J. Hort. Sci. Vol. 1 (1): 1-14, 2006 FOCUS Spices biotechnology K. V. Peter, K. Nirmal Babu' and D. Minoo' Kerala Agricultural University Thrissur, Kerala, India E-mail: kvptr@yahoo.com ABSTRACT In recent times, biotechnological tools have supplemented various conventional approaches in conservation, characterization, improvement and utilization for increasing production and productivity of spices. In many spices, viable micropropagation technologies are available for commercial production and generation of disease - free planting material. Somaclonal variation is important in crops where natural variability is low and a few useful somaclonal variants have been identified in ginger, turmeric and vanilla. Protoplast technology is also available for capsicum, black pepper, fennel, fenugreek, garlic, saffron and peppermint. In vitro cryopreservation, Synseed and Micro-rhizome technologies are available for safe propagation, conservation, movement, and exchange of spices germplasm. Studies are in progress for in vitro production of flavour and colouring compounds like capsaicin, vanillin, anethole, crocin, picrocrocin, saff'ranal, etc. using immobilized and transformed cell cultures. Use of molecular markers for crop profiling, fingerprinting, molecular taxonomy, identification of duplicate hybrids, estimation of genetic fidelity and tagging of genes for marker aided selection (MAS) is gaining importance. Isolation of important and useful genes and development of transgenics is in the preliminary stage. Key words: Spice crops, micropropagation, somaclonal variation, DNA fingerprinting, secondary metabolites INTRODUCTION Spices and herbs are aromatic plants, parts of which are used to flavour cuHnary preparations, in confectionery, and in medicines and perfumery. Spices and herbs are grown throughout the world; different plant species are grown in different regions. India is a rich repository of spices with over 100 species of herbs and spices being grown. Black pepper, cardamom, ginger, turmeric, vanilla, capsicum, cinnamon, clove, nutmeg, tamarind, pimenta, etc., constitute the major spices. Seed spices like coriander, cumin, fennel, fenugreek, dill, caraway, anise and herbal spices like saffron, lavender, thyme, oregano, celery, anise, sage and basil are also important. Crop improvement aims to increase productivity and quality of a target crop to meet increasing human demands. Lack of high yielding, pest and disease resistant varieties, and a limited genetic variability in some crops, is a major production constraint in spices. Use of biotechnological tool stands to play a major role in achieving the above through commercial propagation, development of novel varieties and new breeding lines via somaclonal variation, anther culture, protoplast fusion, bioreactor and recombinant DNA technologies for improving, conserving and utilizing the diversity and increasing the utility of spices. Micropropagation and Plant regeneration High and rapid rate of multiplication coupled with additional advantage of obtaining disease-free planting material makes micropropagation an important and viable alternative to conventional propagation. Black pepper and related species Methods for micropropagation of black pepper have been reported using various explants from both mature and juvenile tissues (Broome and Zimmerman, 1978; Lissamma Joseph et al, 1996). Phenolics and endogenous bacterial contaminants severely hamper establishment in black pepper cultures. Treating explants with fungicides prior to routine sterilization followed by frequent transfer to fresh medium, use of activated charcoal and antibiotics in culture media have been suggested for reducing phenolic interference and systemic contamination. Efficient plant regeneration protocols are essential for genetic manipulation of any crop species. Plants have been successfully regenerated from callus cultures of many Piper species. Plant regeneration 'Indian Institute of Spices Research, Marikunnu P.O., Kozhikode 673 012, Kerala, India mailto:kvptr@yahoo.com Peter et al was reported from shoot tip and leaf, with or without an intervening callus phase, (Bhat et al, 1995). Techniques for somatic embryogenesis in black pepper are reported by Nair and Gupta (2003). Cyclic somatic embryogenesis from maternal tissues like integuments has tremendous potential for automated micropropagation. These systems are useful for transgenic experiments for transfer of Phytophthora resistance. Methods for micropropagation of medicinally important species of piper viz., Piper longum P. chaba and P. betle have also been developed (Sarasan et al, 1993). Plants were regenerated from leaf and stem explants of related species of black pepper like Piper longum, P. betle, P. chaba, P. attenuatum and P. colubrinum through both direct and indirect organogenesis (Bhat et al, 1992; 1995). Somatic embryogenesis is also reported in betelvine (Johri et al, 1996). Cardamom Efficient and commercially viable technology for rapid clonal propagation of cardamom is available (Vatsya et a/, 1987). Many commercial laboratories use micropropagation techniques for large-scale production of clonal material. Successful high-frequency regeneration of plantlets from cardamom has been reported. Attempts on anther and microspore culture were reported to be inconsistent in plant regeneration from anther derived callus on MS medium. Ginger Clonal multiplication of ginger has been reported by many workers (Rout et al, 2001). Micropropagation helps in production of pathogen-free planting material in ginger where diseases often spread through infected seed rhizomes. Regeneration of plantlets through callus has been reported from leaf, vegetative bud, ovary and anther explants. Ginger fails to set fruit in nature. However, in vitro pollination could be effected to overcome prefertilization barriers to develop the 'fruit' and subsequently, plants could be recovered from these fruits (Valsala et al, 1997). Turmeric Technologies for micropropagation mrmeric of for production of disease-free planting material were developed (Nadgauda et al, 1978; Yasuda et al, 1988, Rahman et al, 2004; Prathanturarug etal, 2003,2005). Organogenesis and plant regeneration has been reported in turmeric by various workers (Shetty et al, 1982; Praveen et al, 2005). Renjith et al (2001) reported in vitro pollination and hybridization using two short duration types VK-70 and VK-76 and reported seed set and seed development. This reduces breeding time and helps in recombination breeding which had not been attempted in turmeric earlier. Other Zingiberaceous taxa Protocols for micropropagation of many economically and medicinally important zingiberaceous species like Amomum subulatum (large cardamom), Curcuma aromatica (kasturi turmeric), C. domestica var. 