1 1 CHAPTER 1 INTRODUCTION The genus Nymphaea, commonly known as water-lily, belongs to the family Nymphaeaceae, with perennial or annual rhizomes, floating or submerged leaves and solitary variously coloured showy flowers. Man was probably impressed from the first with the grace and beauty of the water-lilies; for their distribution is worldwide. There is evidence enough that the seeds and roots (tubers) of various species have been used for food by many primitive races, as in Australia, Madagascar, West Africa and Central America; but naturally it remained for more civilized peoples to rise to anything like an aesthetic interest (Conard, 1905). Nymphaeaceae is distributed in almost all tropical and temperate regions of the world and is constituted by 5 genera and about 70 species (Mitra, 1993). They are native mainly to Africa and Asia. Nymphaea is the most diverse genus of the order Nymphaeales. The representative genus Nymphaea of Nymphaeaceae was established by Linnaeus (1753), and comprises about species widespread in freshwater areas of tropical and temperate regions (Borsch et al., 2007). The genus Nymphaea has been classified into five subgenera viz. Anecphya, Brachyceras, Hydrocallis, Lotos and Nymphaea with each subgenus displaying a characteristic distribution (Conard, 1905; Borsch et al., 2007). Subg. Nymphaea, comprising 8 species, ranges throughout the Northern Hemisphere in temperate regions. On the basis of carpel wall fusion, Conard (1905) grouped subgg. Anecphya and Brachyceras as Apocarpiae because of their partially fused carpel, while subgg. Hydrocallis, Lotos and Nymphaea were included in Syncarpiae, because of more complete wall fusion. This classification originated from observations made by Caspary (1866) dividing Nymphaea, on the basis of complete or partial lateral fusion of the carpels, into two main groups, the Symphytopleura and the Lytopleura. Among the 5 genera and about 70 species occurring worldwide, Mitra (1990) reported two genera and ten species from India. In North East (NE) India occurrence of seven species of Nymphaea has been reported (Dkhar et al., 2010). Euryale ferox
2 2 and three species of Nymphaea viz., Nymphaea micrantha, N. pubescens and N. nouchali are reported from Manipur under Nymphaeaceae (Chauhan and Dam, 2000), but as it could be seen from the market and the Nymphaea species growing in the roadside ditches, ponds and lakes, occurrence of more than three Nymphaea species in Manipur was confirmed. Therefore, further work was needed to ascertain the exact number of Nymphaea species in Manipur. The morphological approach for the identification of species is a common method. Nymphaea species have high morphological plasticity. Size of leaves and flowers, and also some qualitative characters of flowers, are thought to be strongly dependent on hydrological, especially temperature, and edaphic conditions (Heslop- Harrison, 1995; Kupriyanova, 1976; Dubyna, 1982). Sometimes same species may appear morphologically different due to different environmental factors. The size of different organs of Nymphaea species also depends on the age of the plant (Dubyna, 1982). So, molecular approach is made to support the morphological data and DNA markers are used for molecular identification. Water-lilies are pollinated either through crossing or selfing, occurring usually through the agency of insects. Self-pollination occurs in N. rudgeana, N. amazonum and N. stellata (Caspary, 1878; Conard, 1905). Night-blooming species of subg. Hydrocallis are probably pollinated by Coleoptera (Cramer et al., 1975; Prance and Anderson, 1976; Prance, 1980; Wiersema, 1987) whereas members of day-blooming subgg. Anecphya, Brachyceras and Nymphaea appeared to be pollinated by Hymenoptera or Diptera (Van der Velde et al., 1978; Schneider and Chaney, 1981; Schneider, 1982a, b; Capperino and Schneider, 1985). Asexual modes of reproduction have been reported in water-lily species, the most predominant being stolon formation, observed in N. lotus of subg. Lotos, N. gardneriana of subg. Hydrocallis, and N. mexicana of subg. Nymphaea. Other forms of asexual reproduction involve the proliferation of floral tissues or leaf tissues which is found to occur regularly in N. lasiophylla and N. prolifera of subg. Hydrocallis (Wiersema, 1987). Unusual occurrences of similar abortive flowers have been reported in other species (Bose, 1961; Mohan Ram and Nayyar, 1974; Majeed, 1977; Mitra and Subramanyam, 1982).
