1. INTRODUCTION. Cinnamon (Cinnamomum verum Bercht. and Presl) and Cassia (Cinnamomum

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1 1. INTRODUCTION Cinnamon (Cinnamomum verum Bercht. and Presl) and Cassia (Cinnamomum cassia (L.) Blume) barks are among the earliest known spices used by the human kind and were among the spices sought after by most of the fifteenth and sixteenth century European explorers (Dao et al., 1999). Frequent references to these spices are available in both pre-biblican and post-biblican writings. The true cinnamon or spice cinnamon is the dried inner bark of Cinnamomum verum. Cinnamomum cassia is known as Chinese cassia. They belong to the family Lauraceae. The term Cinnamomum is derived from the Hebraic and Arabic term kinamon means fragrant spice plant and verum means true. Cinnamomum verum is a native of Sri Lanka and Southern Western Ghats of South India mainly cultivated in Sri Lanka, Seychelles, Madagascar and India. The area under cultivation is estimated to be around 24,000 ha in Sri Lanka and 3,400 ha in the Seychelles producing around 12,000 t and 600 t respectively (Coppen, 1995). Sri Lanka produces more than 90 % genuine cinnamon of the world and is the largest producer of cinnamon accounting for about % of the global production with Seychelles, Madagascar, India and other suppliers collectively contributing the balance. The best quality cinnamon is produced in Negambo district of Sri Lanka. According to the report of Department of Export Agriculture, Sri Lanka, the main cinnamon producing areas are in the coastal belt, Galte (19647 ha), Matara (5477 ha), Rathnapura (3620 ha) and Hambantotoa (1985 ha), and the extent of

2 cinnamon plantations at the end of 2005 was ha. As per the 2005 data the total extent of cinnamon plantations increased by 819 ha during the last 5 years. Leaf and bark oil of cinnamon is obtained by distilling dried cinnamon leaf and bark. The cinnamon oil in world trade is produced from C. verum, C. Cassia and C. camphora. Chinese cassia (Cinnamomum cassia, Syn. C. aromaticum) is indigenous to Southern China and Vietnam. China is the main producer and the harvested area in 1998 was estimated by FAO at 35,000 ha with a production of 28,000 t. Cassia bark and leaf oil are economically important. Cassia buds, the dried unripe fruits, though rare, are also occasionally used as a spice. The leaf and bark oil of cinnamon finds wide scale application in medicine. The bark of C. verum and C. cassia is of great commercial importance due to its aromatic and sweet taste with a spicy fragrance; it also contains a large number of essential oils. A major constituent of cinnamon bark, trans-cinnamaldehyde (C 9 H 8 O) provides the distinctive odour and flavour associated with cinnamon. Oleoresins are the flavour extracts obtained by the solvent extraction of the ground spices. The bark and leaves of Cinnamomum verum and C. cassia are commonly used as spice in home kitchens and their distilled essential oils or synthetic analogues are used as flavouring agent in food and beverage industry. Leaf and bark oils are used in the manufacture of perfumes, soaps, tooth pastes, hair oils and face creams and also as an agent for flavouring liquor and dentifrices. They also find application in pharmaceutical industries. 2

3 According to Bureau of Economics and Statistics, New Delhi, the area under cinnamon and cassia cultivation is 878 ha during The import of cinnamon and cassia to India during was t (worth Rs lakhs) and 8600 t (worth Rs lakhs) respectively. The export of cinnamon and cassia from India during was t and t respectively. The export of cinnamon and cassia in powder form was t and t respectively. The export of cinnamon and cassia oil was 2.4 t and 0.05 t respectively and the export of cinnamon and cassia oleoresin was 1.12 t and respectively during The import of cinnamon and cassia oil to India was t and 1.16 t during Crop improvement programmes in cinnamon were initiated in Sri Lanka by the Ceylon Institute of Scientific and Industrial Research and the Department of Export Agriculture. Eight different types of cinnamon were recognized by growers in Sri Lanka based on leaf morphology, bark pungency, grittiness of bark, leaves, etc., (Wijesekera et al., 1975; Anon., 1996). Department of Export Agriculture of Sri Lanka had identified 19 selections after screening 210 accessions. The Indian Institute of Spices Research (IISR) at Calicut (Kerala, India) has maintained 300 accessions of cinnamon and related taxa. IISR, after evaluating 291 accessions, established five elite lines based on the quality characteristics such as bark oil, oleoresin and leaf oil. One Indian accession IN189 and one Sri Lankan accession SL63 were finally selected based on regeneration capacity, fresh bark yield, dry bark yield, leaf oil, percentage of eugenol in leaf oil and cinnamaldehyde (Krishnamoorthy et al., 1996). 3

