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SPICES BIOTECHNOLOGY Research at the Indian Institute of Spices Research K. V. Peter* and J. Rema Indian Institute of Spices Research, Calicut
The Indian Institute of Spices Research.
ndia, the land of spices is known to be a rich repository of spices and more than 100 species of spices are grown in about two million ha in the country. However, the productivity of many of these crops is considerably low due to various factors such as inadequate availability of high yielding varieties, absence of genotypes resistant to pests and diseases and absence of variability in many of the introduced crops like nutmeg (Myristica fragrans Houtt.) and clove (Syzygium aromaticum Merr. & Perry) besides labor intensive cultural operations. The Indian Institute of Spices Research (IISR) in Calicut, a constituent body of the Indian Council of Agricultural Research (ICAR) is
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a major institute devoted to research on spices. In 1976, it was started as a regional station of the Central Plantation Crops Research Institute (CPCRI), Kasaragod, engaged in research on spices. The National Research Centre for Spices was established in 1986 with its headquarters at Calicut and Cardamom Research Centre at Appangala, Karnataka. Realizing the importance of spices research in India, this research center was upgraded to the Indian Institute of Spices Research, on 1st July, 1995. The Indian Institute of Spices Research conducts and coordinates research on all aspects of spices
improvement, production, protection and post harvest technology of black pepper, cardamom, ginger, turmeric, cinnamon, clove, nutmeg, cambodge, kokam, allspice, vanilla, paprika and herbal spices. It has the mandate to conserve genetic resources of spices and spice agroecosystems; to develop high yielding and high quality spice varieties and to develop sustainable production and protection systems using traditional and non-traditional techniques and novel biotechnological approaches. It also has the mandate to develop post harvest technologies of spices with emphasis on product development and product diversification for domestic and export purposes; to cater to the needs of
*Director, Indian Institute of Spices Research.
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farming community and to serve as a national center for dissemination of technological information on spices. The institute has the world’s largest collection of spices germplasm consisting of 2778 black pepper, (Fig. 1) 293 cardamom, 499 ginger, 698 turmeric (Fig. 2), 465 nutmeg, 30 cambodge, 220 clove, 281 cinnamon, 137 allspice and 28 vanilla (Fig. 3) accessions. It has released four high yielding varieties of black pepper (Sreekara, Subhakara, Panchami and Pournami), one variety of cardamom (CCS-1), five varieties of turmeric (Suvarna, Suguna, Sudharshana, Prabha and Pratibha), one variety of ginger (Varada) and two cinnamon varieties (Navashree and Nithyashree). The rapid and efficient method for clonal multiplication of spices standardized here is being adopted for multiplication and distribution of spices. High production technologies developed at the institute for sustainable high yield of pepper and cardamom have been adopted by the farmers and are contributing to the overall increase in production of spices. Fertilizer dose for optimum yield has been worked out for many of the spice crops and the application of slow release fertilizers, and organic farming recommended for spices contribute to sustainable production of spices. The institute also gives emphasis to production of new and value added products. White pepper and salted ginger are products developed from spices at this institute.
Fig. 1. Black pepper.
Fig. 2. Turmeric.
Pest management Pests and diseases are major constraints in spices production. Techniques were developed for screening black pepper germplasm for reaction to Phytophthora capsici, the
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Fig. 3. Vanilla.
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causal agent of foot rot disease, a major disease of black pepper. Among the several lines screened, P-24 an open pollinated progeny is identified tolerant and productive and is under evaluation in farmer’s fields. An integrated management strategy involving phytosanitation, cultural practices and application of Bordeaux mixture (1%) and copper oxychloride (0.2%) or potassium phosphonate (Akomin) is effective against foot rot disease. An integrated disease management package against foot rot was standardized and is being implemented in over 86 000 ha in Kerala. Field trials showed effectiveness of Trichoderma hamatum and T. harzianum and Gliocladium virens to manage Phytophthora foot rot. VAM has phytotonic effect by increasing both establishment and growth substantially. Isolates of Glomus and Gigaspora were more efficient in promoting growth of black pepper and in suppressing infections by Phytophthora capsici and plant parasitic nematodes. Integrated Disease Management (IPM) involving phytosanitation, soil solarization, seed treatment and soil application of Trichoderma harzianum along with neem cake was highly effective in reducing rhizome rot and increasing ginger yield. An integrated strategy involving removal of affected plants, replanting with disease free planting material and control of vectors was developed for managing the ‘katte’ disease of cardamom. Management strategies developed by the institute against major pests of spices are very effective. Pollu beetle, the most serious pest of black pepper, could be controlled by spraying endosulfan (0.05%) or quinalphos (0.05%). The top shoot borer which damages tender terminal shoots of black pepper was identified as a
serious pest in younger plantations. The pest can be controlled by spraying with monocrotophos (0.05%). Leaf gall thrips and scale insects were identified as important pests of black pepper at higher altitudes and can be controlled by spraying monocrotophos (0.05%) or dimethoate (0.1%) respectively. The shoot borer was identified to be the most serious pest of ginger and turmeric and could be controlled by spraying malathion (0.05%). Besides, a number of potential biocontrol agents have been identified against major insect pests of spices. The institute has a well organized and reasonably equipped biotechnology facility and the facility concentrates on the improvement of production and productivity of spices through various biotechnological means.
