peak resolution factor (Rs), asymmetry factor (As) and tailing factor (Tf), RSD (%) of retention time and peak area response, LOQ, LOD, precision, recovery etc.

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.

Standardization of germination parameters in medicinal and aromatic plants (MAPs)

Natural infection on periwinkle (Catharanthus roseus) by cucumber mosaic virus

A study was conducted to elucidate the germination parameters: germination percent (%), germination energy (%), germination period (days) and mean daily germination (%) in six major medicinal/aromatic plants: Tagetes minuta, T.erecta, T. patula, Papaver somniferum, Plantago ovata and Vetiveria zizanioides. The seed weight (test weight of 1000 seeds) in the six crops ranged from 0.447gm (in certain strain of T. minuta) to 1.784gm (in T. erecta). It was interesting to record that in each of the six crops increase in test weight (seed size) almost linearly enhanced the three seed germination parameters (germination percent, germination energy and mean daily germination). The results led to the conclusion that seed size has greater role in controlling the germination parameters in MAPs.

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.

Mevalonate and non-mevalonate pathways for isoprenoid compounds in MAPs

GENE BANK STATUS/UPDATES

Studies were conducted to follow the biosynthetic pathway of different isoprenoids like menthol, menthone and menthyl acetate (Mentha arvensis), artemisinin and artemisinic acid (Artemisia annua), patchouli alcohols and hydrocarbons (Pogostemon cablin), citronellol, citronellal and geraniol (Cymbopogon winterianus), Citraltrans and citral-cis (Cymbopogon flexuosus) etc. The most recent findings of non-mevalonate (DXP) pathway

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.

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PARTICIPATION IN AWARENESS EXHIBITIONS

• Memorandum of Understanding (MoU) for joint research project between CIMAP and University of Lucknow for studies on insect bioassay of plant extracts.

CIMAP participated in eleven exhibitions for showcasing CIMAP technologies, products, cultivars, seeds etc. The visitors appreciated the R&D efforts of CIMAP in providing technologies to the farmers at the rural level and to the entrepreneurs and industries at semi-urban and urban areas. Visitors took keen interest in CIMAP products and purchased CIMAP publications, products, seeds and propagules. The visitors were supplied with all information on agrotechnologies, processing, value addition and market potential of MAPs and were encouraged to visit CIMAP and its Resource Centres and participate in the training programmes conducted by CIMAP.

• Memorandum of Understanding (MoU) for joint research project with Ocimum Biosolutions, Hyderabad on development of cost effective Microarray chip with periwinkle as a model.

TECHNOLOGY TRANSFER/ CONSULTANCY AGREEMENTS/ CDA/ MoUS SIGNED Technology licensing • Technology transfer on cultivation and distillation of patchouli (Pogostemon cablin Benth) to M/s Chennai Bioflora Pvt. Ltd., Chennai. • Technology transfer for production of Jasmine concrete and absolute to M/s. Sandana Florals, Golden Jubilee Biotech Park for Woman society, Kanchipuram District (Tamil Nadu).

• 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. CNP projects undertaken • 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.

• Licensing of know how and design of improved field distillation unit and CIM-Asvika to M/s Swaraj Herbal Plants Pvt. Ltd., Barabanki, Uttar Pradesh.

• Design/ Drawings for CIMAP improved FDU to Mr Vijayan, Vishakhapatnam.

Pre – licensing agreements

• Survey of ash dyke area and preparation of a pre-feasibility report for NTPC, Unchahar, Raibareli.

• Confidentiality agreement between CIMAP and EMAMI for pre-licensing evaluation of CIMAP products/ formulations: Myconil, HAloe Skin, Toothache lotion, Tooth Powder, Mosnobite, Mosaway, CIM-Paushak, MosRep and, Herby Soft. • 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.

• Survey of land and preparation of a pre-feasibility report for Vaid Organics, Hardoi. • Crop consultancy in patchouli to Caps Ayur Krishi, Bangalore.

HUMAN RESOURCE DEVELOPMENT • 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.

• Confidentiality agreement between CIMAP and Laila Nutraceuticals, Vijayawada for pre-licensing evaluation of nutraceutical formulations and other leads of CIMAP.

• Five Ph.D. degrees were awarded to the staff and students of CIMAP.

• Confidentiality agreement signed with TVC SKY Shop, Mumbai for pre-licensing evaluation of CIMAP herbal formulations.

• More than 112 persons were trained at CIMAP, which included foreign scholars, students, industry representatives and State and Central Government officials.

Collaborations

• First JNU-CIMAP Ph.D. Programme started.

• Memorandum of Understanding (MoU) for joint research project between CIMAP and University of Mysore.

• 100 day CIMAP Certificate Course on Medicinal and Aromatic Plants (100 d CCC) held during 22nd February to 4th Junue, 2007.

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Annual Report 2008 : Highlights

Central Institute of Medicinal and Aromatic Plants

• 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 (AIATrd th 2007) held during 3 -16 September, 2007.

MAJOR FACILITIES • Industrial Technology Center • National Seed Quality Testing Facility at CIMAP • National Quality Referral Laboratory for MAPs and Herbal Products • Installation of Inductively Coupled Plasma: A Facility for Precise Estimation of Trace and Heavy Metals

• CSIR Programme for Youth for Leadership in Science (CPYLS) held during 15 -16 November, 2007.

