| Preface |
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ix | |
| Editors |
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xiii | |
| Contributors |
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xv | |
| Section I: Cultivating and preserving seaweeds |
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Chapter 1 Seaweed in high-energy environments: Protocol to move Saccharina cultivation offshore |
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3 | (34) |
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1.1 Introduction to offshore aquaculture |
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4 | (1) |
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1.2 State of the art of offshore seaweed cultivation |
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5 | (3) |
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1.3 Land-based preparation and site selection |
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8 | (12) |
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9 | (1) |
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1.3.1.1 Site selection: Advance information of the local area ashore and at sea |
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10 | (1) |
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1.3.1.2 Site selection: Site-specific, oceanographic, and water quality parameter |
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11 | (1) |
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1.3.1.3 Site selection: Economic/technical/expansion feasibility |
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12 | (1) |
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1.3.2 Equipment and system design |
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13 | (1) |
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1.3.2.1 System design for the offshore seaweed farm |
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17 | (1) |
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18 | (2) |
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20 | (9) |
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1.4.1 Deployment of the offshore farm |
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20 | (1) |
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1.4.1.1 Deployment of the farm |
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21 | (3) |
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1.4.2 Operation, maintenance, and harvest |
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24 | (1) |
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1.4.2.1 Operation and maintenance at sea |
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24 | (1) |
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25 | (1) |
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1.4.3 Problems and failures |
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26 | (1) |
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1.4.3.1 Prevention of mistakes |
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27 | (2) |
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1.5 Multi-use of offshore installations |
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29 | (3) |
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32 | (5) |
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Chapter 2 Cultivation protocol for Saccharina latissima |
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37 | (24) |
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Kristine Braaten Steinhovden |
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38 | (3) |
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2.1.1 The life cycle of laminariales |
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39 | (2) |
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41 | (1) |
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41 | (2) |
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2.3.1 Collection of sporophytes and induction of sori |
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41 | (1) |
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2.3.2 Sori disinfection, dehydration, spore release, and spore counting |
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42 | (1) |
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2.3.3 Starting gametophyte cultures |
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42 | (1) |
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2.3.4 Maintenance of gametophyte cultures |
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42 | (1) |
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43 | (1) |
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43 | (1) |
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2.4 Experimental procedures |
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43 | (13) |
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2.4.1 Seed supply and sporulation |
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43 | (1) |
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2.4.1.1 Collection of sporophytes and induction of sorus |
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43 | (1) |
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2.4.1.2 Sorus disinfection |
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45 | (1) |
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45 | (1) |
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45 | (1) |
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46 | (1) |
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2.4.2 Gametophyte cultivation |
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46 | (1) |
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2.4.2.1 Starting-up gametophyte cultures |
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46 | (1) |
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2.4.2.2 Maintenance of gametophyte cultures |
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48 | (1) |
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2.4.2.3 Contamination control |
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49 | (1) |
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49 | (1) |
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49 | (1) |
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2.4.3.1 Seeding with spores |
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50 | (1) |
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2.4.3.2 Seeding with gametophytes |
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50 | (1) |
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50 | (1) |
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2.4.3.4 Cultivation under controlled laboratory conditions |
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51 | (1) |
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2.4.3.5 Development of seedlings under optimal conditions |
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52 | (1) |
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53 | (1) |
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53 | (1) |
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2.4.4.2 Deployment at sea |
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53 | (3) |
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56 | (1) |
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57 | (4) |