'Koova', C. aeruginosa, C. caesia , C. amada (mango ginger), Curcuma domestica [C. longa], C. zedoaria Kaempferia galanga, K. rotunda, Alpinia spp., Alpinia conchigera, Alpinia galanga, etc have been developed. (Barthakur and Bordoli, 1992; Chang and Criley, 1993). Yasuda (1988) reported successful callus induction from rhizomes. Prakash et al (2004), Lakshmi and Mythili (2003) and Rahman et al (2004) reported efficient plant regeneration through somatic embryogenesis from leaf base- derived callus of Kaempferia galanga L. Vanilla Micropropagation of vanilla has been standardized for large-scale multiplication of disease-free plants (Cervera and Madrigal, 1981; Kononowicz and Janick, 1984;Geetha and Shetty, 2000). In vitro germination of vanilla seeds and selection of useful genotypes from segregating progenies is also reported. This technique was also used to rescue interspecific hybrids between cultivated V. planifolia and wild V. aphylla through embryo rescue. In vitro propagation of Vanilla tahitiensis (Mary Mathew et al, 2000) and endangered species of Vanilla-V. wightiana , V. andamanica, V. aphylla and V̂ pilifera was also reported to save these species from extinction. Successful plant regeneration from shoot and seed derived callus was reported in vanilla (Davidonis and Knorr, 1991; Nirmal Babu et al, 1997). This efficient system can be used for creation and exploitation of somaclonal variation in this crop where the existing variation is limited. Tree spices Micropropagation protocols have been reported in many tree spices like cinnamon, nutmeg, cassia, clove, camphor, curry leaf, pomegranate, camboge and tamarind (ZhangandStoltz, 1981; Mascarenhas, etal 1987;Mathew andHariharan, 1990;Hazarikaera/, 1995; Mini efaf, 1997; Mallika, et al, 1997; Bhuyan et al, 1997; Huang et al, 1998; Nirmal Babu et al, 2000; Mehta et al, 2000). J. Hort. Sci. Vol. 1 (1): 1-14,2006 Spices biotechnology Plant regeneration through somatic embryogenesis has been reported in Cinnamomum verum and C. camphora. Induction of somatic embryogenesis from zygotic embryos oiSyzygium cumini and nutmeg was reported by Iyer et al,. (2000). Seed and herbal spices Micropropagation protocols for many seed and herbal spices are available. These include coriander, fennel, anise, peppermint, spearmint, celery, thyme, lavender, savory, ocimum, oregano, basil, sage, fennel, parsley, sweet marjoram, dill and garlic (Bhojwani, 1980; Ahuja, 1982; Miura, et al, 1987; Cellarova, 1992; Furmanowa and Ozszowska, 1992; Hunault and Du-Manoir, 1992; Panizza and Tognoni, 1992; Toth and Lacy, 1992; Patnaik and Chand, 1996; Vandemoortele et al, 1996; Sajina et al, 1997; Iyer and Pai, 1998). Plant regeneration has been successfully induced from callus cultures of peppermint, coriander, celery, cumin, fennel, lavender, anise, parsley, poppy, oregano, dill, caraway and sage (Ratnamba and Chopra, 1974; Sehgal, 1978; Chand and Roy, 1981; Jha et al, 1982; Ammirato,1983; Van Eck and Kitt, 1990;1992; Neena Kumari and Sarathy, 1992; Kataeva and Popowich, 1993; Onisei et al, 1994; Okamoto et al, 1994; Donovan et al, 1994; Hunault and Maatar, 1995; Kim et al, 1996; Sajina et al, 1997; Sastry et al, 1997). Propagation through somatic embryogenesis and in vitro flowering and seed set in coriander was reported by Stephan and Jayabalan (2001). In vitro flowering and seed formation in cumin has been reported. Bertaccini et al (2004) used micropropagation for elimination of mite- brone virus and for establishment of virus-free garlic (Allium sativum). Plant regeneration from anther and microspore cultures has been reported in fennel and celery. Capsicum Micropropagation and plant regeneration in chilli was reported using various explants (Agarwal 1988; Anu etal, 2004). Development of haploid capsicum through androgenesis is reported. New approaches for induction of pollen embryogenesis in Capsicum annuum were reported by Gonzalez et al (1996) and Regner (1996). Occurrence of unreduced gametes and ploidy restoration in haploid peppers {Capsicum annuum) was reported. Saffron Reports are available on micropropagation and plant regeneration in saffron. In vitro proliferation of saffron stigma was also reported (Homes et al, 1987; Ilahi et al, 1987; Yang era/, 1996). Field evaluation of tissue cultured plants Black pepper and related species Large-scale field evaluation of tissue cultured black pepper plants, in over 30 ha in all the pepper growing districts of Kerala, indicated that tissue cultured plants were superior to conventional propagules in field establishment, plant height, intemodal length, number of laterals per unit area, number of spikes per unit area, fruit set, mean yield, dry weight, oil content, oleoresin content, etc. Preliminary field performance of micropropagated plantlets of Piper longum, P. chaba and P. betle indicated that these were on par with conventionally propagated plants (Nirmal Babu et al, 2003). Cardamom Large-scale field evaluation of tissue cultured plants of cardamom was carried out by the Spices Board of India and the IISR. Results showed that micropropagated plants performed on par with suckers. Ginger and turmeric Field evaluation of tissue cultured plants of ginger and turmeric indicate that micropropagated plants require at least two crop seasons to develop rhizomes of normal size that can be used as seed rhizomes for commercial cultivation. Tissue cultured plants of kasturi turmeric, mango ginger, Kaempferia galanga, etc. also show a similar pattern. Salvi et al (2002) reported in turmeric that micropropagated plants showed significant increase in shoot length, number of tillers, number and length of leaves, number of gingers and total fresh rhizome weight per plant compared to conventionally propogated plants. Variations among regenerated plants have been reported in Kaempferia galanga. Anu et a/, (2004) reported variation among somaclones and their seedling progeny in Capsicum annuum. Estimation of genetic fidelity in micropropagated pepper using RAPDs Genetic fidelity of micropropagated plants of black pepper was confirmed by Nirmal Babu et al (2003). RAPD (Random Amplification of Polymorphic DN A) profiling and J. Hon. Sci. Vol. 1 (1): 1-14, 2006 Peter et al morphological characterization indicated that the micropropagation protocol can be used for commercial cloning of black pepper. Genetic uniformity of micropropagated Piper longum using RAPD profiling was reported by Ajith ef a/ (1997) and Parani ef a/(1997). In ginger, RAPD profiles did not show any polymorphism among micropropagated plants. However, Nirmal Babu et al (2003) reported RAPD profile differences in micropropagated ginger. Salvi et al (2001) reported that RAPD analysis of regenerated plants in turmeric showed variation. Genetic stability and uniformity of Foeniculum vulgare Mill, plants regenerated through organogenesis and somatic embryogenesis was reported by Bennici et al ( 2004 ). Somaclonal variation Induction and utilization of somaclonal variation was attempted in many spices to develop genotypes resistant to biotic and a biotic stresses. In black pepper, a few Phytophthora foot rot tolerant somaclones were identified through in vitro selection of calli using crude culture filtrate and toxic metabolites isolated from Phytophthora capsici. Attempts to induce somaclonal variation in cardamom resulted in identification of a few Katte virus tolerant somaclones (Nirmal babu et al, 1997). In ginger, field evaluation of somaclones indicated variability and resulted in identification of a few promising, high yielding lines with tolerance to rhizome rot (Nirmal Babu et al, 1996; Nirmal Babu, 1997). RAPD characterisation of these somaclones also showed profile variations indicating genetic differences Isolation of Pythium-tolerant ginger by using culture filtrate as the selecting agent has also been reported. Variants with high curcumin content were isolated from tissue cultured plantlets of turmeric. Root rot disease tolerant clones of turmeric cv. Suguna were isolated using continuous in vitro selection technique against pure culture filtrate of Pythium graminicolum (Gayatri et al, 2005). Variation in essential oil composition of plants regenerated from protoplasts of peppermint was reported. Reports are also available on in vitro selection for salt tolerance in fenugreek, Trigonellafoenum-graecum; in vitro selection for resistance to Altemaria blight in cumin and drought tolerance in coriander through tissue culture has also been showed. Somaclonal variation and virus elimination for improvement of garlic has been reported. Ghosh et al (1997) reported generation of virus free plants by thermotherapy and meristem culture in garlic. MSU - SHK 5, a somaclonally derived Fusarium yellows resistant