Maximising Production of Palmaria Palmata (Linnaeus) Weber & Mohr, 1805

Home , Chondrus crispus, Palmaria palmata

FINAL REPORT TO MASTS under Visiting Fellowship Scheme 2015

Dr J Craig Sanderson

Kai Ho, Tasmanian Sea Vegetables, Associate University Tasmania.

27 July 2015 - 23 October 2015

Maximising Production of Palmaria palmata (Linnaeus) Weber & Mohr, 1805

Scottish Association Marine Sciences Supported by:

 Mara Seaweed  Loch Fyne Oysters  Deakin University and  NAFC Marine Centre University of the Highlands and the Islands.

Acknowledgements

Thanks to Dr Adam Hughes for driving this project from the beginning and overall support while in Scotland. We were grateful to have received funding through MASTS which enabled the project. Dr Phillip Kerrison and the technical staff at SAMS facilitated the seaweed culture making it possible to do more than anticipated within the set time frame. Dr Claire Gachon and Yacine Badis provided inspiration for future research. Dr Liz Cook facilitated off site support. Helpful discussions were made possible through Phycomorph at the meeting in Porto and we were very grateful for Dr Lesley Mc Evoy’s support in the Sheltlands. The final laboratory work could not have been completed without the very capable SAMS intern Sandra Yeregui Osa. ABSTRACT

The aim of this project was to investigate maximising spore release and settlement success for the commercial red algal species Palmaria palmata. Difficulties were encountered in obtaining fertile material to complete the original experimental design so the direction was altered to incorporate investigating mass production of Palmaria palmata through vegetative propagation. This involved obtaining plantlets and tetraspores from fragments and cell aggregates of meristematic and sub-meristimatic tissue. This fulfiled the initial aim of the original project through enabling mass production of Palmaria palmata and has the added bonus of not requiring a source of fertile material meaning it would be possible to conduct at any time of the year. Success in these areas will facilitate the beginning of a future industry supplying Palmaria for edible markets and may lead to opportunities for culturing other algae for more varied uses in the commercial sector.

Vegetative propagation of Palmaria palmata plants was initially sourced through cutting meristems into small pieces approx. 1 x 1 mm. Macerated tissue was then trialled and then very small (100µm) sections of meristematic tissue generated using a microtome. Growth of plant pieces of all sizes was a consistent 10% per day. Culturing of segments of meristems from Osmundia sp and Chondrus crispus for comparison resulted in growth rates of 2 and 3% per day respectively.

Freezing of Palmaria plants was shown to kill all plant tissue except the meristems. Use of a freeze microtome for generating plant fragments from meristems may be worth following up. This method has the added advantage that freezing kills off all associated biota, sterilising the fragments.

The production of new plantlets for Palmaria occurred principally from the meristematic tissue. As part of this project, preliminary work was conducted on generating adventive material from plant tissues other than the meristem using auxins and other shoot producing compounds. Results were confounded by plant infections. Further work needs to be conducted with an emphasis on maintaining axenic culture conditions. Best growth was obtained using commercially available seaweed extract plant growth promoters.

Tips grown from bladelets of Palmaria blades in a commercial aquaculture farm grew from 450 to 58000mm2 over a period of 56 days equivalent to 8.7% per day. This indicates potential growth to full size from spores or very small plant pieces within six months when grown in tanks on land. Reports of growth rates elsewhere for Palmaria palmata of 20+% indicate a potential growth period to full size plants within 2 months.

From tetraspores that were released from fertile plants collected as part of this project, and from Palmaria plants maintained in tanks as part of this project, an infecting oomycete was found that has only recently been identified and yet to be reported in the literature. This may have particular relevance for any industry based on cultivating Palmaria from spores as crashes of cultures of Palmaria have been common place and this fungus may be the cause. The find expands our knowledge in this area. An endophyte of Palmaria palmata was also identified: Acrochaetium secundatum which is a very common contaminant on blades of Palmaria. This identification expanded the range of this ecologically significant microorganism.

