(3) Rinse one fragment in sterilized artificial seawater and place in a laboratory
dish of sterilized artificial seawater. After 10 min or so, many of the sporophytes will be released.
(4) Draw out sporophytes with a micropipette, place two to three droplets on ST3
agar medium, and spread with glass rod.
(5) After one month, the sporling will become a cluster of branching filaments
measuring around 1 mm in diameter, and the mixed bacteria will form a colony
measuring 3–55 mm. Place the seaweed that is not contaminated with bacteria
into a test tube containing 10 ml of ASP 12 NTA medium.
(6) A month later, the seaweed will becoming a microcallus measuring around
3 mm in diameter. For the culturing of this microcallus, sterilization can be
verified through the use of ST3, ASP-B1, or other search media.
Research has already reported successful sterilization of 90% or more with this
single-stage selection method.
Medium use is crucially important for successful seaweed tissue culturing.
Varieties of medium used for seaweed culturing include enriched media, which use
a natural seawater base enriched with nutrient salts and other essential nutrients, and
synthetic media, which use artificial seawater prepared using chemicals alone and
enriched with nutrient salts and other essential nutrients (Table 6.1).
Chief examples of enriched media include the Erd-schreiber (ES) medium,
which has long been used for seaweed culturing. Recent examples include PES,
which has often been used for culturing of green and brown algae; PESI, which is
often used for brown algae culturing; and ESS, which was developed for seaweed
seed production and is useful for lavers, kelps, and sea lettuces. Examples of
synthetic media include ASP, which developed by Provasoli et al. in the U.S.; ASP 1
and ASP 7 have been used frequently for green algae, ASP 2 and ASP 6 for red algae,
and ASP 12 for brown algae. The ASS 1 media was developed as a basic medium for
seaweed tissue culturing and can used to produce excellent results with green, red,
and brown algae; it is also used as a preservation medium for sterilized strains
(Tables 6.2 and 6.3).
Some room for improvement of these media remains, as the current cases are as
yet inadequate for completion of the life cycle or smooth completion of
morphogenesis.
Examples of success with tissue culturing of large seaweed include Chem et al.
in Canada, who in 1978 cultured a tissue fragment measuring several millimeters
taken from the center of the red alga Chrondrus crispus; three months later, this had
grown into an alga measuring several centimeters. That same year, Sega et al. in
Japan used the kelp Laminaria angustata for tissue culturing and succeeded in
producing cloned kelp using cells separated from callus tissue.
Tissue culturing has been attempted with sterilized seaweeds through the
methods described above, but in all cases they can be introduced to an agar medium
to obtain a callus and the cells derived from it. A callus is an amorphous cluster of
cells that appears when part of a parent organism is removed and cultured; under
certain conditions, it does not exhibit differentiability.
6.3 Tissue Culture
149
dish of sterilized artificial seawater. After 10 min or so, many of the sporophytes will be released.
(4) Draw out sporophytes with a micropipette, place two to three droplets on ST3
agar medium, and spread with glass rod.
(5) After one month, the sporling will become a cluster of branching filaments
measuring around 1 mm in diameter, and the mixed bacteria will form a colony
measuring 3–55 mm. Place the seaweed that is not contaminated with bacteria
into a test tube containing 10 ml of ASP 12 NTA medium.
(6) A month later, the seaweed will becoming a microcallus measuring around
3 mm in diameter. For the culturing of this microcallus, sterilization can be
verified through the use of ST3, ASP-B1, or other search media.
Research has already reported successful sterilization of 90% or more with this
single-stage selection method.
Medium use is crucially important for successful seaweed tissue culturing.
Varieties of medium used for seaweed culturing include enriched media, which use
a natural seawater base enriched with nutrient salts and other essential nutrients, and
synthetic media, which use artificial seawater prepared using chemicals alone and
enriched with nutrient salts and other essential nutrients (Table 6.1).
Chief examples of enriched media include the Erd-schreiber (ES) medium,
which has long been used for seaweed culturing. Recent examples include PES,
which has often been used for culturing of green and brown algae; PESI, which is
often used for brown algae culturing; and ESS, which was developed for seaweed
seed production and is useful for lavers, kelps, and sea lettuces. Examples of
synthetic media include ASP, which developed by Provasoli et al. in the U.S.; ASP 1
and ASP 7 have been used frequently for green algae, ASP 2 and ASP 6 for red algae,
and ASP 12 for brown algae. The ASS 1 media was developed as a basic medium for
seaweed tissue culturing and can used to produce excellent results with green, red,
and brown algae; it is also used as a preservation medium for sterilized strains
(Tables 6.2 and 6.3).
Some room for improvement of these media remains, as the current cases are as
yet inadequate for completion of the life cycle or smooth completion of
morphogenesis.
Examples of success with tissue culturing of large seaweed include Chem et al.
in Canada, who in 1978 cultured a tissue fragment measuring several millimeters
taken from the center of the red alga Chrondrus crispus; three months later, this had
grown into an alga measuring several centimeters. That same year, Sega et al. in
Japan used the kelp Laminaria angustata for tissue culturing and succeeded in
producing cloned kelp using cells separated from callus tissue.
Tissue culturing has been attempted with sterilized seaweeds through the
methods described above, but in all cases they can be introduced to an agar medium
to obtain a callus and the cells derived from it. A callus is an amorphous cluster of
cells that appears when part of a parent organism is removed and cultured; under
certain conditions, it does not exhibit differentiability.
6.3 Tissue Culture
149