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Chapter 3 Derivation of clonal stock cultures and hybridization of kelps: A tool for strain preservation and breeding programs |
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61 | (18) |
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62 | (1) |
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63 | (1) |
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64 | (2) |
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3.3.1 Facilities and equipment for collection of sporogenous material and release of spores |
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64 | (1) |
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64 | (1) |
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3.3.1.2 In the laboratory |
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65 | (1) |
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3.3.2 Facilities and equipment list for isolation and propagation of clonal cultures |
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65 | (1) |
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3.3.3 Facilities and equipment list for performing laboratory-scale hybridization programs |
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66 | (1) |
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3.4 Experimental procedures |
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66 | (9) |
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3.4.1 Collection and preparation of fertile sporophytes |
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66 | (1) |
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3.4.2 Induced spore release |
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66 | (3) |
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3.4.3 Conditions to keep gametophytes vegetative |
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69 | (1) |
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3.4.4 Isolation of clonal gametophyte cultures |
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70 | (1) |
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3.4.5 Propagation of clonal cultures |
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71 | (1) |
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3.4.6 Hybridization of clonal gametophyte cultures |
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72 | (3) |
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75 | (1) |
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76 | (1) |
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76 | (3) |
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Chapter 4 Cryopreservation of macroalgae |
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79 | (16) |
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79 | (3) |
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82 | (4) |
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86 | (1) |
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4.3.1 Materials required for both methods |
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86 | (1) |
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4.3.2 Materials for conventional colligative cryopreservation |
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86 | (1) |
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4.3.3 Materials required for a vitrification-based approach |
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87 | (1) |
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4.4 Experimental procedures |
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87 | (3) |
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4.4.1 Method for conventional colligative cryopreservation |
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87 | (1) |
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4.4.2 Method for a vitrification-based approach |
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88 | (2) |
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90 | (1) |
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91 | (1) |
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92 | (3) |
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Chapter 5 Unraveling seaweeds bacteriomes: From field site to computer screen |
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95 | (20) |
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95 | (2) |
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97 | (3) |
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100 | (1) |
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5.4 Experimental procedures |
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100 | (8) |
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100 | (1) |
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101 | (1) |
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5.4.3 16S rRNA amplification |
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102 | (1) |
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5.4.4 16S rRNA amplicon sequencing data analysis |
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103 | (2) |
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5.4.5 Functional prediction based on 16S rRNA gene sequencing |
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105 | (3) |
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108 | (2) |
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110 | (1) |
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110 | (5) |
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Chapter 6 Heavy metal ecotoxicity on the early life history stages of macroalgae |
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115 | (14) |
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115 | (1) |
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116 | (2) |
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118 | (1) |
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6.3.1 Trace metal cleaning |
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118 | (1) |
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6.3.2 Algal material for spore release and toxicity test |
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118 | (1) |
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6.4 Experimental procedures |
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119 | (5) |
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6.4.1 Trace metal clean techniques |
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119 | (1) |
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120 | (1) |
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120 | (2) |
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122 | (2) |
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6.4.5 Adaptation of the protocol to microalgae and adult macroalgal specimens |
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124 | (1) |
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124 | (1) |
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125 | (4) |
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Chapter 7 A simple protocol for a rapid and consistent production of a large number of viable protoplasts from the Ulvophycean species |
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129 | (10) |
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129 | (1) |