3 3 Proliferation of leaf tissues occurs in N. micrantha of subg. Brachyceras (Conard, 1905; Hutchinson and Dalziel, 1966). Hybrids of water-lily are also available. Interspecific hybrids are very common, but intergeneric hybrids are rare and it s a scientific challenge to produce intergeneric hybrids. Till now, only one inter-sub-generic hybrid is produced (Songpanich and Hongtrakul, 2010). Although Nymphaeaceae is small taxonomically, the relationship among genera has not been clear and no single classification has become widely accepted yet. Nymphaea have long been one of the most popular ornamental plants, but they are also used as food and medicine in many parts of the world (Ni et al., 1995). The rhizome, fruit, leaf, petiole, roots, flowers, tubers, and seed are used as edible parts in different ways by people. Various parts of Nymphaea plants are popular in Manipur state of India and are being sold in local markets after collecting from wild and home ponds. The tuberous rhizomes and peduncles of N. nouchali and N. pubescens are eaten as vegetables; seeds on roasting are eaten as puffed grain (Mitra, 1990). Also, aquatic macrophytes are increasingly being recognized as most productive bioresource among plant communities (Auclair et al., 1976). Nymphaea plants are included in the underutilized crop category from the nutritional point of view (Parmia et al., 2013). Information on the nutritional values of edible Nymphaea is limited. Three species namely, N. nouchali Burm. f., N. pubescens Willd. and N. rubra Roxb. ex Andrews are widely used as food, vegetable and traditional medicines. Rhizomes of these three species are taken as food after being boiled; young leaf and peduncle are used in curries and also eaten as a salad. The captivating flower of Nymphaea is commonly used for ornamental and ritual purposes (Sharma et al., 2005). Because of their beautiful, showy or sweetscented flowers, several Nymphaeaceae members common for aquatic gardening (Sharma, 2009). Vibrant colours and sweet fragrances of Nymphaea flowers have been used in many aromatherapy centres, and the flower extract is also used in many cosmetic products (Jirapong et al., 2012). Cut flowers of Nymphaea plants have great demand in the market due to their enchanting flowers with attractive colour and sweet fragrance. Nymphaea flowers have a variety of colours. Few genera of plants offer
4 4 such variety in size and colour of flowers, as is found between the water-lilies (Conard, 1905). Nymphaea is one of the few fascinating groups of aquatic plants recently enchanting plant growers in India and cultivated in aquatic habitats for it s much admired flower colours (Ansari and Jeeja, 2009). Nymphaea flowers are famous especially for their floral colours and fragrance. Day blooming tropical water-lilies has almost every colour except black and most of them have unique fragrance (Wasujit, 1994). Day-flowering Nymphaea species emits attractive smell while, nightflowering Nymphaea species have repelling odour. Information about the chemical composition of the Nymphaea species are in paucity, so this thesis work also includes study about the chemical composition of the fragrance of Nymphaea species. Nymphaea forms an important wetland plant of Manipur (Fig. 1) with aesthetic and economic value. It is used by people as vegetable, food and in traditional medicines. However, the population and habitat area of water lilies are gradually decreasing in Manipur. On the other hand, there is no comprehensive enumeration of Nymphaea species is available in Manipur except a description of three species in the Flora of Manipur (Chauhan and Dam, 2000). Considering this background, the present study was designed with the following objectives. Objectives  Survey, collection and preservation of Nymphaea species found in Manipur.  Morphological and molecular characterization of Nymphaea of Manipur.  Evaluation of nutrient composition of three edible Nymphaea species  Analysis of chemical nature of fragrance of Nymphaea flowers.
5 5 Experimental Design The first step for the study was to undergo survey and collection of plants of the genus Nymphaea from the wetland areas of Manipur. After collection, several replications of each species were subjected to morphological examinations. The selected morphological data include both qualitative and quantitative characters. Qualitative characters include the shape of rhizome and thalamus, colour of sepal and petal and quantitative characters like number of petals, stamens and carpels. Fig. 1 Growth of Nymphaea species in wetland (Ishok, Bishnupur). Three individuals from each population were selected randomly for DNA extraction. Subsequently, the extracted DNA samples were subjected to different techniques such as random amplified polymorphic DNA (RAPD) and inter simple sequence repeats (ISSR). Various parts of edible Nymphaea species were considered for the nutrient analysis. Nymphaea species with different fragrance intensity were selected for the study of the chemical components of fragrance. The sum up of the main objectives and the experimental steps are represented in Fig. 2.
6 EXPERIMENTAL STEPS 6 OBJECTIVES Diversity study Nutrient Evaluation Fragrance Analysis Morphological Molecular Macronutrients Micronutrients Chemical composition Investigation Markers Survey, collection, RAPD and Anthrone method, AOAC method, GC-MS analysis Identification and PCA ISSR AAS, etc Understanding of diversity, the nutrient and fragrance potential of the Nymphaea species of Manipur, NE India leading to its conservation Fig. 2 The objectives and experimental design of the research work.