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Diversity and Micropropagation of Canna from West Bengal and Orissa

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Diversity and Micropropagation of Canna from West Bengal and Orissa Diversity and Micropropagation of Canna from West Bengal and Orissa Thesis submitted to the University of North Bengal For the Award of Doctor of Philosophy in Botany By Tanmayee Mishra Supervisor Dr. Arnab Sen Department of Botany University of North Bengal Raja Rammohunpur, Siliguri January, 2014 This work is dedicated to my Parents Declaration I declare that the thesis entitled “Diversity and Micropropagation of Canna from West Bengal and Orissa” has been prepared by me under the guidance of Dr. Arnab Sen, Associate Professor of Department of Botany, University of North Bengal. No part of the thesis has formed the basis for the award of any degree or fellowship previously. [Tanmayee Mishra] Department of Botany, University of North Bengal RajaRammohunpur, Siliguri-734013 Date: i Abstract Canna, the solitary genus of the family as manmade hybrids. The entire Cannaceae, is popularly known as an cultivated garden ornamentals are ornamental plant with flashy, included under two artificial hybrid brilliantly-colored flowers and large species i.e. Canna x orchiodes L. H. tropical foliage. Various Bailey and Canna x generalis L. H. morphological, cytological and Bailey. taxonomical characteristics of the However, there are number of family Cannaceae is closely related to problems related to the production of other members of Zingiberales like Canna. Seed germination is very Musaceae, Strelitziaceae, difficult and not practiced very often Zingiberaceae, Marantaceae etc. The leading to the problem in breeding of members of the Cannaceae possess a Canna for the production of improved very diverse habitat. The genus Canna varieties. Alternative methods like comprises of about 51 species of protoplast fusion for production of flowering plants. Canna species are hybrid varieties has never been tried native of tropical and subtropical perhaps anywhere in the world. Till humid neotropics (South and North date no work has been done on the America) and with the course of time diversity of Canna found in west they have been introduced in Asian Bengal as well as in the adjacent state paleotropics and subsequently evolved of Orissa. The taxonomy of Cannaceae into semi-native varieties. The is also disputed as the monogeneric pantropical distribution of Canna family Canaceae has recently been species is most possibly the effect of formed and distinguishing features human dispersal. Some of the wild between Canna varieties are poor as species of Canna, namely C. glauca, C. they lack proper molecular indica, C. iridiflora, C. warscwiczii documentation. Some Canna varieties and C. flaccid etc. are popularly known like, Canna edulis and its cultivars are as elemental species of Canna and are rich in edible starch content. It is now involved in producing natural as well established as edible and medicinal ii plant. However, nutritive and polymorphism among them. The band medicinal profiling for most of part of size ranged between 246 bp to 2017 bp. Canna is virtually untouched. The highest correlation was found In the present study an attempt was between Canna edulis and its green made to access the genetic relationship cultivar (96.4%), where as lowest was among the 20 species and cultivars of found between Canna indica and Canna using RAPD, ISSR and PCR- Canna x generalis Cv. “Orange RFLP (for TrnL-TrnF region of Web” (56%). ISSR analysis results chloroplast genomes) techniques. A indicated that the elemental species total of 159 major scorable bands Canna indica and its cultivar Canna ranging from 220 to 1757 bp were indica Cv. “Purpurea” were close to generated from 18 RAPD primers. The each other, which is quite natural. percentage of polymorphism was found Besides, there were three other distinct to be 89.93%. The lowest similarity clades. One was with two elemental was observed between Canna edulis varieties, Canna edulis and Canna and Canna x generalis Cv. “Trinacria edulis green cultivar. Other 16 hybrid Variegata” (49.6%), while the highest cultivars formed 2 different clades in value was recorded between Canna x the dendrogram. generalis Cv. “Dwarf Red” and Canna The dendrogram based on the x generalis Cv. “Dwarf Orange” and combined data sets of both RAPD and between Canna x generalis Cv. “Dwarf ISSR showed considerable similarity Orange” and Canna x generalis Cv. with that obtained from individual “Froken” (94.1%). In the dendrogram, RAPD and ISSR analysis, except for both the cultivars of Canna edulis the highest correlation, that was found came together with a cluster sharing to be between Canna x generalis Cv. node at 89.1%. However, two varieties “Tropical Red” and Canna x generalis of Canna indica were separated out. Cv. “Orange Web” (0.936) while the Regarding the hybrid cultivars, all of lowest was found between Canna x them flocked together in a big cluster generalis Cv. “Dwarf Red” and Canna except Canna x generalis Cv. edulis (0.601). “Trinacria Variegata”. All the three dendrograms (RAPD, Ten ISSR primers resulted in 93 ISSR