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130 | (1) |
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131 | (1) |
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131 | (1) |
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131 | (1) |
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132 | (1) |
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7.4 Experimental procedures |
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132 | (3) |
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135 | (1) |
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136 | (1) |
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136 | (3) |
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Chapter 8 Purification of sporulation and swarming inhibitors from Ulva: Application in algal life-cycle controlling |
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139 | (20) |
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140 | (1) |
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141 | (1) |
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142 | (3) |
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142 | (1) |
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8.3.2 Culturing of U. mutabilis on a large scale |
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142 | (1) |
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8.3.3 Harvesting of Ulva biomass for the extraction and the bioassay-guided fractionation of inhibitors |
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143 | (1) |
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8.3.4 Induction of gametogenesis/sporogenesis and bioassay-guided fractionation of the inhibitors |
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143 | (1) |
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8.3.5 Sporulation inhibitor 1: Extraction |
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144 | (1) |
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8.3.5.1 Sporulation inhibitor 1: Size-exclusion chromatography for cleanup |
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144 | (1) |
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8.3.6 Sporulation inhibitor 2: Extraction and bioassay-guided fractionation |
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144 | (1) |
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8.3.7 Swarming Inhibitor: Extraction and bioassay-guided fractionation |
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145 | (1) |
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8.4 Experimental procedures |
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145 | (10) |
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8.4.1 Cultivation of U. mutabilis under standardized conditions |
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145 | (2) |
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8.4.2 Induction of the gametogenesis/zoo sporogenesis and preparation of the bioassays |
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147 | (2) |
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8.4.3 Sporulation inhibitor 1 |
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149 | (1) |
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8.4.3.1 Extraction of the sporulation inhibitor 1 from thallus |
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149 | (1) |
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8.4.3.2 Extraction of the sporulation inhibitor 1 from (axenic) growth medium |
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150 | (1) |
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8.4.3.3 Bioassay-guided cleanup of sporulation inhibitor 1 with size-exclusion chromatography |
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150 | (2) |
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8.4.4 Sporulation inhibitor 2 |
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152 | (1) |
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8.4.4.1 Extraction of the sporulation inhibitor 2 |
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152 | (1) |
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8.4.4.2 Bioassay-guided cleanup of the sporulation inhibitor 2 |
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153 | (1) |
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153 | (1) |
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8.4.5.1 Extraction and bioassay-guided cleanup of the swarming inhibitor |
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153 | (1) |
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8.4.5.2 Bioassay-guided cleanup of the swarming inhibitor |
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154 | (1) |
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155 | (1) |
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156 | (1) |
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156 | (3) |
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Chapter 9 Preparation of axenic cultures in Ulva (Chlorophyta) |
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159 | (16) |
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159 | (2) |
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161 | (2) |
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163 | (2) |
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163 | (1) |
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9.3.2 Consumables and reagents |
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163 | (1) |
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163 | (1) |
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164 | (1) |
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9.3.3 Ulva culture medium |
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164 | (1) |
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9.4 Experimental procedures |
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165 | (4) |
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9.4.1 Induction of gametogenesis and release of gametes |
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165 | (1) |
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9.4.2 Enrichment of gamete density |
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165 | (1) |
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9.4.3 Purification of gametes from bacteria |
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166 | (1) |
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9.4.4 Measuring gamete density: Flow cytometry |
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167 | (1) |
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9.4.5 Axenicity proof of gametes |
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167 | (1) |
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9.4.5.1 Test of axenicity on agar medium |
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168 | (1) |
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9.4.5.2 Test of axenicity by PCR |
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168 | (1) |
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169 | (1) |
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170 | (1) |
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171 | (4) |
| Section II: Chemical composition |
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Chapter 10 Biochar production from seaweeds |