4 Cinnamon can be propagated either through seeds or clonally by cuttage. When sown immediately after harvest, seeds give 90-94% germination, while on storage for five weeks the viability is completely lost (Kannan and Balakrishnan, 1967). The major drawback of seed propagation is that, cinnamon being a crosspollinated plant, exhibits wide variability in yield (Ponnuswamy et al., 1982; Krishnamoorthy et al., 1992), quality of produce and oil content (Paul and Sahoo, 1993) and other morphological characteristics. Hence, seed propagation is not advisable while clonal propagation is recommended in cinnamon (Weiss, 1997). Single node cuttings with leaves can be rooted in a month s time under high humidity conditions (Anon., 1985). Rema and Krishnamurthy (1993) noted much variability in the rooting response of various cinnamon accessions. Variations in rooting during different seasons have been reported (Anon., 1996). Air layering is also a successful method of vegetative propagation. But seasonal variation in rooting pattern has also been observed in air layers (Ranaware et al., 1995). Chinese cassia is usually grown from seeds, but can be grown from cuttings also. In cassia, vegetatively propagated plants are not used for commercial planting, as such plants are known to give poor quality stem and bark, less vigorous growth and regeneration (Dao, 2004). Ripe fruits from mother trees producing thick bark of good aroma should be selected for propagation A few dozens elite cultivars are recognized to possess considerable market relevance. However, conventional breeding methods are cumbersome because they depend on cross pollination, seed germination and selection as well as vegetative 4

5 regeneration. Modern plant biotechnology and genetic engineering have adequate potential to reduce the time needed for traditional breeding. Haldankar et al. (1994), Pugalendhi et al. (1997) and Joy et al. (1998) reported cinnamon selections for crop improvement. Lack of genetic variability for resistance to major diseases and pests and poor availability of quality seeds make conventional breeding programme ineffective and hence micropropagation is needed in the production of large number of elite lines within a short time span. Tissue culture has emerged as a potent tool for rapid multiplication and propagation of trees (Durzan, 1986) and has been successfully employed for the propagation of various tree species (Mascarenhas and Muralidharan, 1989). The drawbacks of seedlings and cuttages can be overcome to a great extent through biotechnological intervention such as micropropagation which would result in uniform good quality planting material of any elite lines. Successful micropropagation of woody plants is relatively a recent practice (Thorpe, 1990; Bajaj 1997). Plant tissue culture techniques offer many advantages over conventional methods in crop improvement. Clonal multiplication is a reliable method for plant propagation as it yields true-to-type plants. Thus it helps in maintaining uniformity within the population (Mascarenhas and Muralidharan, 1989). Other important applications of plant tissue culture technique are the production of virus-free plants by meristem culture, production of haploids and thereby homozygous dihaploids for plant breeding purposes, somatic embryo production for the mass propagation of plants, somaclonal variation and cell line selection for crop improvement, embryo 5

