Improved maintenance protocols for kelp gametophytes
1. Collection of reproductive sporophytes in field.
2. For each individual, mature sori (reproductive structures) are washed
with running tap water and then with sterile seawater immediately
after collection.
3. A fragment of each blade is excised and stored in silica gel for molecular
identification.
4. Two fragments of equal size (3.8 cm2) of each individual were
incubated at 16°C in 50 mL Falcon tubes containing a glass slide and
sterile seawater.
5. These tubes are stored in a cooler in the dark to avoid germination and
at low temperature (average 10°C, minimum 5°C, and maximum 16°C)
for their transportation to the lab since kelps are known to be sensitive
to higher temperatures.
6. At their arrival in the lab, they are kept in the dark at 10°C.
7. After 12 to 24 hours of incubation, spores are attached on the glass
slide.
8. Sterile seawater is replaced with SFC enriched seawater {Correa, 1988 }
9. Falcon tubes are placed horizontally in culture chambers under
standard culture conditions (10°C, 12:12 light:dark, 25-35 µmol photons
m-2s-1).
10. Culture media is changed once a week and observations are done every
three days approximately.
11. After 15 d in culture, male and female gametophytes are identified
according to their morphological characteristics using a Nikon Eclipse
TE300 inverted microscope (Nikon Corp., Tokyo, Japan). Female
gametophytes are characterized by large cells and filaments with few
branches, whereas male gametophytes are smaller and display highly
branched filaments formed by small cells. These morphological
differences make them unambiguously identifiable under the inverted
microscope.
12. Gametophytes are detached of the glass slide and isolated individually
(one gametophyte per well) in 24 multi-well tissue culture plates with
each well containing 2 mL of Provasoli enriched sea-water.
13. The plates are incubated in a growth chamber at 15°C with a photon
irradiance of 30-35 µmol.m-2s-1
14. The culture medium was changed once a week during the first month
and then once a month.
ASSEMBLE-JRA1-Protocol-26.00
Christophe Destombe, SBR
SBR, France.
Kelp: Reproductive sporophytes collected
in field.
Equippment: Cooler average temperature
10°C, Nikon Eclipse TE300 inverted
microscope,