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List of Figures
i
Page No.
Fig. 1 Pie chart showing distribution of seaweed utilization with
their annual harvest and market value
2
Fig. 2.1 Seaweed species investigated for protoplast isolation and
regeneration studies (A) Ulva fasciata, (B) U. reticulata,
(C) U. rigida, (D) U. beytensis, (E) U. lactuca and (F) U.
taeniata
15
Fig. 2.2 Morphogenesis of Ulva lactuca 19
Fig. 2.3 Morphogenesis of Ulva beytensis 20
Fig. 2.4 Morphogenesis of Ulva taeniata 20
Fig. 2.5 Morphogenesis of Ulva reticulata 20
Fig. 2.6 Morphogenesis of Ulva rigida 20
Fig. 2.7 Morphogenesis of Ulva fasciata 21
Fig. 2.8 Initial protoplasts culture in Petri-dishes. Circle showed
germlings developed after 15 days of culture
22
Fig. 2.9 The final biomass obtained after 45 days of incubation
from initial protoplasts obtained from 100 mg biomass
22
Fig. 2.2.1 Habit of (A) Gracilaria dura and (B) Gracilaria
verrucosa Scale = 2.2 cm
30
Fig. 2.2.2 Thallus showing protoplasts release from G. dura (A) and
G. verrucosa (B) and their respective isolated protoplasts
(C and D) Scale = 50 µm
32
Fig. 2.2.3 Effect of pH (A) Temperature (B) Mannitol concentration
(C) and Incubation period (D). All experiments were
conducted using enzyme mix-F. Different letters above
the bars indicate significant differences at probability
p≤0.05 according to two way anova
34
Fig. 3.1 (A) Tissue explants of Gracilaria dura on agar plate
showing deep pits around them, (B) staining and
appearance of isolated bacterial strain
47
List of Figures
ii
Fig. 3.2 SDS-PAGE analysis of purified agarase. Lane 1
molecular mass markers, lane 2 purified agarase, lane 3
in-vitro activity staining
49
Fig. 3.3 Biochemical characterization of pure agarase describing
effect of (A) pH, (B) temperature, (C) various additives
and (D) NaCl concentrations
51
Fig. 3.4 Enzyme activity profile in presence of (A) detergents and
(B) solvents
51
Fig. 3.5 Analysis of agar hydrolysed products based upon (A)
HPLC coupled with gel permeation chromatography, (B)
LC/Q-Tof mass spectroscopy and (C) 13
C NMR
spectroscopy
52
Fig. 3.6 Agarose gel electrophoresis of plasmid DNA revealing
DNA protective effect of neoagarobiose against the
damage induced by hydroxyl radicals. C: control pUC18
plasmid DNA, T: DNA damage after treatment with
H2O2, S1-S4: DNA protective effect of agar hydrolyzed
product at concentrations 5 to 20 µg/mL
53
Fig. 3.7 Enzymatic treatments showing loosening of thalli (A)
followed by release of protoplasts (B) and cell division
(C)
54
Fig. 3.8 (A) Bioconversion of seaweed galactans into fermentable
sugar galactose and GC/MS analysis of galactose
released after hydrolysis of algal biomass. (B) retention
time of standard galactose (6.8 min), (C) mass
fragmentation pattern of standard galactose, (D) Peak at
6.75 min represents for galactose in the sample after
hydrolysis, and (E) Mass fragmentation of the sample
peak confirming galactose released after hydrolysis.
Gal: Galactose
55
Fig. 4.1 Deposition of cellulosic cell wall around fragile cell
membrane of protoplasts as confirmed with calcofluor
staining. Scale bar = 20µm
66
List of Figures
iii
Fig. 4.2 Regeneration rate of protoplasts of U. reticulata at
different temperatures. Each data is the mean of five
replicates. Vertical bars indicate standard deviation.
Significant differences are indicated with alphabets on
top of bars (p≤0.01)
66
Fig. 4.3 Differentiation of protoplasts of U. reticulata (A) into
variant disc type thalli (B-F), and normal filamentous
thalli (G-K)
67
Fig. 4.4 Regeneration rate of different morphotypes at different
temperatures (A), and their daily growth rates (B).