line in celery has been identified. Microrhizomes Microrhizomes form an important source of disease-free planting material in rhizomatous crops like ginger and turmeric and are ideally suited for germplasm exchange, transportation and conservation. In vitro induction of microrhizomes in ginger, turmeric and Kaempferia is reported by many workers (Bhat et al, 1994; Nirmal Babu, 1997; Raghu Rajan, 1997; Sunitibala et al, 2001; Nirmal Babu et al, 2003). Microrhizome derived plants had more tillers but the plant height was smaller. They gave fresh rhizome yield ranging from 100- 800 g per plant with an estimated yield of 10 kg per 3m^ bed. In vitro formed rhizomes were found to be genetically more stable compared to micropropagated plants (Nirmal Babu et al, 2003). Synthetic seeds Artificial or synthetic seeds can be an ideal system for low-cost plant movement, propagation, conservation and exchange of germplasm. Synthetic seeds were developed by encapsulationg in vitro developed small shoot buds in 3% calcium alginate in black pepper, cardamom, ginger, turmeric, camphor, cinnamon, celery, lavender and fennel. These synthetic seeds could be stored from 7 to 10 months in sterile water with over 80 % viability (Redenbaugh et al, 1986; Pratap 1992;Sharmaefa/, 1994). Protoplast culture The protoplast is a naked cell and absence of the cell wall makes a protoplast suitable for a variety of manipulations that are not normally possible with intact cells. Hence, protoplast is an important tool for parasexual modification of genetic content of cells. Successful isolation and culture of protoplasts was reported in P. nigrum and P. colubrinum (Shaji et al, 1998). Plant regeneration, however, was observed only in P. colubrinum. Protoplasts could be successfully isolated from in vitro grown leaf mesophyll tissues of cardamom, ginger and turmeric. These were cultured upto the microcalli stage (Nirmal Babu, 1997; Geetha et al, 2000). Isolation and fusion of protoplasts in vanilla is reported. Isolation of protoplasts from leaves of nutmeg has been reported by Iyer et al (2000). J. Hort. Sci. Vol. 1 (1): 1-14, 2006 Sf)ices biotechnology Successful isolation and culture of protoplasts was reported in fennel (Miura and Tabata, 1986), fenugreek (Sen and Gupta, 1979), peppermint and garlic (Ayabe etal, 1995) and saffron (Isa et al, 1990). Sub Sang Ki and Park (1995) reported protoplast fusion and culture in garlic. Succ^essful production of interspecific hybrids between peppermint and gingermint was reported by Sato et al (1996). Organogenesis and plant regeneration from isolated protoplasts have been demonstrated in chillies (Pari and Czako 1981; Agarwal, 1988; Prakash et al, 1997). Genetic transformation Preliminary reports are available on Agrobacterium - mediated gene transfer in P. nigrum (Sasi'kumar and Veluthambi, 1996). They obtained primary transfofmants for kanamycin resistance in cotyledons using Agrobacterium tumefaciens binary vector strtiins LB A 4404 and EHA 105. Sim et al (1998) reported Agrobacterium- mediated transfer of GUS gene to black pepper. Nirraal Babu et al (2005) reported Agrobacterium - mediated transformation of black pepper with the gene for osmotin, a PR (Pathogenesis related) protein known to induce resistance to Phytophthora. Preliminary experiments to standardize optimum conditions for gene delivery and efficiency of the plasmid vector pAHC25 and promoter Ubi-1 and transformation of cardamom using biolistic process resulted in transient expression of GUS gene in bombarded callus tissue. A few reports are available on Agrobact^rium- mediated genetic transformation of capsicum (Liu ^t al, 1990;Shivegowdae?a/, 2002). Regeneration of transgenic pepper plants resistant to TMV and CMV has been reported. Molecular characterization and development of mapping populations In recent times, there is increased emphasis on using molecular markers for characterization of genotypes for genetic fingerprinting, to identify and clone important genes, for marker assisted selection and in understanding inter-relationships at the molecular level. Black pepper In black pepper, molecular markers like RAPD, AFLP and ISSR were used for assessment of genefic variability to characterize important cultivars, varieties, related species to develop fingerprints and to study inter relationships (Pradeep Kumar et al, 2001). A mapping population was developed for preparation of the genetic map in black pepper (Nirmal Babu et al, 2003). Male parent-specific RAPD markers were used by Johnson et al (2005) to identify hybrids. Jaramillo and Manos (2001) used phylogenetic analysis of sequences of the Internal Transcribed Spacers (ITS) of nuclear ribosomal DNA based on a world wide sample of the genus Piper. In long pepper {Piper longum), Banerjee et al (1999) reported male sex associated RAPD markers. Genetic diversity among landraces of a dioecious Piper betle using molecular markers was reported by Anjali et al (2004). Cardamom Molecular techniques like RAPD, RFLP and ISSR polymorphism were used to characterize cardamom germplasm collections comprising important cultivars, varieties and related genera to develop fingerprints and to study inter-relationships. The study indicated no duplicates in the 100 lines characterized and that the Kerala and Kamataka populations were divergent as they formed two separate clusters in the phylogram. RAPD and ISSR profiling of 11 species representing 5 major, related tribes of cardamom indicated that Ammomum is closest to the cultivated cardamom (Nirmal babu et al, 2005). A protocol for isolation and molecular characterization of DNA from market samples of cardamom was standardized and can be used to identify different grades of commercial cardamom and to identify adulttrents if any (IISR Annual Report, 2004). Ginger RAPD profiling of various ginger cultivars and related species is in progress at the Indian Institute of Spices Research to study the inter- relationships and to identify core collections in the germplasm. Ninetysix accessions of ginger were analysed and interrelationships studied. Polymorphism detected is moderate to low in ginger. RAPD profiling of ginger somaclones and selected 'variants' among micropropagated, callus regenerated and microrhizome derived plants indicated differences in RAPD profiles. Phylogenetic analysis of the tribe Zingibereae (Zingiberaceae) was performed by Ngamriabsakul et al (2003) using nuclear ribosomal DNA (ITS 1,5.8S and ITS2) and chloroplast DNA. The study suggested that the tribe Zingibereae and the genus Curcuma are monophyletic. Kress et al (2002) studied phylogeny of the gingers (Zingiberaceae) using DNA sequences of the nuclear / Hort. Sci. Vol. 1 (1): 1-14,2006 Peter et al internal transcribed spacer (ITS) and plastid matK regions and proposed a new classification of the Zingiberaceae. Turmeric Sasaki et al (2004) used single nucleotide polymorphism (SNP) analysis of the tmK gene to identify Curcuma plants. Sasikumar et al (unpublished) studied over 96 Indian cultivars and related species of turmeric using RAPD profiling for establishing interrelationship. RAPD analyses showed good polymorphism among the 96 accessions studied. Five species of Curcuma were characterized using 12 primers. Intra species polymorphism in {curcuma was high compared to the interspecies polymorphism (IISR 2003, 2004). An efficient protocol for isolation of high molecular weight DNA from dried. Powder samples of turmeric, including market samples, is described by Remya et al (2004). This will help in PCR-based detection of adulteration in marketed turmeric powder. Cao et al (2001) and Sasaki et al (2(X)2) used sequence analysis of Chinese and Japanese Curcuma drugs on the 18S rRNA gene and tmK gene and the application of amplification-refractory