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175 | (12) |
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Elizabeth Garrido-Ramirez |
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175 | (2) |
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177 | (1) |
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177 | (3) |
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10.3.1 Extraction and pre-drying of seaweed biomass |
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177 | (1) |
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10.3.2 Sample preparation by pyrolysis |
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177 | (3) |
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10.3.3 Biochar generation by pyrolysis |
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180 | (1) |
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10.3.4 Storage of biochar samples |
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180 | (1) |
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10.4 Experimental procedures |
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180 | (2) |
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10.4.1 Extraction and pre-drying of seaweed biomass (the case of Macrocystis pyrifera) |
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180 | (1) |
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10.4.2 Sample preparation for pyrolysis |
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180 | (1) |
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10.4.3 Biochar generation by pyrolysis |
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181 | (1) |
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10.4.4 Storage of biochar samples |
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181 | (1) |
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182 | (1) |
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183 | (1) |
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183 | (4) |
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Chapter 11 Identification and quantification of laminarins in brown algae |
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187 | (12) |
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187 | (2) |
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189 | (2) |
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191 | (1) |
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11.3.1 Extraction of laminarin from macroalgal material |
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191 | (1) |
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11.3.2 Precipitation of alginate |
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191 | (1) |
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11.3.3 Liquid chromatography-mass spectrometrical method measurement |
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192 | (1) |
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11.4 Experimental procedures |
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192 | (3) |
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11.4.1 Laminarin extraction |
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192 | (1) |
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11.4.2 LC-MS measurements |
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193 | (1) |
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11.4.3 Laminarin reference and quantification |
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194 | (1) |
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195 | (1) |
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196 | (1) |
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197 | (2) |
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Chapter 12 Determination of carbohydrate composition of macroalgae |
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199 | (12) |
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200 | (1) |
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201 | (1) |
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202 | (1) |
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202 | (1) |
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202 | (1) |
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203 | (1) |
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12.4 Experimental procedures |
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203 | (5) |
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12.4.1 Preparation seaweed sample |
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204 | (1) |
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204 | (1) |
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204 | (1) |
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205 | (1) |
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12.4.3 Analysis monomeric carbohydrates |
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205 | (1) |
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12.4.3.1 Sample preparation |
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205 | (1) |
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206 | (2) |
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208 | (1) |
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208 | (2) |
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208 | (1) |
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12.5.2 Seaweed hydrolysis |
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209 | (1) |
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12.5.3 High-performance anion-exchange chromatography with pulsed amperometric detector |
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209 | (1) |
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210 | (1) |
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210 | (1) |
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Chapter 13 Quantification of proteins in seaweeds |
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211 | (14) |
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212 | (1) |
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212 | (1) |
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213 | (4) |
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13.3.1 Kjeldahl method: Total protein determination |
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213 | (1) |
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13.3.2 Dumas method: Total protein determination |
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213 | (3) |
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13.3.3 Amino acid determination |
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216 | (1) |
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13.3.4 Nitrogen-to-protein-conversion factor determination |
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216 | (1) |
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13.3.5 Lowry method: Soluble protein determination |
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216 | (1) |
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13.3.6 Bradford method: Soluble protein assay |
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217 | (1) |
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13.4 Experimental procedures |
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217 | (5) |
