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