mutation system analysis for authentication. Vanilla In the absence of classical phenotypic markers in perennial crops like vanilla, molecular markers such as RAPD and AFLP were used to establish genetic similarities and interrelationships in cultivars, seed progenies, somaclones and interspecific hybrids. Isoenzyme, RAPD and AFLP polymorphisms, supplemented by morphological characters, have been used to study the existing variability in cultivated vanilla, species interrelationships, identification of interspecific hybrids, and, fingerprinting of important genotypes. The study indicated limited variability among the cultivated collections of V̂ planifolia grown in India. Vanilla tahitensis was found to be closest to V. planifolia. Significant variations exist among selfed seed progenies of V. planifolia. This variation was further magnified when plant regeneration was through callus or when explants were grown in colchicine containing medium. Progeny obtained from crosses between V. planifolia and V. aphylla is truly hybrid, and thus, in vitro technology can be used for generation of variability in crop improvement (Minoo et al, 2006). In tree spices, Shibu et al (2000) identified sex - specific DNA markers for identifying female trees in nutmeg. Yapwattanaphun et al (2004) used ITS sequence data to elucidate phylogenetic relationship in mangosteen {Garcinia mangostana) and its wild relatives {Garcinia spp.). Molecular characterization and preparation of molecular maps has been done in Capsicum. Amedo-Andres et al (2002) developed RAPD and SCAR markers linked to the Pvr4 locus for resistance to PVY in capsicum. Blum et al (2002) reported mapping of the locus for pungency in Capsicum. Kang et al (2001) developed interspecific {Capsicum annuum x C. Chinese) F2 linkage map in pepper using RFLP and RAPD markers. Caranta et al (1999) developed CAPS marker for the Pvr 4 locus for pyramiding potyvirus resistance genes in pepper. Isolation of candidate genes Work on isolation of genes responsible for agronomically important characters, especially for biotic and a biotic stresses, has also been attempted in spices. In black pepper, progranunes on isolation, cloning of genes and validation is in progress and a few putative, genomic and cDNA fragments associated with resistance genes have been isolated (IISR 2004, 2005; Johnson et al, 2005). Molecular cloning of a cDNA fragment encoding the defense related protein a-l,3-glucanase in black pepper {P. nigrum L.) and methyl glutaryl CoA reductase in Piper colubrinum has been reported. Bhat et al (2(X)5) reported isolation and sequencing of CMV coat protein gene with reference to black pepper. Chen et al (2005) reported cDNA cloning and characterization of a mannose-binding lectin from Zingiber officinale Roscoe (ginger) rhizomes. Molecular cloning of mannose-6-phosphate reductase and its developmental expression in celery was studied by Everard et al (1997). Wang and Kumar (2004) reported that heterologous expression of Arabidopsis ERS1 causes delayed senescence in coriander. Huh et al (2001) utilized the candidate gene approach to identify phytoene synthase as the locus for mature fruit color in red pepper {Capsicum spp). Tai and Staskawicz (2000) constructed yeast artificial chromosome (YAC) library of hot pepper {Capsicum annuum L.) and identified clones from the Bs2 resistance locus. Bai et al (2004) reported successful cloning and expression of Crocus sativus phytoene desaturase gene and preparation of antiserum. Tsaftaris et al (2004) reported / Hort. Sci. Vol. 1 (1): 1-14,2006 Spices biotechnology isolation of three homologous API-like MADS-box genes in Crocus sativus L. and characterized their expression. Conservation of genetic resources In vitro conservation Genetic resources of most spices are conserved either in seed gene banks or in field repositories. Storage of germplasm in seed banks is not practical in some crops as these are vegetatively propagated and seeds are either recalcitrant or heterozygous. Conservation of germplasm in in vitro gene banks and cryobanks is a viable and safe alternative. Conservation of pepper, cardamom, ginger, turmeric, vanilla, seed and herbal spice germplasm in vitro in gene banks by slow growth was reported (Dekkers et al, 1991; Nirmal Babu et al, 1996, 1997,1999). Conserved material of all the species developed into normal plants without any deformities and was morphologically similar to the mother plants. RAPD profiling of the conserved plants too showed genetic integrity. Suspensions of embryogenic cell lines of fennel, conserved at 4 °C for upto 12 weeks, produced normal plants upon transfer to normal laboratory conditions (Umetsu et al, 1995). Conservation of genetic resources in in vitro gene banks is now an established convention and gene banks for conservation of spice germplasm functions at the IISR and at the National Bureau of Plant Genetic Resources, New Delhi. About 500 accessions of spice germplasm are currently conserved the in vitro repository of IISR. Cryopreservation Cryopreservation of black pepper and cardamom seeds in liquid nitrogen (LN^) has been reported. Plants could be successfully regenerated from cryopreserved seeds of capsicum and anise, and, technologies for cryopreservation of black pepper, cardamom, ginger, turmeric and vanilla germplasm - using vitrification and encapsulation methods is available. Choudhary and Chandel, (1995); eported cryopreservation of vanilla pollen for conservation of the haploid genome and for assisted pollination between species that flower during different seasons and successful fertilization was effected using cryopreserved pollen. Production of secondary metabolites Use of biotechnology for biosynthesis of secondary metabolites particularly in plants of pharmaceutical significance holds an interesting alternative to conventional production of plant constituents. In vitro proliferation of the stigma of saffron, Crocus sativus and chemical analysis of metabolites produced through tissue cultures has been reported by Himeno et al (1988); Koyama et al (1987). In vitro metabolite production from saffron tissue cultures has also been demonstrated by Venkataraman et al (1989) and Vishwanath era/(1990). Production of flavour components and secondary metabolites in vitro using immobilised cells is an ideal system for spice crops. Production of saffron and capsaicin was reported using cell cultures (Johnson et al, 1996). Reports on in vitro synthesis of crocin, picrocrocin and saffranal from saffron stigma (Himeno and Sano, 1995) and colour components from cells derived from pistils (Hori et al, 1988) are available for further scale up. Johnson et al (1996) reported biotransformation of ferulic acid vanillamine to capsaicin and vanillin in immobilised cell cultures of Capsicum frutescens. Callus and cell cultures have been established in nutmeg, clove, camphor, ginger, lavender, mint, thyme, celery, etc. Cell immobilization techniques have been standardized in ginger, sage, anise and lavender (Ilahi and Jabeen, 1992). Production of essential oils from cell cultures (Ernst, 1989) and accumulation of essential oils by Agrobacterium tumefaciens transformed shoot cultures of Pimpinella anisum has been reported (Salem and Charlwood, 1995). Regulation of the shikimate pathway in suspension culture cells of parsley (Conn and McCue, 1994) and production of anethole from cell cultures of Foeniculum vulgare (Hunault et al, 1989) is also reported. Growth of shoot cultures and production of monoterpene by transformed shoots of Mentha citrata and Mentha piperita in flasks and fermentors was reported. Chavez et al (1996) reported biosynthesis of the sesqiterpene phytoalexin capsidol in elicited root cultures of chilli pepper. Production of rosmarinic