ABSTRACT INTRODUCTION PART 1 Investigation to maximise spore release and settlement success for the commercial red algal species Palmaria palmata (Linnaeus) Weber & Mohr, 1805 PART 2 Mass culture of Palmaria palmata from meristematic tissue. PART 3 Growth and adventive tissue accelerating compounds. REFERENCES Appendix 1 Identification of meristematic tissue through freezing. Appendix 2 Growth of Palmaria fronds from excised adventitious shoots (tips). Appendix 3 Palmaria Oomycete infection Appendix 4 Palmaria Endophyte/epiphyte Appendix 5 Phycomorph Workshop Porto, Portugal September 2015. Appendix 6 Shetlands visit, September 2015. Appendix 7

MASTS, Marine Alliance for Science and Technology for Scotland meeting October 2015. INTRODUCTION

In many developed countries significant proportions of the population are obese or overweight. There is compelling evidence from literature that seaweeds should become more common in global diets. Seaweeds are high in protein, dietary fibre, long-chain omega-3 fatty acids and a suite of vitamins, minerals and antioxidants. Regular consumption can reduce obesity and associated illnesses, such as diabetes and heart disease, whilst potentially enhancing the health of societies now and for future generations.

Worldwide seaweed production via aquaculture has doubled in the past 10 years and now accounts for a total annual harvest of 23.8 Mt valued at over US$6 billion globally. Other than culture Porphyra, which is the most valuable edible seaweed species in the world, culture of red seaweeds is secondary to the easier to culture brown seaweeds, despite red seaweeds generally being of higher nutritional value. Demand for red seaweeds currently exceeds supply.

Red seaweeds are also the source of both unique chemical compounds and various gelling agents which are widely used as food additives.

The red alga Palmaria palmata (L.) O. Kuntze is a conspicuous vegetation component in the low intertidal and upper subtidal zones along both sides of the North Atlantic Ocean, from the Arctic to cold-temperate regions. The alga has been harvested and used for centuries in the coastal areas of Northern Europe and North America. Pressure on wild stocks has encouraged investigation into mass culture methods which has met some success for tank and rope cultivation.

The original aim of this project was to further exploit the current knowledge of Palmaria life history traits to provide mass spore release on cue at any time of the year. A number of substrates for on-growing Palmaria were to be investigated.

Problems were encountered sourcing tetrasporic plants for this investigation and the project brief was expanded to investigate mass production of Palmaria palmata through vegetative propagation as outlined in a relatively recent publication by Titlyanov et al. (2006) entitled ‘New methods of obtaining plantlets and tetraspores from fragments and cell aggregates of meristematic and sub-meristimatic tissue of the red alga Palmaria palmata’. This fulfils the initial aim of the original proposed project through enabling mass production of Palmaria. It also has the added bonus of not requiring a source of fertile material and can be conducted at any time of the year. Success in these areas will facilitate the beginning of a future industry supplying Palmaria for edible markets and may lead to other opportunities for culturing red algae for more varied uses in the commercial sector.

This proposed project fits within the MASTS ‘Productive Seas’ research theme. Aquaculture is pivotal to the rural economy of some areas within Scotland and is likely to expand into the production of other non-food products and services through biofuels, marine biotechnology and genomics. Research into growing Palmaria complements aquaculture production of seaweeds for biofuels and is a nutrient extractive aquaculture operation as distinct from fin fish aquaculture. PART 1

Investigation to maximise spore release and settlement success for the commercial red algal species Palmaria palmata (Linnaeus) Weber & Mohr, 1805

Palmaria palmata fronds are usually fertile in the first six months of the year although this can vary depending on geographic location. In the vicinity of Oban, fronds may become fertile as early as October-November (SAMS personnel pers com). The seasonality of reproduction did not agree with the timing of this project (August-October). Pang and Luning 2006 have induced tetraspory in Palmaria palmata under conditions of short day (8:16 dark: light) and low temperature: 10oC within 5 weeks. This was trialled as part of the project but there were difficulties associated with maintaining these conditions. Inducing tetraspory is also dependent on having tetrasporic plants and as tetrasporic plants have the same appearance the same as male plants, there is the possibility that all plants collected were males. Inducing tetraspory was not successful.

Careful examination of field populations of P. palmata at Ellenabeich adjacent to Easdale Island (56°17'45.9"N 5°39'15.2"W) and nearby Seaview (56°17'19.8"N 5°38'04.2"W) revealed small numbers of fertile plants. These plants usually appeared to have been damaged in some way, either through exposure to sun or herbivory and going fertile may have been a stress response. These plants were used as a source of tetraspores.