6 rescue to overcome the pre- and post-fertilization barriers, slow growth technique and cryopreservation for germplasm conservation, somatic hybridization, production of secondary metabolites, development of novel transgenic plants, etc. There are only a few reports on micropropagation of Cinnamomum spp. So far there are no reports on in vitro shoot multiplication from mature trees of Cinnamomum verum. There are no earlier reports on callus regeneration and somatic embryogenesis in Cinnamomum verum. The maintenance of genetic integrity among micropropagated plants vis-avis explant source(s) will be one of the most crucial concerns (Larkin and Scowcroft, 1981). In plant propagation, the most crucial aspect is to retain genetic integrity with respect to the mother plant (Jin et al., 2008). This is more important for tree species and other perennial crops where the life span is long and the performance of in vitro derived plants can be ascertained only after their long juvenile stage (Brown and Sommer, 1982; Gamborg, 1993). Tissue cultured and woody species take extensive evaluation time to access genetic fidelity (Vendrame et al., 1999). The use of molecular techniques distinctly facilitates and shortens the process of evaluation (Jain 2001). The use of meristem tip culture is considered least likely to increase genetic instability (Nehra and Kartha, 1994) as demonstrated by RAPD analysis of regenerated plants of Digitalis obscura (Gavidia et al., 1996), Achillea sp. (Wallner et al., 1996) oak (Barrett et al., 1997) and alfalfa (Piccioni et al., 1997). In spite of this fact, some RAPD differences were found among regenerated plants of poplars 6

7 (Rani et al., 1995), Pelargonium sp. (Cassells et al., 1997) and Piper longum (Parani et al., 1997). Occurrence of variability among tissue cultured plants has been investigated extensively and reported in oil palm (Corley et al., 1986), blackberry (Harry et al., 1983), Coleus (Marcotrigiano et al., 1990), strawberry (Moore et al., 1991) and plantain (Vuylsteke et al., 1998). Micropropagated plants from the cultures of preformed structures such as shoot tips and axillary buds have been reported to maintain clonal fidelity (Ahuja, 1987; Wang and Charles, 1991; Ostry et al., 1994) but there is still a possibility of generating somaclonal variants employing this method (Rani and Raina, 2000). Because tissue culture system itself act as a mutagenic inducer (Jain 2001), it is necessary to control genetic stability of micropropagated plants. Rani and Raina (2000) showed that some species are inherently more unstable than others during propagation and therefore, genetic diagnostics, especially at the DNA sequence level, should be made an integral component of any micropropagation system aimed at producing true-to-type plants. However, Rani et al. (1995) reported variations to the extent of 26% in micropropagated plants of Populus deltoides. Occurrence of somaclonal variation is a potential drawback when the propagation of an elite tree is intended, where clonal fidelity is required to maintain the advantages of desired elite genotypes (Rahman and Rajora, 2001). On the other hand, stable somaclonal variations of specific type may be advantageous for the improvement of certain traits (Antonetti and Pinon, 1993; Karp, 1995; Jain et al., 1998). Somatic embryogenesis is frequently regarded as the best system for the propagation of superior genotypes mostly because both root and shoot meristems are 7

8 present simultaneously in somatic embryos (Jin et al., 2008). Before somatic embryogenesis is used for the above purpose, the genetic fidelity of cultures needs to be determined (Vendrame et al., 1999). Random amplified polymorphic DNA (RAPD) based detection of genetic polymorphism (Welsh and Mc Clelland, 1990; Williams et al., 1990) has found successful application in describing somaclonal variability in regenerated individuals of several plant species (Isabel et al., 1993; Hashmi et al., 1997). RAPD analysis could also be useful for studying the genetic influence of different hormonal combinations during morphogenesis (Soniya et al., 2001). The present investigation is an attempt in this direction and undertaken with the following objectives 1. Standardization of micropropagation protocols for Cinnamomum verum and Cinnamomum cassia. 2. To develop a callus regeneration protocol for Cinnamomum verum. 3. To develop a protocol for direct somatic embryogenesis in Cinnamomum verum. 4. To standardize the hardening of in vitro developed plantlets and field establishment. 5. To compare the genotype of the regenerated plants with that of the mother plant using RAPD markers. 8