Alphabets at top of bars indicate significant differences
among themselves (p≤0.01)
68
Fig. 4.5 Development of normal filamentous thalli from disc type
germling. (A) Plate showing fully developed normal
filamentous and disc type morphotypes, (B) swarmers
released from the disc type morphotype, (C) regenerated
normal filamentous thalli from the swarmers
69
Fig. 5.1.1 Maximum likelihood tree deciphering phylogeny of
different Ulva sp. based on rbcL gene
83
Fig. 5.1.2 Maximum likelihood tree deciphering phylogeny of
genus Ulva sp. based on nrITS region
84
Fig. 5.1.3 Maximum likelihood tree deciphering phylogeny of
genus Ulva sp. based on tufA gene
85
Fig. 5.1.4 Maximum likelihood tree deciphering phylogeny of
genus Ulva based on ndhJ gene
86
Fig. 5.1.5 Maximum likelihood tree deciphering phylogeny of
genus Ulva based on accD gene
87
Fig. 5.1.6 Maximum likelihood tree deciphering phylogeny of
genus Ulva based on rpoC gene
88
Fig. 5.1.7 Number of parsimony informative characters in each of
the six regions included in the analysis of the
Ulvophyceaen species data set
89
List of Figures
iv
Fig. 5.2.1 1H NMR spectra of (1) Ulva lactuca, (2) Gracilaria dura
and (3) Sargassum tenerrimum acquired during month of
April-May using (a) organic solvent extract and (b)
aqueous extract
98
Fig. 5.2.2 1H NMR spectra of (1) Ulva lactuca, (2) Gracilaria dura
and (3) Sargassum tenerrimum acquired during month of
December using (a) organic solvent extract and (b)
aqueous extract
99
Fig. 5.2.3 1H NMR spectra acquired (a) immediately after
extraction and (b) after an incubation of 12 h at 20˚C for
(1) Ulva lactuca, (2) Gracilaria dura and (3) Sargassum
tenerrimum
100
Fig. 5.2.4 1H NMR spectra of Ulva lactuca (a) after freeze drying
and (b) direct aqueous extract. Arrows indicated the
peaks found missing after freeze drying
101
Fig. 5.2.5 Two dimensional correlation spectroscopy (Left Column)
TOCSY and (Right Column) HSQC for (Top Row) Ulva
lactuca, (Middle Row) Gracilaria dura and (Bottom
Row) Sargassum tenerrimum
103
Fig. 5.2.6 1H NMR spectra for samples collected during different
periods (a) March 2011, (b) December 2011 and (c)
April-May 2012 for algal species (1) Ulva lactuca, (2)
Gracilaria dura and (3) Sargassum tenerrimum
104
Fig. 5.2.7 Characterization of lignin monomers from Ulva lactuca
using DFRC method. (a) GC retention time of the
derivatized extract and mass fragmentation pattern of (b)
guaiacyl and (c) syringyl units released. Inset image
represent the mass fragmentation of respective units
determined by Martone et al. (2009)
105
Fig. 5.2.8 Metabolic model framed by mapping the functional
relevance of the identified metabolites providing
information on to the central metabolic processes of
marine macrophytic algae
106
Fig. 5.3.1 Mass fragmentation of indole-3-acetic acid (IAA) with
diagnostic transition (m/z) from 175.18 to 130.12
117
List of Figures
v
Fig. 5.3.2 Mass fragmentation of indole-3-butyric acid (IBA) with
diagnostic transition (m/z) from 203.01 to 158.03
118
Fig. 5.3.3 Mass fragmentation of abscisic acid (ABA) with
diagnostic transition (m/z) from 265.23 to 164.98
119
Fig. 5.3.4 Mass fragmentation of gibberellic acid (GA3) with
diagnostic transition (m/z) from 346.46 to 253.0
120
Fig. 5.3.5 Mass fragmentation of salicylic acid with diagnostic
transition (m/z) from 138.14 to 93.08
121
Fig. 5.3.6 Mass fragmentation of kinatine riboside with diagnostic
transition (m/z) from 348.16 to 216.12
122
Fig. 6.1.1 Protoplast fusion (A) Mixture of protoplasts, (B) Pearl-
chain formation after AC impulse, (C) Fused protoplast
after DC impulse, (D, E and F) Protoplasts with two
chloroplasts
142
Fig. 6.1.2 Thallus morphology of fusing partner and regenerated
putative hybrids
144
Fig. 6.1.3 Chromosome count of (A) U. fasciata (2n=6), (B) Hybrid
1 (2n=8), (C) Hybrid 2 (2n=12), (D) Hybrid 3 (2n=12)
and (E) U. reticulata (2n=10)
145
Fig. 6.1.4 Bright-field images with superposition of the nucleus
fluorescence (blue) and chloroplast autofluorescence (in
red)
145
Fig. 6.1.5 Relative fluorescence intensity of nuclei stained with
DAPI for fusing partners and regenerated putative
hybrids
146
Fig. 6.1.6 Isoenzyme profile of lipoxygenase in hybrid plants and
the fusing parents. UF: U. fasciata, UR: U. reticulata and
H: Hybrids
146
Fig. 6.1.7 RAPD patterns of regenerated plants from the fusion of
the protoplasts from U. fasciata and U. reticulata. UF: U.