Approximately 300g wet weight Palmaria plants were physically cleaned and placed in 2l of sterilised nutrient enhanced (KTH), aerated seawater. Each day, over four days, the plants were transferred into a new solution of nutrients and seawater. The seawater the plants came from was left to settle for 2-4 hours and the excess water poured off. The remaining medium was examined for spores. Due to the poor condition of some of the plants, there was much detritus amongst the spores. In the absence of clean starter spores, these were trialled for tumble culture. These spores were added to 10l car buoys in filter sterilised seawater with KTH medium added. The medium was replaced weekly. These were initiated on 10/9/2015 and finally examined on 19/10/2015. They revealed spores of approximately 100 x 300µm in size amongst many contaminants. PART 2

2/ MASS CULTURE OF PLANTS FROM MERISTIMATIC TISSUE

Mass culture of Palmaria palmata was investigated using fragments and cell aggregates of meristematic and sub-meristimatic tissue. This methodology follows that of Titlyanov et al 2006a and Titlyanov et al 2006b. See also reviews of Reddy et al 2008 and Titlyanov and Titlyanov 2010. The same technique was also trialled for Osmundea and Mastocarpus stellatus for comparison. The aim is to culture significant quantities of Palmaria from vegetative tissue and progress towards a means of scaling up cultures using this methodology.

METHOD

Using fragments of plants for vegetative reproduction was trialled on three occasions. Firstly with meristem and thallous segments approx. 1x1 mm (with Osmundea for comparison, Section 1), the second for macerated meristematic tissue (with Chondrus crispus for comparison, Section 2) and the third with meristematic, side, and thallous tissue and meristematic tissue cut using a microtome (see diagram below for plant part locations, Section 3). In all trials, the fragments were added to 500ml culture flasks, with aeration under light of intensity 120 µ mol-2s-1 . Media was changed weekly when measurements of the fragments was also conducted. Media consisted of filter sterilized, tindalized, nutrient enhanced according to the ‘KTH’ recipe. ‘KTH’ is a modified f/2 recipe using readily available commercial sources of NO3 and PO4. Measurements consisted of taking pictures for later analysis of area of the fragments and weighing the filter gauze used to separate the fragments with and without samples for wet weight of plant pieces. Area of the fragments was determined using Image J. The rates of growth of the fragments were calculated by the formula:

µ = ln(Area2/Area1) x n-1 x 100 where µ is the relative growth rate (% x day-1), Area2 and Area1 are final and initial size, ‘n’ is the number of days. Figure showing parts of Palmaria frond mentioned in the text. Side and thallous tissue = ‘sub-meristimatic’ tissue.

meristem

thallous side

Section 1

In the first trial, growth was compared for tissue of sizes approximately 1 x 1 mm obtained from meristems and sub-meristimatic tissue (‘M’ for meristem, ‘T’ for thallus, see diagram). Plants used were small plants less than 15cm tall obtained from Otter Ferry culture tanks. There were approximately 10 fragments in each culture flask. Five replicates of each. The experiment ran from 12 August 2015 to 7 October 2015. RESULTS

See Figures

Graphs showing growth data for section 1 plants – average area per plant fragment. 3500 3500 M1 T1 3000 Palmaria Meristem 3000 Palmaria Thallous M2 T2 2500 2500

2 M3 2 T3 ^ 2000 ^ 2000 m m T4 m m M4

a 1500 a 1500 e e

r r T5 A 1000 M5 A 1000

500 500

0 0 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 / / / / / / / / / / / / / / / / 8 8 8 9 9 9 0 0 8 8 8 9 9 9 0 0 / / / / / / / / / / / / 1 1 1 1 4 4 4 3 3 3 / / 4 4 4 3 3 3 / / 1 2 1 2 3 3 1 2 1 2 3 3 1 1 70 Osmundea Meristem 60

50 2 ^ 40 O1 m m

a 30 e r

A O3 20 O4 10 O5 0 5 5 5 5 5 5 5 5 1 1 1 1 1 1 1 1 / / / / / / / / 8 8 8 9 9 9 0 0 / / / / / / 1 1 4 4 4 3 3 3 / / 1 2 1 2 3 3 1

Graphs showing total fragment area versus weight data for section 1 plants

2.5 Palmaria 0.05 Osmundea 0.05 2 y = 0.0007x 0.04 R² = 0.039 y = 0.0005x 0.04 1.5 R² = 0.9478 0.03 m m g g

t 0.03 t h h g g

i 1

i 0.02 e e W

W 0.02 0.5 0.01 0.01 0 0.00 0 1000 2000 3000 4000 0 20 40 60 80 Area mm^2 Area mm^2 Table showing relative growth rates for Section 1 plant fragments.