increasing threat from disease epidemics, man’s interference with the environment, spread of a few high yielding varieties all lead to rapid erosion of gene pools and also make in situ and ex situ conservation methods insufficient and inadequate to conserve the germplasm. In vitro conservation attains significance in such a contest. Germplasm of black pepper, cardamom, ginger, turmeric and many herbal/minor spices are conserved by slow growth strategy (Babu et al., 1994) and cryopreservation techniques. At present the in vitro gene bank conserves about 434 accessions of different spices (Babu et al., 1996b).
Clonal multiplication
For preserving the nation’s spices wealth, survey, collection and conservation of germplasm of spices are given priority. In vitro conservation of germplasm is an alternative to in situ and ex situ genebanks for assembling the spices wealth. The
The gap between demand and supply of ‘elite’ planting materials of spices is on the increase in black pepper, vanilla and cardamom. Conventional techniques of multiplication alone are not sufficient to meet this widening gap. Micropropagation protocols have been standardized for 32 spices (Table 1) (Figs. 4 – 8) at this institute (Ravindran et al., 1996). These technologies could be taken up by commercial agencies for production of planting materials.
Fig. 4. Micropropagation of black pepper.
Fig. 5. Micropropagation of cardomom.
Conservation of spices
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Fig. 6. Micropropagation of vanilla.
Fig. 8. Micropropagation of Piper bekle.
Fig. 9. Piper barberi planted out in pots.
Conservation and propagation of endangered spices
Fig. 7. Micropropagation of camphor.
Piper barberi (Fig. 9), P. schmidtii, P. wightii and Vanilla aphylla are considered endangered. The micropropagation and conservation technology developed at the institute has enabled to conserve these species in in vitro genebank in addition to their large scale micropropagation. (Babu et al., 1996a).
Developing ‘synseed’ technology for germplasm exchange of spices The ‘synseed’ technology has been standardized in black pepper, camphor (Fig. 10), cinnamon and turmeric for the first time (Sajina et al., 1996). This technology helps in preserving germplasm and for their long distant transport saving space, time and cost.
Fig. 10. Synthetic seeds of camphor.
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Table 1. Crops for which micropropagation protocols are available. Herbal spices Apium graveolens Lavendula angustifolia Marjorana hortensis Mentha piperita Origanum vulgare M. spicata Origanum vulgare Ocimum basillum Ocimum sanctum Petroselinum crispum Pimpinella anisum Salvia officinalis Thymus vulgaris
Piper species Piper nigrum P. longum P. chaba P. betles P. colubrinum P. barberi
Zingiberaceous species Ammomum subulatum Curcuma longa C. aromatica C. amada Elettaria cardamomum Kaempferia galanga Kaempferia rotunda Zingiber officinale
Tree spices Cinnamomum verum C. camphora
Seed spices Carum carvi Anethum graveolens Foeniculum vulgare
Orchid Vanilla planifolia
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Protocols for regeneration of spices from callus In order to create and exploit the somaclonal variability and for the production of transgenic spices, regeneration protocols from calli of different explants of black pepper, ginger, cinnamon (Fig. 11) and vanilla, have been standardized. Regeneration of whole plantlets from callus is important for production of transgenic plants of spices. Transgenic lines may be the only answer to produce disease resistant varieties of black pepper, ginger, cardamom etc., as the conventional techniques are not very effective due to lack of exploitable genetic variability. In ginger, ten escapes were identified as promising when the somaclones were screened against Pythium aphanidermatum, (Babu et al., 1995), the causal organism of dreaded soft rot. These lines are also promising in yield. Callus regeneration protocols to develop somaclones were developed in 15 spices namely, black pepper, Piper barberi, cinnamon, Indian long pepper, cardamom, sage, Java long pepper, ginger, lavender, betel vine, turmeric, fennel, Piper colubrinum, vanilla, and anise. These efficient plant regeneration systems are highly suitable for generation of variability through somaclonal variation and as a pre-requisite for any further genetic engineering experiments.
Fig. 11. Somatic enbryogenesis in cinnamon.