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13.4.1 Kjeldahl method: Total protein determination |
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217 | (1) |
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217 | (1) |
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217 | (1) |
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218 | (1) |
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218 | (1) |
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13.4.2 Dumas method: Total protein determination |
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218 | (1) |
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218 | (1) |
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13.4.3 Amino acid determination |
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219 | (1) |
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13.4.3.1 Preparation of reagents |
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219 | (1) |
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13.4.3.2 Preparation of eluents |
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219 | (1) |
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13.4.3.3 Preparation of standards |
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219 | (1) |
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220 | (1) |
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220 | (1) |
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13.4.4 Nitrogen-to-protein-conversion factor determination |
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220 | (1) |
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220 | (1) |
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13.4.5 Lowry method: Soluble protein determination |
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221 | (1) |
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13.4.5.1 Preparation of the standard solution |
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221 | (1) |
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13.4.5.2 Preparation of Folin-Ciocalteau reagent |
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221 | (1) |
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13.4.5.3 Test tube protocol |
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222 | (1) |
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13.4.6 Bradford method: Soluble protein assay |
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222 | (1) |
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222 | (1) |
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222 | (1) |
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223 | (1) |
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223 | (2) |
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Chapter 14 Comprehensive phytohormone quantification in the red alga Pyropia yezoensis by liquid chromatography-mass spectrometry |
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225 | (12) |
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226 | (1) |
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226 | (1) |
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227 | (1) |
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14.3.1 Extraction and purification of phytohormones |
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227 | (1) |
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14.3.2 Liquid chromatography-mass spectrometry |
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228 | (1) |
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14.4 Experimental procedures |
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228 | (6) |
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229 | (1) |
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14.4.2 Partial purification of phytohormones with solid-phase extraction |
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229 | (2) |
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14.4.3 Liquid chromatography-mass spectrometry analysis |
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231 | (3) |
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234 | (1) |
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234 | (2) |
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236 | (1) |
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Chapter 15 Total phenolic content and antioxidant capacity analysis of seaweed biomass |
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237 | (12) |
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237 | (1) |
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238 | (1) |
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239 | (2) |
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239 | (1) |
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240 | (1) |
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240 | (1) |
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15.3.3.1 Total phenolic content analysis of seaweed biomass |
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240 | (1) |
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15.3.3.2 Antioxidant capacity analysis of seaweed biomass: 2,2-Diphenyl-1-picrylhydrazyl assay |
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241 | (1) |
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15.4 Experimental procedures |
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241 | (6) |
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15.4.1 Total phenolic content analysis of seaweed biomass |
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241 | (1) |
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15.4.1.1 Folin-Ciocalteu assay |
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241 | (1) |
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15.4.1.2 Prussian blue assay |
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243 | (2) |
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15.4.2 Antioxidant capacity analysis of seaweed biomass: 2,2-Diphenyl-1-picrylhydrazyl assay |
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245 | (2) |
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247 | (1) |
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247 | (1) |
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247 | (2) |
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Chapter 16 Extraction of phycocyanin and phycoerythrin pigments |
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249 | (18) |
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250 | (1) |
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251 | (1) |
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252 | (2) |
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252 | (1) |
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16.3.2 Algal conditioning |
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253 | (1) |
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16.3.3 Buffer preparation |
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254 | (1) |
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16.4 Experimental procedures |
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254 | (5) |
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16.4.1 Phycobiliproteins extraction |
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254 | (1) |
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254 | (1) |