DATE 12/8/15 19/8/15 26/8/15 2/9/15 10/9/15 21/9/15 29/9/15 7/10/15 Initiated O1 2.22 2.10 3.08 -1.31 0.58 3.38 O2 -14.17 4.96 2.63 5.73 -3.45 0.54 O3 2.64 1.75 O4 6.30 5.13 O5 7.74 AVERAGE -0.75 4.34 2.85 2.21 -1.43 1.96 T1 -16.41 18.97 7.02 4.60 2.99 3.35 T2 15.25 11.19 13.88 8.73 6.82 4.37 T3 -8.42 T4 -21.96 T5 2.26 1.66 2.66 5.55 5.39 AVERAGE -5.86 10.61 10.45 5.33 5.12 4.37 M1 20.33 9.85 6.17 7.29 9.02 9.88 M2 -2.39 7.68 6.23 8.34 8.93 10.92 M3 7.98 6.07 4.76 9.40 15.48 13.00 M4 7.62 6.26 M5 4.13 4.11 AVERAGE 7.53 6.79 5.72 8.34 11.14 11.27

Example of change in size as a result of growth of fragments, Section 1.

M3 26/8/15 M3 07/10/15 DISCUSSION

Growth of Palmaria meristematic plant fragments averaged close to 10% and thallous fragments 5%. Osmundea fragments: 2-3%. A couple of the thallous cultures were ended prematurely as the pieces disintegrated. One of the thallous cultures (T2) had growth comparable to the meristematic cultures. This sort of variation in growth for the sub- meristimatic areas is consistent with variation found by Titlyanov et al 2006. Titlyanov et al 2006 recorded up to 30% growth although this was only recorded for the second week. Their cultures also levelled out at approximately 10% for meristematic tissue (6% for sub- meristimatic tissue).

Titlyanov et al 2006 claim plant regeneration from cell aggregates of 10-100,000 cells. In an attempt to test this claim, macerated tissue of varying sizes was trialled in Section 2.

Section 2

In the second trial, growth was compared for tissue that was macerated from the meristems of Palmaria and Mastocarpus stellatus. Plants used were obtained from Easdale (56°17'45.9"N 5°39'15.2"W and nearby Seaview (56°17'19.8"N 5°38'04.2"W). There were multiple fragments in each culture flask. Three replicates of each flask. The experiment ran from 29 August 2015 to 7 October 2015.

Graphs showing growth data for section 2 plants

600 Pal1 500 Pal2 Pal3 400

2 Mast1 ^

m Mast2 300 m Mast3 a e r Sampled

A 200

100

0 5 5 5 5 5 5 1 1 1 1 1 1 / / / / / / 8 9 9 9 0 0 / / / / 1 1 4 3 3 3 / / 2 1 2 3 3 1 Graphs showing area v weight data for section 2 plants

0.35 0.12 Palmaria palmata Mastocarpus stellatus 0.3 0.1 y = 0.01x 0.25 y = 0.0095x R² = 0.6591 0.08 R² = 0.7585 ) ) g g 0.2 ( (

t t 0.06 h h g g i

i 0.15 e e 0.04 W W 0.1 0.05 0.02 0 0 0 10 20 30 0 2 4 6 8 10 12 Area mm^2 Area mm^2

Table showing relative growth rates for Section 2 plant fragments.

DATE 19/8/15 26/8/15 2/9/15 10/9/15 21/9/15 29/9/15

Pal1 2.28 13.15 8.30 Pal2 14.59 6.19 11.81 Pal3 7.00 10.66 9.91 Average 7.96 10.00 10.01 Mast1 6.43 3.38 6.36 Mast2 2.05 3.52 3.79 Mast3 1.25 1.28 11.47 Average 3.25 2.73 7.21** **Influenced by epiphytes

Example of change in size as a result of growth of fragments, Section 2.