Suspension cultures were established in nutmeg, ginger, cinnamon, lavender, camphor, sage, clove, fennel, allspice, dill, coriander and anise. These plant cells cultured in vitro could be utilized for production of primary and secondary metabolites of economic value. Protocols once standardised for scaling up of these metabolites would have tremendous industrial application.
tional breeding and selection rather slow in many spices. Recent advances made in developing techniques for transfer of foreign DNA into plant cells have aroused much interest in the possibility of utilizing recombinant DNA technology in crop improvement. Among the more important and frequently used techniques of gene transfer are Agrobacterium or viral vector-based
Isolation of protoplasts
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Protoplasts were successfully isolated from cardamom, black pepper, P. colubrinum (resistant to Phytophthora capsici), ginger, capsicum and vanilla, as a prerequisite for genetic manipulation studies. The protoplasts of P. colubrinum were successfully regenerated to plantlets.
Anther culture Production of secondary metabolites in culture Spices are ideal group of crops for production of secondary metabolites in culture. At the institute, in vitro proliferation of mace (the spice from nutmeg rich in myristicin) was successfully standardized. (Babu et al., 1992).
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Plants were successfully regenerated from anther derived callus cultures of diploid and triploid ginger (IISR, 1996). Anther culture forms a source for development of haploids.
In order to create and exploit the somaclonal variability and for the
production of transgenic spices, regeneration protocols from calli of different explants of black pepper, ginger, cinnamon and vanilla, have been
Future line of work Long juvenile phase coupled with long breeding cycle makes conven-
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standardized.
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transformation and the transformation by direct uptake of naked DNA. Of these, the Agrobacterium – mediated gene transfer is the most successful in plants especially in dicots. Though preliminary studies were carried out on Agrobacterium transfer system in P. nigrum and on transformation of ginger using biolistic process to study the optimum conditions for gene delivery and the efficiency of the plasmid vector pAHC 25 and promoter Ubi-1 (maize ubiquitin) for transformation and gene expression in ginger and cardamom embryogenic callus culture, the work has to be intensified in future programs. Work on DNA isolation from black pepper, vanilla and ginger and RAPD profiling in black pepper has been initiated. Use of molecular markers for genetic characterization of germplasm is given emphasis in future programs. Spices have rich secondary metabolites. In vitro production of secondary metabolites and their scaling up for industrial production is another area to be given emphasis in spices research. Biocontrol of diseases and pests of spices is receiving attention. Biotechnology research to manage Phytophthora diseases of spices and Ralstonia wilt is also gaining momentum.
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In vitro production of secondary metabolites
and their scaling up for industrial production is another area to be given emphasis in
References IISR (1996), Research Highlights, Indian Institute of Spices Research, Calicut. Babu, K., Geetha, S. P., Manjula, C., Ravindran, P. N. and Peter, K. V. (1994). Medium term conservation of cardamom germplasm — An in vitro approach. In: Proceedings of the Second Asia-Pacific Conference on Agricultural Biotechnology, p. 57. Babu, K., Rema, J., Sree Ranjini, D. P. Samsudeen, K. and Ravindran, P. N. (1996a). Micropropagation of an endangered species of Piper (P. barberi Gamble) and its conservation. Journal Plant Genetic Resources 9 (1): 179 – 182. Babu, K., Geetha, S. P., Manjula, C., Sajina, A., Minoo, D., Samsudeen, K., Ravindran, P. N. and Peter, K. V. (1996). Biotechnology – Its role in conservation of genetic resources of spices. In: Biotechnology for Development, Das, M. R. and Mundayoor Satish, eds (State Committee on Science Technology and Environment, Kerala) pp 198 – 212. Babu, K., Nair, R. R., George, J. K. and Ravindran, P. N. (1992). Piper barberi Gamble – A redescription of the species with a note on the karyotype. Journal Spices Aromatic Crops 1: 88 – 93. Babu, K., John Zachariah, T., Minoo, D., Samsudeen, K. and Ravindran, P. N. (1992). In vitro proliferation of nutmeg aril (mace) by tissue culture. Journal Spices Aromatic Crops 1: 142 – 147. Babu, K., Samsudeen, K and Ravindran, P. N. (1995). Biotechnological approaches for crop improvement in ginger, Zingiber officinale Rose. In: Proceedings of All India Symposium on Recent Advances in Biotechnological Applications on Plant Tissue and Cell Culture (Central Food Technological Research Institute, Mysore) p 11. Ravindran, P. N., Peter, K. V., Nirmal Babu, K., Rema, J., Samsudeen, K., Minoo, D., Geetha, S. P., Sajina, A., Mini, P. M., Manjula, C. and John, C. S. (1996). Biotechnological approaches in spice crops — present scenario and future prospects. In: Biotechnology for Development, Das, M. R. and Mundayoor Satish, eds. (State Committee on Science Technology and Environment, Kerala) pp 175-197. Sajina, A., Minoo, D., Geetha, S. P., Samsudeen, K., Rema, J., Nirmal Babu, K., Ravindran, P. N. and Peter, K. V. (1996). Production of synthetic seeds in spices. In: Proceedings of National Seminar on Biotechnology of Spices and Aromatic Crops (Calicut) p 9.
spices research.
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