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254 | (1) |
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255 | (1) |
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16.4.1.4 Ultrasonic treatment |
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255 | (1) |
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16.4.1.5 Enzymatic hydrolysis |
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255 | (1) |
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16.4.2 Phycobiliprotein purification |
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256 | (1) |
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16.4.2.1 Adsorption bed chromatography |
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256 | (1) |
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16.4.2.2 Ammonium sulphate precipitation |
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257 | (1) |
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16.4.2.3 Ion-exchange column |
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257 | (1) |
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16.4.2.4 Hydroxyapatite chromatography |
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257 | (1) |
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16.4.2.5 Aqueous two-phase extraction |
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258 | (1) |
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258 | (1) |
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259 | (1) |
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260 | (2) |
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262 | (5) |
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Chapter 17 Quantification and localization of reactive oxygen species in marine macrophytes |
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267 | (12) |
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268 | (1) |
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269 | (4) |
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17.2.1 ROS detection reaction mechanism using dihydroethidium and dichlorodihydrofluorescein diacetate |
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269 | (2) |
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17.2.2 Histochemical localization mechanism using nitro blue tetrazolium and DAB staining |
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271 | (2) |
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273 | (1) |
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17.3.1 Reactive oxygen species visualization by microscopy |
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273 | (1) |
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17.3.2 Reactive oxygen species quantification using dihydroethidium and dichlorodihydrofluorescein diacetate |
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273 | (1) |
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17.3.3 ROS histochemical localization |
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273 | (1) |
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17.4 Experimental procedures |
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274 | (1) |
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17.4.1 Reactive oxygen species visualization by microscopy |
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274 | (1) |
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17.4.2 ROS quantification using DHE and DCFH-DA |
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274 | (1) |
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17.4.3 Histochemical localization for reactive oxygen species detection |
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274 | (1) |
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275 | (1) |
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17.5.1 Reactive oxygen species visualization |
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275 | (1) |
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17.5.2 Reactive oxygen species quantification |
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275 | (1) |
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17.5.3 Reactive oxygen species histochemical localization |
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276 | (1) |
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276 | (1) |
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276 | (3) |
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Chapter 18 Metabolomics of intra- and extracellular metabolites from micro- and macroalgae using GC-MS and LC-MS |
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279 | (22) |
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280 | (1) |
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281 | (2) |
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283 | (4) |
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283 | (1) |
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283 | (2) |
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285 | (1) |
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285 | (1) |
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18.3.5 Instrumental setup |
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286 | (1) |
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18.4 Experimental procedures |
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287 | (8) |
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18.4.1 Sampling and metabolic quenching |
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288 | (1) |
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18.4.1.1 Filtration of planktonic single-celled algae |
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288 | (1) |
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18.4.1.2 Collection of algal gametes (of Ulva spp.) |
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288 | (1) |
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18.4.1.3 Collection of algal thalli |
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289 | (1) |
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18.4.2 Solid-phase extraction of extracellular metabolites |
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289 | (1) |
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18.4.3 Extraction of intracellular metabolites |
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290 | (1) |
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18.4.3.1 Cell disruption by ultrasound treatment |
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290 | (1) |
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18.4.3.2 Cell disruption with a bead mill |
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290 | (1) |
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18.4.4 Two-step-derivatization for GC-MS analysis |
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291 | (1) |
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292 | (1) |
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18.4.6 Data analysis for GC-MS data |
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292 | (3) |
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295 | (2) |
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297 | (1) |
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297 | (4) |