Pal2 10/9/15

Pal2 7/10/15 DISCUSSION

Again growth rates for Palmaria meristematic tissue approached 10%. Growth rates of Mastocarpus stellatus over a similar period was 2% except for the final measurement (7%) but this was confounded by the presence of epiphytes (Ectocarpus sp. and possibly a Petalonia like alga ?). Cutting the Palmaria into very small pieces was found to be very difficult and the resultant fragments very variable. For this reason in Section 3 a microtome was trialled to cut small pieces. Section 3

In the third trial, growth was compared for:

1/ Apex or meristematic tissue (10 fragments each replicate)

2/ Side tissue (10 fragments each replicate)

3/ Branch tissue (10 fragments each replicate)

4/ 2.5mm sections of thallous (5 pieces each replicate)

5/ 5mm sections of thallous (3 pieces each replicate)

6/ microtome generated fragments for meristem: approx. 500µm (‘one turn’; 10 fragments each replicate) and

7/ approx.100 µm (1/3 turn; 10 fragments each replicate).

The Palmaria tissue used for this experiment was sourced from identified tetrasporic plants. This was so that tetraspores may be generated within 5-6 weeks as per Titlyanov et al 2006. The two different sized sections of thallous (2.5mm and 5mm square) were trialled to see if there was a difference in the amount of time it would take before they became reproductive. Two replicates of each. The experiment ran from 11 September 2015 to 15 October 2015.

The microtome generated segments were produced using a standard microtome with a sliced carrot (Daucus carota) to hold sections of meristem. This enabled cutting sections a lot smaller than it was possible to do by hand, and of a consistent size. The carrot was kept refrigerated until used and the microtome blade was chilled with ice prior to use. This experiment was initiated on 11 September 2015.

Table showing relative growth rates for Section 3 plant fragments. ‘Turn’ refers to a turn of the microtome wheel equivalent to approx. 0.01mm.

DATE 11/9/15 21/9/15 29/9/15 7/10/15 14/10/15 21/10/15 ApexM1 start 4.78 2.52 ApexM2 Start 5.88 5.38 AVERAGE 5.33 3.95 SideM1 Start 2.32 0.78 SideM2 Start -5.70 4.48 AVERAGE -1.69 2.63 BranchM1 Start 4.04 3.50 BranchM2 Start 1.96 3.52 AVERAGE 3.00 3.51 1Turn 1 Start 35.73 10.92 7.71 6.11 8.09 1Turn 2 Start 35.00 12.16 10.71 6.67 4.45 AVERAGE 35.36 11.54 9.21 6.39 6.27 1/3Turn 1 Start 22.80 9.46 6.90 8.10 4.06 1/3Turn 2 start 22.42 7.13 9.18 7.01 6.91 Graphs showing growth data for section 3 plants

180 AVERAGEPalmaria palmata 22.61 8.29180 2.5mm 8.04 1 7.55 5.48 160 ApexM1 160 2.5mm 2 140 ApexM2 140 5mm 1 120 SideM1 120 2 2 ^ 100 SideM2 ^ 5mm 2 m 100 m m m BranchM1

80 Sampled a

a 80 e e

r BranchM2 r

A 60 Sampled A 60 40 40 20 20 0 0 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 / / / / / / / / / / / / / / / / / / 9 9 9 9 9 0 0 0 0 9 9 9 9 9 0 0 0 0 / / / / / / / / / / 1 1 1 1 1 1 1 1 8 3 8 3 8 / / / / 8 3 8 3 8 / / / / 1 1 2 2 3 8 3 8 1 1 2 2 3 8 3 8 1 1 1 1 600 1Turn 1

500 1Turn 2 1/3Turn 1 400 2

^ 1/3Turn 2 m 300 m Sampled a e r

A 200

100

0 5 5 5 5 5 5 5 5 5 5 1 1 1 1 1 1 1 1 1 1 / / / / / / / / / / 9 9 9 9 9 0 0 0 0 0 / / / / / 1 1 1 1 1 8 3 8 3 8 / / / / / 1 1 2 2 3 8 3 8 3 1 1 2

Example of change in size as a result of growth of fragments, Section 3.

