WEBVTT
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You already learned about the biology of
these two brown algal models, Ectocarpus
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and Saccharina, and in this chapter you
will see some techniques which are
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necessary to study them, and the
first one is to produce algal material
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because without any algal material,
studying them is impossible.
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So this is the absolute first step
before any downstream experiment.
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This will be done by two PhD students,
Giannis and Samuel from Roscoff,
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who will guide you through
the experimental protocols.
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So hello everyone, my name is Giannis
Theodorou and I'm on my third year
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of my PhD and I will show you how to
maintain and cultivate Saccharina lattissima
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for our experiments and we
start with our material,
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getting it from the wild
and bringing it to the lab.
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In our lab, we work with wild
Saccharina acquired from the sea.
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We look for a big blade with
fertile spots called sori.
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Once we find a good sample,
we bring it back to the lab.
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Then we begin the cultivation protocol.
We cut the part with these fertile spots
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and we wipe them with a bit of
distilled water on a paper towel.
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Then we scratch this part
to remove the epiphytes.
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We cut the tissue into small pieces, place
them in sterile seawater to release the spores.
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The spores are only visible
under the microscope.
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So hello, my name is Samuel Boscq, I'm
working as a first year PhD student at
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Benedicte's lab, so working on microalgae
models, and so now that we have the spores
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I'm going to present to you the
two possibilities that we have.
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So either we put them under white light
to create some new sporophytes that
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will develop and will be
usable for our experiments,
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or we put them under red light to
create some stock for later use.
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First the spores solution is filtered to
remove microalgae and other microorganisms.
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The filtered spores obtained are
put under low-intensity white light,
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and they germinate, and white light
triggers the induction of gametogenesis.
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As a result, the gametophyte is small,
one single cell, when the oogonium
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and the antheridia are differentiated.
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Male gametes will fertilise the female
oogonium, resulting in a zygote,
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which is elongating soon after germination.
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The zygote divides and
makes the early embryo.
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So after our embryos have developed
for 1-2 months and being a few millimetres
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long, we are placing them in a bottle
so that they can continue growing
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since they are needing space in exchange
for to be able to develop further.
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We need this development further so that
we can study their anatomy, their histology,
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and also possibly pass them through some
ecophysiological stresses to see their responses.
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Once the embryos are a bit over a
centimeter long, we collect them
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and put them into a large plastic bottle.
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Then, to provide a source of nutrients
and a medium for culture, we add
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filtered natural seawater enriched with
nutrients, such as Provasoli solution for example.
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Afterward, we connect the bottle to an
airflow to keep the level of oxygen steady
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as well as creating agitation in the culture.
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The embryos kept this way can be
cultivated to a dozen centimeters long
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before they require to be
transferred in a larger system.
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Another useful technique to cultivate
algae is to create stock cultures.
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Why make a stock? It allows us to keep
a given genotype for several years
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in lab conditions. It also allows us
to amplify the culture when a lot of
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organisms are necessary. How do we
create the stock? We cultivate the
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gametophytes in seawater-filled petri dishes
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under red light and let them grow
for a few months to several years.
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At a certain point, the algae reach quite
a large size, the space in the Petri dish
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is too small for them to continue to grow.
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In which case, we will collect them and
grind them thoroughly to smaller pieces.
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Then we generously dilute them in seawater.
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The fragmented algae in the new
dishes will then continue to grow,
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and thus multiply the stock.
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For long-term storage, seawater needs to
be changed every month to renew the level
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of nutrients. The stock thus made is
now usable for any type of experiment.