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Chapter 19 Preparative extraction of exometabolites from seaweed surfaces |
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301 | (8) |
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301 | (1) |
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302 | (2) |
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304 | (1) |
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19.4 Experimental procedures |
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304 | (3) |
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19.4.1 Development of extraction protocol |
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304 | (1) |
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19.4.2 Determination of algal surface/mass ratio |
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305 | (1) |
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306 | (1) |
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307 | (1) |
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307 | (2) |
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Chapter 20 Disruption-free solid-phase extraction of surface metabolites from macroalgae |
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309 | (14) |
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309 | (1) |
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310 | (1) |
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311 | (1) |
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311 | (1) |
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20.3.2 Solid-phase surface extraction method |
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312 | (1) |
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20.4 Experimental procedures |
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312 | (3) |
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20.4.1 Extraction procedure |
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313 | (2) |
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315 | (1) |
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315 | (2) |
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317 | (1) |
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317 | (6) |
| Section III: Cellular and molecular characterization |
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Chapter 21 The immunodetection and in situ imaging of cell-wall polysaccharides in brown algae |
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323 | (12) |
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323 | (1) |
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21.2 State of the art: Imaging cell walls in brown algae |
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324 | (1) |
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325 | (2) |
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21.4 Experimental procedures |
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327 | (5) |
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21.4.1 Preparation of algal material and fixation procedures |
|
|
327 | (1) |
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21.4.2 Embedding protocol for London resins White resin |
|
|
328 | (1) |
|
21.4.3 Embedding protocol for Lowicryl resin |
|
|
329 | (1) |
|
21.4.4 Sectioning of resin-embedded samples |
|
|
330 | (1) |
|
21.4.5 Imaging algal cell walls using general stains |
|
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330 | (1) |
|
21.4.6 Immunolabeling for fluorescence microscopy |
|
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330 | (1) |
|
21.4.7 Immunolabeling for electron microscopy |
|
|
331 | (1) |
|
21.4.8 Pretreatments before immunolabeling |
|
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331 | (1) |
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332 | (1) |
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333 | (1) |
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333 | (2) |
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Chapter 22 Atomic force microscopy based analysis of cell-wall elasticity in macroalgae |
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335 | (14) |
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335 | (1) |
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22.1.1 Biomechanical characterization of macroalgal cell walls |
|
|
335 | (1) |
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336 | (2) |
|
22.2.1 Mechanical characterization of macroalgal cell walls |
|
|
336 | (2) |
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|
|
338 | (1) |
|
22.4 Experimental procedures |
|
|
338 | (7) |
|
22.4.1 Preparation of algal material for atomic force microscopy analysis |
|
|
338 | (1) |
|
22.4.2 Choosing a force for indentation |
|
|
339 | (2) |
|
22.4.3 Influence of osmoticum upon macroalgal mechanics |
|
|
341 | (1) |
|
22.4.4 Selection of tip size |
|
|
342 | (1) |
|
22.4.5 Performing atomic force microscopy based elasticity measurements |
|
|
343 | (2) |
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345 | (1) |
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346 | (1) |
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346 | (3) |
|
Chapter 23 Dynamic and microscale mapping of cell growth: Case of Ectocarpus filament cells |
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349 | (16) |
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350 | (1) |
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351 | (2) |
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353 | (1) |
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353 | (1) |
|
23.3.2 Ectocarpus culture |
|
|
353 | (1) |
|
23.3.3 Equipment and software |
|
|
353 | (1) |
|
23.4 Experimental procedures |
|
|
354 | (6) |
|
23.4.1 Preparation of living algal material for time-lapse microscopy |
|
|
354 | (1) |
|
23.4.1.1 Gametes release on glass-bottom Petri dish, multiwell plate or free coverslips |
|
|
354 | (1) |
|
23.4.1.2 Preparation of "homemade" glass bottom Petri dishes |
|
|
354 | (1) |
|
23.4.2 Cell-surface deformation monitoring using time-lapse microscopy |
|
|
355 | (1) |
|
23.4.2.1 Prior preparation of the equipment |
|
|
356 | (1) |
|
23.4.2.2 Cell-surface labeling with fluorescent microspheres |
|
|
356 | (1) |
|
23.4.2.3 Measuring irreversible deformations during growth (plastic strain) |
|
|
356 | (1) |
|
23.4.2.4 Measuring deformations induced by hypo- or hypertonic shock (mainly elastic deformations) |
|
|
357 | (2) |
|
23.4.3 Picture analysis: Quantification of cell-surface marker displacement |
|
|
359 | (1) |
|
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360 | (2) |
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362 | (3) |
|
Chapter 24 Actin fluorescent staining in the filamentous brown alga Ectocarpus siliculosus |
|
|
365 | (16) |
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365 | (2) |
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367 | (2) |
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369 | (4) |
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|
|
369 | (1) |
|
24.3.2 Solutions and recipes |
|
|
369 | (1) |
|
24.3.2.1 Preparation of Rhodamine-Phalloidin (R415) or AlexaFluor568-Phalloidin (A12380, life technology) |
|
|
369 | (1) |
|
24.3.2.2 Preparation of buffers |
|
|
370 | (1) |
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|
|
372 | (1) |
|
|
|
373 | (1) |
|