11/9/2015 14/10/2015 DISCUSSION

Again consistent growth rates were obtained for the meristematic tissue (Apex and microtome generated sections) of nearly 10%/day. Titlyanov et al 2006 recorded up to 30%/day in their experiment but this was only for one week and after 4 weeks their growth rates also approached 10%/day. The table below shows a comparison of the culture conditions between their study and the current. There are a few differences but these appear to make little difference to the resulting growth. 10% appears to be a standard growth rate for Palmaria fragments and compares well for growth right through to mature fronds in the laboratory and in outside tank culture (S3eed project pers com).

Table 1 Comparison of culture conditions used in this study versus Titvanov et al 2006.

Titlyanov et al 2006 Sanderson et al 2015 Container volume 2 l 0.5 l Light 70 µ mol-2s-1 120 µ mol-2s-1 Temperature 9 - 10oC 9-10oC Photoperiod 12:12 12:12 Nutrients Von Stosch KTH Water change Weekly Weekly Air Bubbled Bubbled Plant origin Portugal via Germany? Easdale Plant genotype Selected fast growing? Random

Growth rates of the side and inner thallous fragments are again less than meristematic tissue and probably approaching only 3% at best. These fragments appeared to have been compromised. As the aim of this section was to have tetrasporic plants so that tetraspores may be generated, the plants used had been held over in the laboratory and do not appear to have been in the optimal condition. The pieces showed evidence of deterioration and infection and this is reflected in the lower measured growth rates.

The plants generated by the microtome appear healthy and have shown 10% growth (at least for individual segments) consistently until experiment completion (5 weeks).

If fragments other than the meristem could be encouraged to grow into plants as well, then the whole thallous could be cut into multiple pieces resulting in a much greater biomass from source plants. As Palmaria fronds can show adventitious growth from the edges as evidenced in Appendix 2, there must be internal auxins that initiate meristematic activity. As a final trial (underway at project completion) various auxins were added to the seawater containing plant fragments to see if meristematic growth could be generated in sub-meristimatic thallous tissue. As the sides appear to have the capacity to ignite into meristems, both side and meristematic tissue were trialled. PART 3

Growth and adventive tissue accelerating compounds for Palmaria

To encourage meristematic like growth in areas of the thallous other than the meristem. A number of compounds were trialled. These were:

 Glycerol (as per Robain et al 1990)  2,4-dichlorophenoxyacteic acid  Maxi crop (seaweed based plant growth stimulant)  Growing Success – organic rooting gel (another seaweed based plant growth enhancer)  Baby Bio – contains 1-naphthylacetic acid

METHOD

Thallous pieces were cultured as previously. Treatments were:

Gly Glycerol (as per Robain et al): 80% seawater + .25M glycerol

Ch 2,4-dichlorophenoxyacteic acid: 0.5µM

A Maxi crop (seaweed based plant growth stimulant): 0.1ml/l litre

B Growing Success – organic rooting gel (another seaweed based plant growth enhancer): 0.1ml/l litre

C Baby Bio – contains 1-naphthylacetic acid: 0.1ml/l litre

Cont standard culture conditions: ‘Control’

RESULTS

Measurement of growth for the different treatments as depicted in the figure below suggest that the seaweed growth additives were as good as if not better than the control. The two shoot promoters Gly and Ch appeared to be the poorer performers. Plant fragments often took on a mottled appearance and may have been compromised through infection.

CONCLUSION

At the concentrations and for the period trialled, Gly and Ch did not appear to enhance adventive tissue. The seaweed growth promoters may be an effective means of accelerating growth. Further work needs to be done including a greater emphasis on axenic conditions. Growth Treatments 14

10 2 ^

m 8 m

a 6 e r A 4

0 18/10/15 23/10/15 28/10/15 2/11/15 7/11/15 12/11/15 17/11/15

A B C Ch Cont Gly REFERENCES

Clayden SL and Saunders GW 2014 A study of two Acrochaetium complexes in Canada with distinction of Rhododrewia gen. nov. (Acrochaetiales, Rhodophyta) Phycologia Volume 53 (3), 221–232.

García-Jiménez P, Robaina RR, Luque A, Tsekos I 1996 Glycerol-activated cellular division and biosynthetic activity during growth and morphogenesis of carpospore seedlings of Grateloupia doryphora (Cryptonemiales, Rhodophyta). Phycologia 35: 261-269.