and combined) were similar to scorable bands showing 88.17% each other in many ways, such as iii grouping of garden cultivars as a also it is an important plant for its somaclone complex, separation of medicinal values. In the present study, elemental species from the hybrid the antioxidant diversity among 20 cultivars and the closeness of four different Canna accessions was dwarf cultivars etc. The only exception determined on the basis of two is the position of C. indica Cv. parameters i.e. temperature changes Purpurea. While in ISSR tree C. indica and solvent types. The correlation of Cv. Purpurea went with C. indica, it phytochemical characteristics and positioned separately in RAPD tree and antioxidative properties of rhizome ISSR-RAPD combined tree. were taken up firstly through thermal All the bands generated through PCR changes of the aqueous extracts i.e. amplification of Taberlet (TrnL-TrnF) cold aqueous (CAE) and hot aqueous region following restriction digestion (HAE) extracts and secondly in two were found to be monomorphic. different solvent extracts i.e. in Therefore, chloroplast genome may not aqueous (HAE) and methanolic (ME) be considered for studying extracts. Total phenol and flavonoid polymorphism among various species contents of various Canna samples in and cultivars of Canna. different extracts were ranged between The phylogenetic tree constructed with 22.67 to 158.22 mg GAE/g and 14.32 Clustal Omega and Phylip 3.69 to 65.02 mg QE/g respectively. In both program of Taberlet region of various the cases methanolic extract was found members of Zingiberales recovered all to be better than aqueous extracts the major families intact. Families like (ME˃HAE˃CAE). The DPPH radical Musaceae, Zingiberaceae, Cannaceae, scavenging activity was found to be Marantaceae, Strelitziaceae, highest in methanolic extract (84.96%). Heliconiaceae and Lowiaceae were For ferric reducing power assay (FRP), flocked together. The sequences of methanol was proved to be the most different Canna varieties done by us effective solvent to assess the reductive were perfectly placed with other potential for Canna rhizome. Both sequences (Canna) retrieved from the DPPH and FRP assay followed the public domain. same trend as that of phenols and Canna is an important plant not only flavonoids (ME˃HAE˃CAE). The from the ornamental point of view but Hydrogen peroxide scavenging activity iv of various Canna samples in different activity (78.41%) and hydroxyl radical extracts was quite different from other inhibition activity (39.04%) were antioxidant properties. Cold aqueous observed in bioactive diethyl ether : extract had highest H2O2 scavenging ethyl acetate (1:1) fraction. Inhibition activity (89.11%) followed by hot of lipid peroxides was maximum in aqueous extract and methanolic extract. ethyl acetate fraction (67.89%). Separation and isolation of antioxidant Further, thin layer chromatography, molecules of Canna edulis (an edible confirms the distribution of phenolic Canna) was performed on the basis of compounds in the above bioactive their percolation in various polar and fractions. Thus it can be concluded that non polar solvents through silica gel the antiradical scavenging activity of column chromatography. Canna rhizome may be due to the Investigations were done to find out presence of polyphenolic compouns DPPH radical scavenging activity, total like phenols, flavonoids, flavonols and total proanthocyanidin proanthocynidins etc. contents, nitric oxide scavenging GC-MS (Gas chromatography-Mass activity, hydroxyl radical scavenging spectroscopy) analysis of C. edulis leaf activity and anti lipid peroxidation extract identified the presence of 3 activity of different solvent fractions of major compounds namely 22,23- C. edulis rhizome. Out of 29 fractions Dibromostigmasterol acetate $$ 22,23- studied, 6 fractions showed inhibition Dibromostigmast-5-en-3-yl acetate, N- above 75%, and were used for further Heptyl-N'-{9-[2-(heptyl-methyl- phytochemical screening. Diethyl carbamoyl)-acetylamino]-nonyl}-N- ether : ethyl acetate (1:3) fraction methyl-malonamide and Cholest-5-ene, showed the maximum inhibition 3.beta.-chloro- $$ Cholesteryl chloride percent (93.08%). Highest amount of $$ Cholest-5-ene, 3-chloro-, (3.beta.)- total flavonol (i.e.37.12 mg/ml $$ Cholesterol chloride $$ 3.beta.- quercetin equivalent per 100 mg Chlorocholest-5-ene, which have rhizome extract) and total antibacterial, antifungal and proanthocyanidins (i.e. 0.012 mg/ anticoccidal activities. catechin/g dry weight) were recorded In order to produce genetically in diethyl ether : ethyl acetate (1:3) modified and improved varieties of fraction. Maximum NO scavenging Canna, various in vitro techniques had v been tried. Banana micropropagation Protoplast fusion technique was tried to medium (BM) supplemented with 3% generate genetically hybrid varieties. In sucrose, 0.7% agar, and 0.17% vitro generated leaves and shoots of NH4NO3 and different plant growth Canna indica and Canna edulis were regulators like BAP (2 mg/l) and NAA used as the source for isolation of (0.5 mg/l)) was found to be effective in protoplasts. Viable protoplasts inducing callus.
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