24.4 Experimental procedures |
|
|
373 | (2) |
|
24.4.1 Preparation of poly-L-lysine-coated coverslips |
|
|
373 | (1) |
|
24.4.2 Preparation of Ectocarpus samples |
|
|
373 | (1) |
|
24.4.2.1 Filaments grown on coverslips |
|
|
373 | (1) |
|
24.4.2.2 Free-floating filaments |
|
|
374 | (1) |
|
24.4.3 Actin staining procedure |
|
|
374 | (1) |
|
|
|
375 | (1) |
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|
|
376 | (1) |
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|
|
376 | (5) |
|
Chapter 25 Cryofixation of brown algae for transmission electron microscopy |
|
|
381 | (10) |
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381 | (2) |
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|
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383 | (1) |
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|
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384 | (1) |
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|
|
384 | (1) |
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|
|
384 | (1) |
|
25.4 Experimental procedures |
|
|
384 | (3) |
|
25.4.1 Preparation of material |
|
|
384 | (1) |
|
25.4.2 Cryogen and substitution fixative |
|
|
385 | (1) |
|
|
|
386 | (1) |
|
25.4.4 Freeze substitution |
|
|
386 | (1) |
|
25.4.5 Infiltration and embedding of samples into resin |
|
|
386 | (1) |
|
|
|
387 | (1) |
|
|
|
387 | (1) |
|
|
|
388 | (3) |
|
Chapter 26 Probing the subcellular topography of seaweeds: Transmission electron microscopy, immunocytochemistry, and correlative light microscopy |
|
|
391 | (20) |
|
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|
|
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|
|
392 | (1) |
|
|
|
393 | (2) |
|
26.2.1 General considerations |
|
|
393 | (1) |
|
26.2.2 Protocol considerations |
|
|
394 | (1) |
|
|
|
395 | (4) |
|
26.3.1 Fixation, dehydration, and embedding |
|
|
395 | (2) |
|
|
|
397 | (1) |
|
|
|
397 | (1) |
|
26.3.4 Uranyl acetate/Lead citrate staining |
|
|
397 | (1) |
|
26.3.5 Immunogold labeling |
|
|
398 | (1) |
|
26.4 Experimental procedures |
|
|
399 | (8) |
|
|
|
399 | (1) |
|
|
|
399 | (1) |
|
|
|
400 | (1) |
|
|
|
400 | (1) |
|
|
|
401 | (2) |
|
26.4.6 Uranyl acetate/Lead citrate staining |
|
|
403 | (1) |
|
26.4.7 Immunogold labeling |
|
|
404 | (1) |
|
26.4.8 Considerations for light microscopy and correlative studies |
|
|
405 | (2) |
|
|
|
407 | (2) |
|
|
|
409 | (2) |
|
Chapter 27 Coralline algae preparation for scanning electron microscopy and optical microscopy |
|
|
411 | (18) |
|
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|
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|
|
411 | (1) |
|
|
|
412 | (1) |
|
|
|
412 | (2) |
|
27.3.1 Materials for optical microscopy |
|
|
412 | (1) |
|
27.3.2 Materials for scanning electron microscopy |
|
|
413 | (1) |
|
27.4 Experimental procedures |
|
|
414 | (11) |
|
27.4.1 Choice of the portion to cut and orientation of the sample |
|
|
414 | (6) |
|
27.4.2 Optical microscopy |
|
|
420 | (3) |
|
27.4.3 Scanning electron microscopy |
|
|
423 | (2) |
|
|
|
425 | (2) |
|
|
|
427 | (1) |
|
|
|
427 | (2) |
|
Chapter 28 Extraction of high quality RNA from brown algae for transcriptomic analysis |
|
|
429 | (12) |
|
|
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|
|
429 | (1) |
|
|
|
430 | (2) |
|
|
|
432 | (1) |
|
|
|
432 | (1) |
|
28.3.2 Sample homogenization |
|
|
432 | (1) |
|
|
|
432 | (1) |
|
|
|
432 | (1) |
|
|
|
432 | (1) |
|
28.3.4 RNA quality control |
|
|
433 | (1) |
|
28.4 Experimental procedures |
|
|
433 | (3) |
|
28.4.1 Sample homogenization |
|
|
433 | (1) |
|
|
|
433 | (2) |
|
28.4.3 RNA quality control |
|
|
435 | (1) |
|
28.4.3.1 Measuring RNA concentration and purity |
|
|
435 | (1) |
|
28.4.3.2 RNA integrity control |
|
|
436 | (1) |
|
|
|
436 | (1) |
|
|
|
437 | (1) |
|
|
|
437 | (4) |
|
Chapter 29 Induction of sexual reproduction in Spirogyra cultures for laser capture microdissection of gametes and zygotes |
|
|
441 | (12) |
|
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|
|
441 | (2) |
|
|
|
443 | (1) |
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|
|
444 | (1) |
|
|
|
444 | (1) |
|
29.3.1.1 Algae cultivation |
|
|
444 | (1) |
|
29.3.1.2 Laser microdissection |
|
|
444 | (1) |
|
|
|
445 | (1) |
|
29.4 Experimental procedures |
|
|
445 | (4) |
|
29.4.1 Cultivation of Spirogyra and induction of sexual reproduction |
|
|
445 | (1) |
|
29.4.1.1 Media preparation |
|
|
445 | (1) |
|
29.4.1.2 Vegetative growth |
|
|
446 | (1) |
|
29.4.1.3 Sexual reproduction induction |
|
|
446 | (1) |
|
29.4.2 Fixation of thallus before laser capture microdissection |
|
|
447 | (2) |
|
29.4.3 Laser capture microdissection |
|
|
449 | (1) |
|
|
|
449 | (2) |
|
|
|
451 | (1) |
|
|
|
451 | (2) |
|
Chapter 30 Cloning and expression strategies for the postgenomic analysis of brown algae |
|
|
453 | (16) |
|
|
|
|
|
454 | (1) |
|
|
|
455 | (1) |
|
|
|
456 | (3) |
|
30.3.1 Strains and vector |
|
|
457 | (1) |
|
|
|
457 | (1) |
|
|
|
457 | (1) |
|
30.3.2 General stock solutions |
|
|
457 | (1) |
|
30.3.3 PCR, cloning and transformation procedures |
|
|
457 | (1) |
|
30.3.4 Protein expression and purification |
|
|
458 | (1) |
|
|
|
458 | (1) |
|
|
|
459 | (1) |
|
30.4 Experimental procedures |
|
|
459 | (5) |
|
30.4.1 Bioinformatics analysis |
|
|
459 | (1) |
|
|
|
459 | (1) |
|
30.4.2.1 Amplification of gene of interest |
|
|
459 | (1) |
|
30.4.2.2 PCR fragment purification |
|
|
460 | (1) |
|
30.4.2.3 PCR fragment digestion |
|
|
460 | (1) |
|
30.4.2.4 pFO4 vector preparation |
|
|
460 | (1) |
|
|
|
461 | (1) |
|
30.4.2.6 Transformation in E. coli DH5a |
|
|
461 | (1) |
|
|
|
461 | (1) |
|
30.4.2.8 Plasmid extraction and glycerol stock |
|
|
462 | (1) |
|
30.4.2.9 Transformation of expression strain, PCR screening, and glycerol stock |
|
|
462 | (1) |
|
30.4.3 Small-scale test expression |
|
|
462 | (1) |
|
30.4.3.1 Cultivation was performed in two phases |
|
|
462 | (1) |
|
30.4.3.2 Lysis of cells and detection of proteins |
|
|
462 | (1) |
|
30.4.4 Large-scale expression and purification |
|
|
463 | (1) |
|
30.4.4.1 Expression culture |
|
|
463 | (1) |
|
30.4.4.2 Affinity chromatography |
|
|
463 | (1) |
|
30.4.4.3 Size-exclusion chromatography |
|
|
463 | (1) |
|
30.4.4.4 Analysis of the chromatography |
|
|
463 | (1) |
|
|
|
464 | (2) |
|
|
|
466 | (3) |
|
Chapter 31 Polyethylene glycol-mediated transformation in the green macroalga Ulva mutabilis (Chlorophyta): A forward genetics approach |
|
|
469 | (16) |
|
|
|
|
|
|
|
|
|
470 | (1) |
|
|
|
471 | (3) |
|
|
|
474 | (1) |
|
31.3.1 Equipment, consumables, and reagents |
|
|
474 | (1) |
|
31.3.1.1 Equipment and consumables |
|
|
474 | (1) |
|
|
|
474 | (1) |
|
31.3.2 Ulva culture medium |
|
|
475 | (1) |
|
31.4 Experimental procedures |
|
|
475 | (6) |
|
31.4.1 Appropriate vector preparation for transformation of U. mutabilis |
|
|
475 | (1) |
|
31.4.2 Preparation of gametes of U. mutabilis for transformation |
|
|
475 | (1) |
|
31.4.3 Transformation of U. mutabilis gametes |
|
|
476 | (1) |
|
31.4.4 Cultivation of transgenic Ulva lines and selection by phleomycin treatment |
|
|
477 | (1) |
|
31.4.5 Determination of the transformation efficiency and further single-strain cultivation |
|
|
478 | (1) |
|
31.4.6 Confirmation of stable transformation |
|
|
478 | (3) |
|
|
|
481 | (1) |
|
|
|
482 | (1) |
|
|
|
482 | (3) |
| Index |
|
485 | |