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
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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