• Digital Photos and Art Lab

• CIMAP Winter School (CWS-2007) on "Recent Techniques in Structural and Functional Genomics" held during 1st -10th December, 2007.

• Botanical Reference Substances (BRS)

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• CIMAP Winter Training (CWT-2008) on “Techniques and Tools of Biotechnology and Bioinformatics for Graduate and Master Students” held during 20th January-20th February, 2008. • A Unique "Know each other" meet of Scientists-Students and Teachers during CPYLS at CIMAP during 19th20th February, 2008.

IMPORTANT EVENTS

PERFORMANCE INDICATORS Entrepreneurship Training Programmes CIMAP organized Thirty two entrepreneurship training programmes in different parts of the country through which persons were provided technical knowledge and guidance for cultivation, processing, marketing etc. for quality production of medicinal and aromatic plants. In House Publications

• National Technology Day (11th 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 rural prosperity (5th – 11th 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. • CIMAP Day Function: MAPs show the way to adopt Integrative agriculture mode (Iam) (11 August, 2007) th

• fgUnh lIrkg ¼14 flrEcj] 2007½ % lheSi esa fgUnh fnol lekjksg & tu&tu rd igWqpk;s fgUnh esa foKku • lh-,l-vkbZ-vkj- LFkkiuk fnol % ^gfjr [ksrh ls izkS|ksfxdh iFk&LoLFk lalkj dh vksj* ¼26 flrEcj] 2007½ • MINT-2007: Interface on Mint (9 October, 2007) launched by Minister of State for commerce Sh. Jairam Ramesh, Government of India, Jointly organized by CIMAP, Lucknow and Spice Board, Cochin th

• jk"Vªh; oSKkfud laxks"Bh % ^ikni foKku }kjk LoLFk vkSj le`) lekt ds fy;s gfj;kyh dh [kkst* ¼29 uoEcj] 2007½% jktHkk"kk ds c<+rs

One MAP journal (one volume; four numbers), six farm bulletins, two popular magazines (Hindi & English), two information folders, two souvenirs, one-proceeding, one compilation of Institute Seminars (Gyanankur), Annual Report Highlights, Annual Report,one Planner and two issues of Newsletter were published this year. Plant varieties & Agrotechnologies Two plant varieties were released and two new agrotechnologies were developed. Sponsored Projects Thirteen sponsored projects completed during this financial year, thirty nine sponsored projects were progressing according to plan and Six R&D projects and seven consultancy projects were taken up. CIMAP has been working as nodal laboratory and participating laboratory in CSIR's network projects for mission mode research. Patents A total of seventeen patents (twelve foreign) were granted to the Institute this year. Publications 100 original research articles, 19 book chapters and 48 popular articles were published by S&T staff of CIMAP.

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In House Seminars

• fle&mRlo fdlku esyk 2008 ¼31 tuojh] 2008½ • National Science Day : Convergence of Science and Academia at CIMAP (28th February, 2008).

Forty three Seminar presentations and invited talks were made by the scientists of CIMAP.

• CIMAP Annual Day (26th March, 2008) : Exhibitions, field visit and lecture by eminent scientist.

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Annual Report 2008 : Highlights

External Cash Flow CIMAP earned an EBR of Rs 351.868 lakh, vis-à-vis CSIR grant of lab budget of Rs 1937.867 lakh and network programme budget of Rs. 112.124 lakh.

AWARDS & RECOGNITIONS • First Nina Saxena Excellence in Technology Award – 2007 to team CIMAP consisting of Dr.(s) SPS Khanuja, Shilpi Paul, Ajit K. Shasany, Anil K. Gupta, Mahendra P. Darokar, M.M. Gupta, RK Verma, Govind Ram, Anirudha Kumar, RK Lal, RP Bansal, Anil K. Singh, R.S. Bhakuni, Sudeep Tandon for development of complete technology package for the variety of Artemisia annua by IIT, Kharakpur in their Foundation Day Programme on 18th August, 2007. • Team CIMAP comprising of Vandana Srivastava, AS Negi, JK Kumar, MM Gupta, SPS Khanuja won the most cited paper 2003-2006 award by Bioorganic & Medicinal Chemistry journal for their paper entitled, “Plantbased anticancer molecules: A Chemical and biological profile of some important leads” published in Bioorganic and Medicinal Chemistry, Volume 13, Issue 21(2005), pages 5892-5908. • Team CIMAP comprising of Dr.(s) MM Gupta, GD Bagchi, Karuna Shankar, J Singh, SPS Khanuja received a letter of appreciation from Dr. Nityanand, Chairman of Indian Pharmacopeia Commission, Govt. of India for significant scientific contribution to IP-2007 by Team CIMAP on 11 August 2007 at CDHPC meeting of IPC at Mumbai. • CIMAP recognized as Designated National Repository (DNR) by National Biodiversity Authority (NBA). • Dr. RS Sangwan elected Fellow of the National Academy of Sciences, Allahabad. • Dr. DD Patra elected Fellow of the National Academy of Agricultural Sciences, New Delhi. • Dr. DD Patra Awarded ISCA Platinum Jubilee Award, 2008 conferred by Indian Science Congress Association, Kolkata • Dr Dwijendra Singh elected fellow of the Royal Entomological Society, London.

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