acid in suspension cultures of Salvia officinalis has been discussed by Hippolyte et al (1992). Phenyl propanoid metabolism in suspension cultures of Vanilla planifolia was studied by Funk and Brodelius (1990 a, b). Reports on production of phenolic flavour compounds using cultured cells and tissues of vanilla are also available (Dorenburg and Knorr, 1996). In vitro production of petroselinic acid was reported from cell suspension cultures of coriander (Kim et al, 1996). Kintzios et al (2004) reported scaling up of micropropagation in Ocimum basilicum L. in an airlift bioreactor and accumulation of rosmarinic acid thereof. Though the feasibility of in vitro production of spice J. Hon. Sci. Vol. 1 (1): 1-14,2006 Peter et al principles has been demonstrated, methodology for scaling up and reproducibility needs to be developed before it can reach commercial levels. Once standardized, this technology can have tremendous potential in industrial production of important compounds like capsaicin, vanillin, crocin, picrocrocin, saffranal, myristicin, anethole, menthol and curcumin. Micropropagation technology is available for rapid cloning of many spices. Technology for conservation of genetic resources in in vitro gene banks is another useful development. Molecular characterization of germplasm has made reasonable progress. Identifying markers for important agronomic characters will help in marker assisted selection to shorten breeding time. Application of recombinant DNA technology for production of transgenics resistant to biotic and abiotic stress has a long way to go in spices improvement. Although programmes have been initiated in many laboratories on in vitro secondary metabolite production, these techniques need to be refined and scaled up for possible industrial application. Considering commercial possibilities, intensification of biotechnological activities in spices is needed in the coming decades. REFERENCES Agarwal, S. 1988. Shoot tip culture of pepper and its micropropagation. Curr Sci., 57:1347 - 1348. Ahuja, A., Verma, M and Grewal S. 1982. Clonal propagation of Ocimum species through tissue culture. Ind. J. Exptl. Biol., 20:455-458. Ajith, A., Parani, M., Rio, C.S., Latha, R. and Balakrishna, R 1997. Micropropagation and genetic fidelity studies in Piper longum L. p. 94-97. In: Biotechnology of Spices, Medicinal & Aromatic Plants. Edison, S., K.V. Ramana, B. Sasikumar, K.N.Babu, S. J. Eapen. (eds.), Indian Society of Spices, Calicut, Kerala, India. Ammirato, P.V. 1983. The regulation of somatic embryo development in plant cell cultures : Suspension culture techniques and hormone requirements. Bio/Tech, 1:68-74. Anjali,V., Nikhil, K and Ranade, S.A. 2004. Genetic diversity amongst landraces of a dioecious vegetatively propagated plant, betelvine (Piper betle L.). /. Biosci., 29:319-328. Anu, A., Nirmal Babu, K and Peter, K.V. 2004. Variations among somaclones and its seedling progeny in Capsicum annuum. PI. Cell Tiss. Org. Cult., 76:261- 267. Amedo-Andres, M.S., Gil Ortega, R., Luis Arteaga,M and Hormaza, J.I. 2002. Development of RAPD and SCAR markers linked to the Pvr4 locus for resistance to PVY in pepper {Capsicum annuum L.). Theor. Appl. Genet., 105:1067-1074. Ayabe, M., Taniguchi, K and Sumi, S.I. 1995. Regeneration of whole plants from protoplasts isolated from tissue cultured shoot primordia of garlic {Allium sativum L.).Pl. Cell Rep., 15:17-21. Bai. J., Miao, C , Xu, Y., Tang, L., Wang, Z. T & Chen, F. 2004. Cloning, expression of Crocus sativus phytoene desaturase gene and preparation of antiserum against it. Wuhan University J. Natural Sci., 9:252-258. Banerjee, N.S., Manoj, P and Das, M.R. 1999. Male sex- associated RAPD markers in Piper longum L. Curr Sci., 77:693-695. Barthakur, M.P and Bordoloi, D.N. 1992. Micropropagation of Curcuma amada (Roxb.). J. Spices and Aromatic Crops, 1:154-156. Bennici, A., Anzidei, M & Vendramin, G.G. 2004. Genetic stability and uniformity of Foe«JCM/Mm vulgare Mill. regenerated plants through organogenesis and somatic embryogenesis. PI. Sci., 166:221-227. Bertaccini, A., Botti, S., TabaneUi, D., Dradi, G, Fogher, C , Previati, A., da Re, F., Nicola, S., Nowak, J and Vavrina, C.S. 2004. Micropropagation and establishment of mite-brone virus-free garlic {Allium sativum). Acta Hort. 631:201-206. Bhat, A.I., Haresh, P.S and Madhubala, R 2005. Sequencing of coat protein gene of an isolate of Cucumber Mosaic Virus infecting black pepper in Ind. J. PI. Biochem. Biotech., 14:37- 40. Bhat, S.R., Chandel, K.RS and Malik, S.K. 1995. Plant regeneration from various explants of cultivated Piper species. PI. Cell Rep., 14:398-402. Bhat, S.R., Chandel, K.S.P and Kacker, A. 1994. In vitro induction of rhizome in ginger Zingiber officinale Rose. Ind. J. Exptl. Biol., 32:340 - 344 Bhat, S.R., Kackar, A and Chandel, K.RS. 1992. Plant regeneration from callus cultures of Piper longum L. by organogenesis. PI. Cell Rep., 11:525-528. Bhojwani, S.S. 1980. In vitro propagation of garlic by shoot proliferation. Sci. Hort., 13:47-52. Bhuyan, A.K., Pattnaik, S and Chand, P.K. 1997. Micropropagation of curry leaf tree {Murraya koenigii (L.) Spreng.) by axillary proliferation using intact seedlings PI. Cell Rep., 16:779- 782. Blum, E., Liu, K., Mazourek, M., Yoo, E.Y., Jahn, M and Paran, I. 2002. Molecular mapping of the C locus J. Hort. Sci. Vol. 1 (1): 1-14,2006 8 Spices biotechnology for presence of pungency in Capsicum. Genome, 45:702-705. Broome, O.C and Zimmerman, R.N. 1978. In- vitro propagation of black pepper. Hortl. Sci., 43:151 -153. Buyukalaca, S and Mavituna, F. 1996. Somatic embryogenesis and plant regeneration of pepper in liquid media. PI. Cell, Tiss. Org. Cult., 46:227-237. Cao, H., Sasaki, Y., Fushimi, H., Komatsu, K and Cao, H. 2001. Molecular analysis of medicinally used Chinese and Japanese Curcuma based On 18S rRNA and tmK gene sequences. Bio. Pharmaceutical Bull., 24:1389-1394. Caranta, C , Thabuis, A and Palloix, A. 1999. Development of CAPS marker for the Pvr 4 locus: a tool for pyramiding potyvirus resistance genes in pepper. Genome, 42:1111-1116. Cellarova, E .1992. Micropropagation of Mentha. In Biotechnology in Agriculture and Forestry, Vol 19. High Tech and Micropropagation III (Ed. Y P S Bajaj) pp:262-275. Cervera, E and Madrigal, R. 1981. /n vitro propagation of Vanilla (Vanilla planifolia A.). Environ. Experi. Botany, 21:441. Chan, L.K and Thong, W.H. 2004. In vitro propagation of Zingiberaceae species with medicinal properties. /. PI. Biotech. 6:181-188. Chand, S and Roy, S.C. 1981. Induction of organogenesis in callus cultures of Nigella sativa L. Ann. Bot., 48:1-4. Chang, B.K.W and Criley, A. 1993. Clonal propagation of pink ginger in vitro. Hortl. Sci., 28:1203. Chavez Moctezume, M.P and Lozoya Gloria, E. 1996. Bio synthesis of sesquturpenic phytoalexin capsidol in elicited root cultures of chilli pepper. PI. Cell Rep., 15:360-367. Chen, Z.H., Kai, G.Y., Liu, X.J., Lin, J., Sun, X.F and Tang, K.X. 2005. cDNA cloning and characterization of a mannose-binding lectin from Zingiber officinale Roscoe (ginger) rhizomes. J. Biosci., 30:213-220 . Conn, E.E and McCue, K.F. 1994. Regulation of the shikimate pathway in suspension cultured cells of parsley (Petroselinum crispum L.) In Advances in Plant Biotechnology (Ryu D. D. Y. and Furusaki S. eds.) Elsevier Science, Netherlands pp : 95-102. Davidonis, G and Knorr, D. 1991. Callus formation and shoot regeneration in Vanilla planifolia. Food Biotech., 5:59 66. Dekkers, A.J., Rio, A.N and Ghosh, C.J. 1991. In vitro storage of multiple shoot culture of gingers at ambient temperatures of 24 29°C. Sci. Hortic, 47:157-167. Donovan, A., Collin, H.A., Isaac, S and Mortimer, A.M. 1994. Analysis of potential sources of variation in tissue culture derived celery plants. Ann. Appl. Bot., 124:383-398. Domenburg, H and Knorr, D. 1996. Production of phenolic flavour compounds with cultured cells and tissues of vanilla species. Food Biotech., 10:75-92. Ernst, D. 1989. Pimpinella anisum L. (Anise): Cell culture, somatic embryogenesis and production of anise oil. In Biotechnology in Agriculture and Forestry Vol 7- Medicinal and Aromatic Crops II (Y P S Bajaj Ed.), Springer-Verlag Berlin pp:381-397. Everard, J.D., Cantini, C , Grumet, R., Plummer, J and Loescher, W.H. 1997. Molecular clonning of mannose-6-phosphate reductase and its developmental expression in celery. PL Physiol., 113:1427. Pari, M and Czako, M. 1981. Relationships between position and morphogenic responses of pepper hypocotyl explants cultured in vitro. Scientia Horticulturae. 