Yokoya NS, Ávila M, Piel MI, Villanueva F, Alcapan A 2014 Effects of plant growth regulators on growth and morphogenesis in tissue culture of Chondracanthus chamissoi (Gigartinales, Rhodophyta). J Appl Phycol 26:819–823

Pang SJ and Luning K 2006 Tank cultivation of the red alga Palmaria palmata: Year-round induction of tetrasporangia, tetraspore release in darkness and mass cultivation of vegetative thalli. Aquaculture 252 (1):20-30

Reddy CRK, Jha B, Fujita Y, Ohno M 2008 Seaweed micropropagation techniques and their potentials: an overview Journal of Applied Phycology Volume: 20(5) 609-617

R. R. Robaina, G. Garcia-Reina & A. Luque 1990 The effects of the physical characteristics of the culture medium on the development of red seaweeds in tissue culture. Hydrobiologia 204/205: 137-142.

Titlyanova EA , Titlyanova TV, Kadel P, Lüning K 2006a New methods of obtaining plantlets and tetraspores from fragments and cell aggregates of meristematic and submeristematic tissue of the red alga Palmaria palmata. JMBE 339 (1): 55-64

Titlyanova EA , Titlyanova TV, Kadel P, Lüning K 2006b Obtaining plantlets from apical meristem of the red alga Gelidium sp. Journal of Applied Phycology, 18(2): 167-174

Titlyanov, E. A.; Titlyanova, T. V. 2010 Seaweed cultivation: Methods and problems Russian Journal of Marine Biology 36(4): 227-242 APPENDIX 1

IDENTIFICATION OF MERISTIMATIC TISSUE THROUGH FREEZING.

This part of the project follows the methodology of Titlyanov et al 2006 where plants were frozen and then placed in culture media. Meristimatic tissue of Palmaria appears to survive the process whereas thallous tissue dies. Titlyanov et al 2006 use the meristems as a source of fragments for the generation of new plants.

METHOD

Individual plants of Palmaria palmata, Chondrus crispus, Mastocarpus stellatus and Osmundea sp were wrapped in tinfoil and placed in a freezer at -80oC for 30 mins. They were taken out and allowed to equilibrate to room temperature before being placed in filtered sterilised seawater with KTH nutrient media and kept at 10oC. After one week the media was changed daily.

RESULTS

Meristems of Palmaria were obvious after the freezing, while the fate of Chondrus crispus, Mastocarpus stellatus and Osmundea sp. was indeterminate.

DISCUSSION

A combination of freezing and the microtome may enable the generation of many meristematic fragments. This may be possible through use of a freeze microtome.

Freezing of Palmaria example, start, 1 week, 2 weeks showing surviving meristem. Examples are circled. APPENDIX 2

Adventitious shoots (tips) were harvested from Palmaria fronds taken from tanks at Otter Ferry where Palmaria was being cultured. 700g of tips were used as a starter in a small tank. A representative sample of plants was taken after 56 days. Growth rate was calculated from change in area as previous.

Change in size of cultured Palmaria ‘tips’.

15/10/2015 20/8/2015

8.7 %/day APPENDIX 3

An obligate intracellular monothallic oomycete parasite of Palmaria tetraspores was found as part of this project extending our knowledge of this infection

Infected Palmaria spores by internal oomycetes

50µm 50µm

Pictures: Yacine Badis

. APPENDIX 4

Palmaria (picture 1) infected by Acrochaetium secundatum (pics 2 &3)

Acrochaetium secundatum morphotype virgatulum

See Clayden and Saunders 2014 Phycologia Volume 53 (3), 221– 232.

Pictures: Yacine Badis APPENDIX 5

PHYCOMORPH, Oporto Portugal

17/18 September 2015

An opportunity to go to a Phycomorph conference was taken up in September

The main aim of PHYCOMORPH is to unify a scattered European research landscape to enable a step-change in the basic knowledge of macroalgal reproduction and development, and to ensure appropriate and efficient transfer to R&D and Innovation Institutes dedicated to the development of aquaculture techniques, in tune with current needs in Europe and worldwide. Consequently, PHYCOMORPH will undertake for the first time a strictly interdisciplinary approach, combining molecular/developmental biology, genetics, and analytical chemistry.

For more information, see: http://www.phycomorph.org.