fasciata, UR: U. reticulata and H: Hybrids. Arrowheads
showed the bands specific to one parent and introgressed
into the genome of the regenerated hybrids
147
List of Figures
vi
Fig. 6.1.8 ISSR patterns of regenerated plants from the fusion of the
protoplasts from U. fasciata and U. reticulata. M:
Molecular weight marker, UF: U. fasciata, UR: U.
reticulata and H: Hybrids. Arrowheads showed the bands
specific to one parent and introgressed into the genome of
the regenerated hybrids
148
Fig. 6.1.9 (A) AFLP patterns of regenerated plants from the fusion
of the protoplasts from U. fasciata and U. reticulata. UF:
U. fasciata, UR: U. reticulata and H: Hybrids.
Arrowheads showed the bands specific to one parent and
introgressed into the genome of the regenerated hybrids
(B) Principal co-ordinate based distribution of parent and
the regenerated hybrids
149
Fig. 6.1.10 MSAP patterns of regenerated plants from the fusion of
the protoplasts from U. fasciata and U. reticulata. UF: U.
fasciata, UR: U. reticulata and H: Hybrids, H: Genomic
DNA was digested with HpaII and EcoRI, M: Genomic
DNA was digested with MspI and EcoRI
151
Fig. 6.1.11 Mitochondrial coxIII PCR amplification pattern.
Arrowheads represent the bands specific to the fusing
partners and shared by regenerated hybrids
152
Fig. 6.1.12 Cladogram for different chloroplast, nuclear regions
showing distribution of hybrids with respect to the fusing
partners based on nucleotide diversity analysis by
neighbour joining method
153
Fig. 6.1.13 Analysis of daily growth rate (%) of regenerated hybrids
and the fusing partners at different temperatures
154
Fig. 6.1.14 Endogenous phytohormone content in regenerated
hybrids and fusing partners
159
Fig. 6.1.15 Score plot (A) and loading plot (B) from the principal
component analysis (PCA) for NMR spectroscopic data
for parental thalli and regenerated hybrids
162
Fig. 6.1.16 Relative proportion of metabolities deciphering the
metabolism energy status of the regenerated hybrids and
the parents
163
List of Figures
vii
Fig. 6.2.1 Protoplast fusion (A) Mixture of protoplasts, (B & C)
Pearl-chain formation after AC impulse, (D & E) Fused
protoplast with two chloroplasts after DC impulse
164
Fig. 6.2.2 The putative hybrids generated after fusion of (A) M.
oxyspermum and (B) U. reticulata, (C-N) putative
hybrids
165
Fig. 6.2.3 Relative fluorescence intensity of putative hybrids and
the fusing partners as determined from confocal
microscopy. Above image showed the total genomic
DNA. UR: U. reticulata, M: M. oxyspermum and H
series: Putative hybrids
166
Fig. 6.2.4 ISSR profile of regenerated putative hybrids along with
the fusing partners. Arrow head showed the bands shared
with either of the fusing partner
167
Fig. 6.2.5 AFLP profile of putative hybrids and fusing partner.
Arrowheads showed the bands shared with either of the
partner
168
Fig. 6.2.6 AFLP based clustering of regenerated hybrids and fusing
partners. UR: U. reticulata, M: M. oxyspermum, H1-9:
Hybrids
168
Fig. 6.2.7 (A) MSAP profile for protoplast fusing partners and
regenerated putative hybrids and (B) their clustering. M;
M. oxyspermum, UR: U. reticulata and H1-9: Hybrids
169
Fig. 6.2.8 Growth rate (DGR %) of the hybrids and the fusing
partners
171
Fig. 6.2.9 (A) Loading and (B) score plot of the regenerated hybrids
and the parents by PCA analysis of fatty acid groups
174