15:207. Funk, C and Brodelius, P.F. 1990a. Phenyl propanoid metabolism in suspension cultures of Vanilla planifolia Andr. II. Effects of precursor feeding and metabolic inhibitors. Plant Physio., 94:95-101. Funk, C and Brodelius, P.F. 1990b. Phenyl propanoid metabolism in suspension cultures of Vanilla planifolia Andr. Ill Conversion of 4- methoxycinnamic acids into 4 - hydroxybenzoic acids. Plant Physio., 94:102-108. Furmanowa, M and Olszowska, 0.1992. Micropropagation of Thyme {Thymus vulgaris L.) pp : 230-242 In Biotechnology in Agriculture and Forestry Vol.19 High-Tech and Micropropagation III (Y P S Bajaj Ed) Springer Verlag, Heildelberg. Gayatri, M.C., Roopa, D.V and Kavyashree, R. 2005. Selection of turmeric callus for tolerant to culture filtrate of Pythium graminicolum and regeneration of plants, PI. Cell, Tiss. Org. Cult., 83:33-40. Geetha, S and Shetty, S.A. 2000. In vitro propagation of Vanilla planifolia, a tropical orchid. Curr. Sci., 79:886-889. George, P.S and Ravishankar, G.A. 1997. In vitro multiplication of Vanilla planifolia using axillary bud explants. PI. Cell Rep., 16:490-494. Ghosh, D.K., Ahlawat, Y.S and Dutta, G.M. 1997. Production of virus free garlic plants by thermotherapy and meristem culture. Ind. J. Agril. Sci., 67:591-594. Gonzalez-Melendi, P., Testillano, P.S., Ahamadian, P., J. Hon. Sci. Vol. 1 (1): 1-14,2006 Peter et al Fadon, B and Risueno, M.C. 1996. New in situ approaches to study the induction of pollen embryogenesis in Capsicum annuum. European J. Cell Bio., 69-313-1,U. Hazarika, B.N., Nagaraju, V and Parthasarathy, V.A. 1995. Micropropagation of Murraya koenigii Spreng. Ann. PI. Physiol., 9:149-151. Himeno, H and Sano, K. 1995. Synthesis of crocin, picrocrocin and safranal by saffron stigma like structures proliferated in vitro. Agril. Bio. Chem.,. 51:2395-2400. Hippolyte, I., Marin, B., Baccou, J.C and Jonard, R. 1992. Growth and rosmarinic acid production in cell suspension cultures of Salvia officinalis L. PI. Cell Rep., 11:109-112. Homes, J., Legros, M and Laziul. 1987. In vitro multiplication of Crocus sativus L Acta Hort.. 212:675-676 Hori, H., Enomoto, K and Nakaya, H. 1988. Induction of callus from pistils of Crocus sativus L. and production of colour components in the callus. PI. Tiss. Cult. Lett., 5:72 -77. Huang, L.C., Huang, B.L and Murashige, T. 1998. A micropropagation protocol for Cinnamomum camphora. In Vitro Cell. Dev. Biol- Plant 34:141- 146. Huh, J.H., Kang, B.C., Nahm, S.H., Kim, S., Lee, M.H and Kim, B.D. 2 0 0 1 . A candidate gene approach identified phytoene synthase as the locus for mature fruit color in red pepper (Capsicum spp.). Theor. Appl. Genet., 102:524-530. Hunault, C , Desmarest, P and Du Manoir, J. 1989. Foeniculum vulgare Miller: Cell cultures, regeneration and the production of anethole. pp. 185 - 209. In Biotechnology and Forestry Vol 7. Medicinal and Aromatic Plants II (Y. P. S. Bajaj ed) Springer-Verlag, Heidelberg.. Hunault, G and Du Manoir, J. 1992. Micropropagation of fennel, pp: 199-216. In: Biotechnology in Agriculture and Forestry Vol.19 High-Tech and Micropropagation III (Y P S Bajaj Ed). Springer- Verlag, Heidelberg. Hunault, G and Maatar, A. 1995. Enhancement of somatic embryogenesis frequency by gibberellic acid in fennel. PI. Cell Tiss. Org. Cult., 41:171-176 IISR. 2003. Annual Report 2002-2003, Indian Institute of Spices Research, Calicut IISR. 2004. Annual Report 2003-2004, Indian Institute of Spices Research, Calicut IISR. 2005. Annual Report 2004-2005, Indian Institute of Spices Research, Calicut Ilahi, I. and Jabeen, M. 1992. Tissue culture studies for micropropagation and extraction of essential oils from Zingiber officinale Rose. Pak. J. Bot., 24:54-59. Ilahi, I., Jabeen, M and Firdous, N.C. 1987. Morphogenesis with saffron tissue culture. J. PI. Physiol., 128:221 - 223. Isa, T., Ogasawara, T and Kaneko, H. 1990. Regeneration of saffron protoplasts immobilised in Ca - Alginate beads. Jap. J. Breed., 40:153-159. Iyer, I.R., Jayaraman, C and Gopinath, P.M. 2000. Isolation of protoplasts from leaves of nutmeg Myristica fragrans Houtt. Biomedicine, 20:200-202. Iyer, I.R., Jayaraman, C , Gopinath, RM and Sita, GL. 2000. Direct somatic embryogenesis in zygotic embryos of nutmeg (Myristica fragrans Houtt.) Trop. Agri., 77:98-105. Iyer, P.V and Pai, J.S. 1998. Micropropagation of sweet marjoram (Marjorana hortensis Moench) J. Spices and Aromatic Crops, 7:47-49. Jaramillo, M.A and Manos, P.S. 2001. Phylogeny and patterns of floral diversity in the genus Piper (Piperaceae). Amer. J. Bot., 88:706-716. Jha, T.B., Roy, C and Mitra, GC. 1982. In vitro culture of Cuminum cyminum: regeneration of flowering shoots from calli of hypocotyl and leaf explants. PI. Cell Tiss. Org. Cult., 2:11-14. Johnson, GK., Ganga, G, Sandeep Varma, R., Sasikumar, B and Saji, K.V. 2005. Identification of hybrids in black pepper (Piper nigrum L.) using male parent- specific RAPD markers. Curr. Sci., 88:1-2. Johnson, T.S., Ravishanker, GA and Venkataraman, L.V. 1996. Biotransformation of ferulic acid vanillamine to capsacin and vanillin in immobilised cell cultures of Capsicum frutescens. PI. Cell. Tiss. Org. Cult., 44:117-123. Johri, J.K., Aminuddin and Aruna, P. 1996. Regeneration of betelvine (Piper betle L.) through somatic embryogenesis. Ind. J. Exptl. Biol, 34:83-85. Kado, C.I. 1991. Molecular mechanisms of crown gall tumorigenesis. Critical Rev. PL Set., 10:1-33. Kang, B.C., Nahm, S.H., Huh, J.H., Yoo, H.S., Yu, J.W., Lee, M.H and Kim, B.D. 2001. An interspecific (Capsicum annuum L. x C.chinese) F2 linkage map in pepper using ^FLP and RAPD markers. Theor Appl. Genet., 102:531-539. Kataeva, N.V and Popowich, E.A. 1993. Maturation and rejuvenation of Coriandrum sativum shoot clones during micropropagation. Plant Cell Tissue & Organ Culture, 34:141-148. J. Hort. Sci. Vol. 1 (1): 1-14,2006 10 Spices biotechnology Kim, S.W., Park, M.K and Liu, J.R. 1996b. High frequency plant regeneration via somatic embryogenesis in cell suspension cultures of coriander. Pi. Cell Rep., 15:751-754. Kintzios, S., Kollias, H., Straitouris, E and Makri. 2004. Scale-up micropropagation of sweet basil (Ocimum basilicum L.) in an airlift bioreactor and accumulation of rosmarinic acid. Biotech. Lett., 26:521-523. Kononowicz, H and Janick, J. 1984. In vitro propagation of Vanilla planifolia. HortScience. 19:58-59. Koyama, A., Ohmori, Y., Fujioka, H., Miyagawa, H., Yamasaki, K and Kondaa, H. 1987. Formation of stigma like structures and pigments in cultured tissues of Crocus sativus. Jap. J. Pharmacog. 41:226-229. Kress, W.J., Prince, L.M. and Williams, K.J. (2002) The phylogeny and a new classification of the gingers (Zingiberaceae): evidence from molecular data. Amer J. Bot., 89:1682-1696. Lakshmi, M and Mytiiili, S. 2003. Somatic embryogenesis and plant regeneration from callus cultures of Kaempferia galanga - a medicinal plant. J. Medicinal Aromatic PI. ScL, 25:947-951 Lissamma Joseph., Nazeem, P.A., Mini, S.T., Shaji Philip and Mini Balachandran. 1996. In vitro techniques for mass multiplication of black pepper (Piper nigrum L.) and ex vitro performance of the plantlets. J. Plantation Crops (Supplt) 24:511-516. Liu, W., Parrot, W.A., Hildebrand, D., Collins, G.B and Williams, E.G. 1990. Agrobacterium induced gall formation in bell pepper (C.annuum L.) and formation of shoot like structures expressing induced genes. PI. Cell Rep., 9:360-364. Madhusudhanan, K and Rahiman, B. A. 1997. In vitro response of Piper species on activated charcoal supplemented media, p. 16-19. In: Biotechnology of Spices, Medicinal and Aromatic Crops, Edison, S. Ramana, K. V., Sasikumar, B., Nirmal Babu, K. and Santhosh J. E. (eds.), Indian Society for Spices. Mallika, V.K., Rekha, K., Marymol, M., Manjula, M and Vikraman Nair, R. 1997. In vitro shoot initiation from explants of field grown trees of nutmeg (Myristica fragrans Houtt.). p.29-34. In: Biotechnology of Spices, Medicinal and Aromatic Crops. Edison S, Ramana K V, Sasikumar B, Nirmal Babu K and Santhosh J E (eds.), Indian Society for Spices. Mary, M., Rio, Y,S., Kuruvilla, K.M., Lakshmanan, R., Gilu, L.G., Madhusoodanan, K.J and Potty, S.N. 2000. Multiple shoot regeneration in kokum and camboge. J. Spices and Aromatic Crops, 9:151-152. Mary, M., Rio, Y.S., Gilu, L.G, Lakshmanan, R and Madhusoodanan, K.J. 2(X)0a. In vitro propagation of Vanilla tahitensis Moore. J. Spices and Aromatic Crops, 9:171-173. Mascarenhas, A.F., Nair, S., Kulkami, V.M., Agrawal, D.C., Khuspe, S.S and Mehta, U.J. 1987. Tamarind, p. 316- 330. In: Cell and Tissue Culture in Forestry, Vol.3 J M Bonga & D J Durzan, (eds.) Martinus Nijhoff, Dordecht. Mathew, M.K and Hariharan, M. 1990. In vitro multiple shoot formation in Syzygium aromaticum. Ann. Bot. 65:277 279. Mehta, U.J., Krishnamurthy, K.V and Hazra, S. 2000. Regeneration of plants via adventitious bud formation from mature zygotic embryo axis of tamarind (Tamarindus indica L.). Cum Sci., 78:1231-1234. Mini, P.M., John, C.Z., Samsudeen, K., Rema, J., Nirmal Babu, K and Ravindran, P.N. 1997. Micropropagation of Cinnamomum verum (Bercht & Presl), p.35-38. In. Biotechnology of Spices, Medicinal and Aromatic Crops. Edison S, Ramana K V, Sasikumar B, Nirmal Babu K and Santhosh J E (eds.), Indian Society for Spices. Minoo, D., Sajina, A., Nirmal Babu, K and Ravindran, P.N. 1997. Ovule culture of vanilla and its potential in crop improvement., p. 112-118. In: Biotechnology of Spices, Medicinal and Aromatic Plants, Edison, S., Ramana, K.V., Sasikumar, B., Nirmal Babu, K. and Santhosh, J. Eapen (eds.). Indian Society for Spices, Calicut, India. Miura, Y and Tabata, M. 1986. Direct somatic embryogenesis from protoplasts of Foeniculum vulgare. PI. Cell Rep., 5:310 -313. Miura, Y , Fukui, H and Tabata, M. 1987. Clonal propagation of chemically uniform fennel plants through somatic embryoids. Planta Medica, 53:92- 94. Nadgauda, R.S., Mascarenhas, A.F., Hendre, R.R and Jagannathan, V. 1978. Rapid clonal multiplication of turmeric Curcuma longa L. plants by tissue culture. Ind. J. Exptl. Biol., 16:120 122 . Nair, R.R and Gupta, S.D. 2003. Somatic embryogenesis and plant regeneration in black pepper {Piper nigrum L.): Direct somatic embryogenesis from tissues of germinating seeds and ontogeny of somatic embryos. J. Hort. Sci. and Biotech., 78:416-421. Neena Kumari and Saradhi, P.P. 1992. Regeneration of plants from callus cultures of Oreganum vulgare L. PI. Cell Rep., 11:476-479. Ngamriabsakul, C , Newman, M.F. and Cronk, Q.C.B. / Hort. Sci. Vol. 1 (1): 1-14, 2006 11 Peter et al (2003) The phylogeny of tribe Zingibereae (Zingiberaceae) based on its (nrDNA) and trnl-f (cpDNA) sequences. Edinburgh J. Bot., 60:483-507. Nirmal Babu, K., Anu, A., Remasree, A.B and Praveen, K. 2000. Micropropagation of curry leaf tree Murraya koenigUL. (Spreng).PL CellTiss. Org. Cult,61:199- 203. Nirmal Babu, K., Geetha, S.P., Minoo, D., Ravindran, P.N and Peter, K.V. 1999. In vitro conservation of germplasm. pp : 106-129, In: Biotechnology and its application in Horticulture. S P Ghosh (ed). Narosa Publishing House, New Delhi. Nirmal Babu, K., Minoo, D., Geetha, S.P, Samsudeen, K., Rema, J., Ravindran, PN and Peter, K.V. 1998. Plant biotechnology - it's role in improvement of spices. Indian J. Agril. Sci., 68 (Special Issue.):533-547. Nirmal Babu, K., Ravindran, PN and Peter, K.V. 1997. Protocols for micropropagation of spices and aromatic crops. Indian Institute of Spices Research, Calicut, Kerala. Nirmal Babu, K., Rema, J., Sree Ranjini, D.P., Samsudeen, K and Ravindran, PN. 1996b. Micropropagation of an endangered species of Piper, P. barberi Gamble and its conservation. J. PI. Genet. Resources 9:179-182. Nirmal Babu, K., Sajina, A., Minoo, D., John, C.Z., Mini, P.M., Rema, J and Ravindran, P.N. 2003. Micropropagation of camphor tree Cinnamomum camphora (Presl.). PI. Cell Tiss. Org. Cult., 74:179-183. Nirmal Babu, K.,. George, J.K., Anandaraj, M., Venugopal, M.N., Nair, R.R 2005. Improvement of selected spices through Biotechnology tools - Black pepper, Cardfamom, Ginger, Vanilla. Final Report, Department of Biotechnology, Government of India. pp. 111. Nirmal Babu, K. 1997. In vitro studies in Zingiber officinale Rose. Ph.D Thesis. Calicut University, Kerala, India. Okamoto, A., Sakurazawa, H and Arikawa, K. 1994. Regeneration of plantlets from celery (Apium graveolens L.) callus using a fermentor. J. Fermentation and Bioengineering 77:208 -211. Onisei, T., Toth, E.T and Amariei, D. 1994. Somatic embryogenesis in lavender tissue culture I. Isolation and characteristics of an embryogenic callus line. J. Herbs, Spices and Medicinal Plants 2:17-29. Panizza, M and Tognoni, F. 1992. Micropropagation of lavender {Lavandula officinalis Chaix X Lavandula latifolia villars cv. Grosso). pp : 295-305. In: Biotechnology in Agriculture and Forestry. Vol.19. High-Tech and Micropropagation III. Y. P. S. Bajaj (ed.) Springer-Verlag, Heidelberg. Parani, M., Anand, A and Parida, A. 1997. Application of RAPD finger printing in selection of micropropagated plants of Piper longum for conservation . Cum Sci., 73:81-83. Patnaik, S and Chand, PK. 1996. In vitro propagation of medicinal herbs Ocimum americanum L. syn. O. canum Sims (hoary basil) and Ocimum santum L. (holy basil). PL Cell Rep., 15:846-851. Pradeep Kumar, T., Karihaloo, J.L and Archak. S. 2001. Molecular characterization of Piper nigrum cultivars using RAPD markers. Cum ScL, 8:246-248. Prakash, A.H., Rio, K.S and Kumar, M.U. 1997. Plant regeneration from protoplasts of Capsicum L. cv. California Wonder. J. Biosci., 22:339-344. Prakash, S., Elangomathavan, R., Seshadri, S., Kathiravan and Ignacimuthu, S. 2004. Efficient regeneration of Curcuma amada Roxb. plandets from rhizome and leaf sheath explants. PL Cell Tiss. Org. Cult., 78:159- 165. Pratap, K.R (1992) Artificial Seeds. Vatika. 1:27-30. Prathanturarug, S., Soonthomchareonnon, N., Chuakul, W., Phaidee, Y. and Sarakamp, P. 2005. Rapid micropropagation of Curcuma longa using bud explants pre-cultured in thidiazuron-supplemented liquid medium. PL Cell Tiss. Org. Cult., 80:347-351. Praveen, K. 2005. Variability in Somaclones of Turmeric (Curcuma^onga L.)Ph.D Thesis. Calicut University, Kerala, India. Rahman, M.M., Amin, M.N., Jahan, H.S and Ahmed, R. 2(X)4. In vitro regeneration of plantlets of Curcuma longa Linn: a valuable spice plant in Bangladesh. Asian J. PL Sci., 3:306-309. Rao, S.R and Ravishankar, G.A. 2000. Vanilla flavour: production by conventional and biotechnological routes. /. ScL FoodAgri., 80:289-304. Ratnamba, S.P and Chopra, R.N. 1974. In vitro induction of embryoids from hypocotyls and cotyledons of Anethum graveolens seedlings. Z Pflanzenphysiol. 73:452-455. Redenbaugh, K., Brian, D.P., James, W., Mary, E., Peter, R and Keith, A.W. 1986. Somatic seeds: Encapsulation of asexual plant embryos. Biotech., 4:9-83. Regner, F. 1996. Anther and microspore culture in capsicum In: VitroHaploid Production in Higher Plants, pp. 77- 89. Jain S M, Sopory S K and Veilleux R E (eds.) Vol. 3. Kluwer Academic Publishers, The Netherlands. Remya, R., Syamkumar, S. and Sasikumar, B. 2004 Isolation J. Hort. Sci. Vol. 1 (1): 1-14,2006 12 Spices biotechnology and amplification of DNA from turmeric powder . British Food J., 106:673-678. Renjith, D., Valsala, P.A. and Nybe, E.V. 2001. Response of turmeric (Curcuma domestica Val.) to in vivo and in vitro pollination. J. Spices and Aromatic Crops, 10:135-139. Rout, G.R., Palai, S.K., Samantaray, S and Das, R 2001. Effect of growth regulator and culture conditions on shoot multiplcation and rhizome formation in ginger {Zingiber officinale Rose.) in vitro. In Vitro Cell. Devtl. Biol. - Plant, 37:814-819. Sajina, A., Mini, RM., John, C.Z., Nirmal Babu, K., Ravindran, RN and Peter, K.V. 1997. A note on micropropagation of large cardamom. J. Spices and Aromatic Crops 6:145-148 Salem, K.M.S.A and Charlwood, B.V. 1995. Accumulation of essential oils by Agrobacterium tumefaciens transformed shoot cultures of Pimpinella anisum. PI. Cell Tiss. Org. Cult., 40:209-215. Salvi, N.D., George, L. and Eapen S. 2001 Plant regeneration from leaf base callus of turmeric and random amplification polymorhic DNA analysis of regenerated plants. PI. Cell Tiss. Org. Cult., 66:113- 119. Salvi, N.D., George, L. and Eapen, S. 2002 Micropropagation and field evaluation of micropropagated plants of turmeric. PI. Cell Tiss. Org. CM//., 68:143-151. Sano, K and Himeno, H. 1987. In vitro proliferation of saffron (Crocus sativus L.) stigma. PI. Cell Tiss. Org. Cult., 11:159-166. Sarasan, V., Elizabeth, T., Beena, L and Nair, GM. 1993. Plant regeneration in Piper longum L. (Piperaceae) through direct and indirect adventitious shoot development. J. Spices and Aromatic Crops 2:34 40. Sasaki, Y., Fushimi, H and Komatsu, K. 2004. Application of single-nucleotide polymorphism analysis of the tmK gene to the identification of Curcuma plants. Biological and Pharmaceutical Bulletin 27:144-146. Sasaki, Y, Fushimi, H., Cao, H., Cai, S.Q. and Komatsu, K. 2002 Sequence analysis of Chinese and Japanese Curcuma drugs on the 18S rRNA gene and tmK gene and the application of amplification-refractory mutation system analysis for their authentication. Biol. Pharm. Bull, 25:1593-9. Sasikumar, B and Veluthambi, K. 1996b. Transformation of black pepper (Piper nigrum L.) using Agrobacterium Ti plasmid based vectors. Ind. Perfumer, 40:13-16. Sastry, E.V.D., Sanjeev, A.,Rajendra, K and Sharma, R.K .1997. In vitro responses of fennel (Foeniculum vulgare Mill.), p. 49-50. In: Biotechnology of Spices, Medicinal and Aromatic Crops. Edison S, Ramana K V, Sasikumar B, Nirmal Babu, K. and Santhosh, J.(eds.) Indian Society for Spices. Sato, H., Yamada, K., Mii, M., Hosomi, K., Okuyama, S., Uzawa, M., Ishikawa, H and Ito, Y. 1996. Production of an interspecific somatic hybrid between peppermint and gingermint. PL Sci., (Limerick) 115:101-107. Sehgal, C.B. 1978. Differentiation of shoot buds and embryoids from inflorescence of Anethum graveolens in cultures. Phytomor, 28:291-297. Sen, B and Gupta, S. 1979. Differentiation in callus culture of leaf of two species of Trigonella. Physiologia Plantarum 45:425-428. Shaji, P., Anandaraj, M and Sharma, Y. R. 1998. Comparative study of protoplast isolation and development in Piper nigrum (black pepper) and P. colubrinum. pp. 51-53. In: Developments in plantation crops research, NM Mathew and CK Jacob(eds.), AUied Publishers, New Delhi. Sharma,T.R., Singh, B.M and Chauhan, R.S. 1994. Production of encapsulated buds of Zingiber officinale Rose. PI. Cell Rep., 13:300-302. Shibu, M.P., Ravishankar, K.V., Lalitha, A., Ganeshaiah, K.N and Shanker, R.U. 2000. Identification of sex specific DNA markers in the dioecious tree, nutmeg (Myristica fragrans Houtt.). PL Genet. Resources Newslett., 121:59-61. Shivegowda, S.T., Mythili, J.B., Saiprasad, G.V.S., Gowda, T.K.S., Anand, L and Gowda, R. 2002. In vitro regeneration and transformation in chilli pepper (Capcisum annuum L.). J. Hortl. Sci. Biotech., 77:629-634. Sim, S.L., Jafar, R., Power, J.B and Davey, M.R. 1998. Development of an Agrobacterium-mediated transformation system for black pepper (Piper nigrum L.). Acta Hort., 461:349-354. Stephan, R and Jayabalan, N. 2001. Propagation of Coriandrum sativum L. through somatic embryogenesis. Ind. J. Exptl. BioL, 39:387-389. Suh Sang Ki and Park, H. 1995. Protoplast isolation, fusion and culture of garlic (Allium sativum L.). J. Korean Soc. Hort. ScL, 36:614-619. Sunitibala, H., Damayanti, M and Sharma, G.J. 2001. In vitro propagation and rhizome formation in Curcuma longa Linn. Cytobios, 105:71-82. Tai, T and Staskawicz, B.J. 2000. Construction of yeast J. Hon. Sci. Vol. 1 (1): 1-14, 2006 13 Peter et al artificial chromosome library of pepper {Capsicum annuum L.). and identification of clones from the Bs2 resistance locus. Theor. Appl. Genet., 100:112- 117. Toth, K.F and Lacy, M.L. 1992. Micropropagation of celery (Apium graveolens var. dulce). pp:218-228. In: Biotechnology in Agriculture and Forestry Vol.19 High-Tech and Micropropagation III.Y. P. S.Bajaj(ed.) Springer - Verlag, Heidelberg. Tsaftaris, A.S., Pasenti, K., Iliopoulos, I and Polidoros, A.N. 2004. Isolation of three homologous API-like MADS-box genes in Crocus (Crocus sativus L.). and characterization of their expression. PI. Sci., 166:1235-1243. Umetsu, H., Wake, H., Saitoh, M., Yamaguchi, H and Shimomura, K. 1995. Characteristics of cold preserved embryogenic suspension cells in fennel Foeniculum vulgare Miller. J. Plant Physiol., 146:337-342. Valsala, P.A., Nair, S.G and Nazeem, P.A. 1997. In vitro seed set and seed development in ginger. Zingiber officinale Rose. p. 106-108. In: Biotechnology of Spices, Medicinal and Aromatic Plants, Edison, S., Ramana, K.V., Sasikumar, B., Nirmal Babu K and Santhosh J. Eapen (eds.j. Indian Society for Spices, Calicut, India, Van Eck, J.M and Kitto, S.L. 1990. Callus initiation and regeneration in Mentha. HortScience. 25:804-806. Van Eck, J.M and Kitto, S.L. 1992. Regeneration of peppermint and orangemint from leaf disc. PI. Cell Tiss. Org. Cult., 30:41-49. Vandemoortele, J.L., Billard, J.P., Boucaud, J and Caspar, T. 1996. Micropropagation of parsley through axillary shoot proliferation . PI. Cell Tiss. Org. Cult., 44:25-31. Vatsya, B., Dinesh, K., Kundapurkar, A.R and Bhaskaran, S. 1987. Large scale plant formation of cardamom {Elettaria cardamomum) by shoot bud cultures. PI. Physiol. Biochem., 14:14 -19. Venkataraman, L.V., Ravishanker, G.A., Sarma, K.S and Rajasekaran, T. 1989. In vitro metabolite production from saffron and capsicum by plant tissue and cell cultures, p. 147-151. In: Tissue Culture and Biotechnology of Medicinal and Aromatic plants Kukreja et al (eds.), CIMAP, Lucknow, India. Vishwanath, S., Ravishankar, GA and Venkataraman, L.V. 1990. Induction of crocin, crocetin, picrocrocin and safranal synthesis in callus cultures of saffron - Crocus sativus L. Biotech. Appl. Biochem., 12:336- 340. Wang, Y and Kumar, P.P. 2004. Heterologous expression of Arabidopsis ERSl causes delayed senescence in coriander. PI. Cell Rep., 22:678-683. Yapwattanaphun, C , Subhadrabandhu, S., Honsho, C , Yonemori, K., Chinawat, Y and Suranant, S. 2004. Phylogenetic relationship of mangosteen (Garcinia mangostana) and several wild relatives (Garcinia spp.) revealed by ITS sequence data. / . Amer. Soc. Hortl. Sci., 129:368-373. Yasuda, K., Tsuda, T., Shimiju, H and Sugaya, A. 1988. Multiplication of Curcuma sp. by tissue culture. Planta Medica 54:75-79. Zhang, B and Stoltz, L.P. 1981. In vitro shoot formation and elongation of dwarf pomegrante. Hort. Sci., 26:1084. J. Hort. Sci. Vol. 1 (1): 1-14,2006 14