among these, the high pH-high calcium, PEG, and electricpulse methods have been
shown to be reproducible. Recent years have seen greater available of electrical cell
fusion devices using electrofusion, or fusion through electrical stimulation. The
pomato, created from the fusion of potato and tomato protoplasts, may be the
best-known example of cell fusion in higher plants. Many other hybrids have also
been produced through cell fusion recently, most notably with members of the
nightshade family (Cheney et al. 1986).
Examples of cell fusion use in seaweed remain rare, with only minor reports
from studies of freshwater algae. A recent successful and reproducible example in
seaweeds was obtained from a syncytium produced through the PEG and electrofusion methods. It remains difficult, however, to distinguish whether complete
fusion has occurred even within seaweeds from the same genus and species, or
between different species and families. Another issue concerns whether the new cell
resulting from fusion will be capable of developing into a new seaweed organism.
Development of new techniques for syncytium (hybrid) culturing and selection may
become an important issue in the future (Waaland 1975).
Let us consider an actual example of seaweed cell fusion using protoplasts.
Figure 6.8 shows the principles of cell fusion through the PEG method. Even with
this fusion method, cell fusion occurs randomly, and cells of different species do not
fuse at a 1:1 ratio. The fusion rate is around 10% for the same species and 15% for
different species, but because fusion occurs within the same species or with two or
more cells, target cells must be selected from these for culturing. In some cases,
traits from both types may not be expressed in the fused cell, and the target trait
may not be expressed. Many issues remain to be addressed in the selection of fused
cells and subsequent regeneration through culturing, and continued research is
expected to result in functional improvements going ahead.
As an example of the PEG method, cell fusion involving the red alga laver and
the green alga sea lettuce occurs according to the following sequence (Fig. 6.8).
1. Protoplasts are separated from laver (a red alga) and sea lettuce (a green alga).
2. Protoplasts are mixed at a 1:1 ratio in a Tris-sorbitol buffer solution and adjusted
to 10 per milliliter.
3. Two to three droplets of the mixture are transferred to an agar-coated glass slide
and allowed to naturally draw for several minutes.
4. After verification that the protoplasts have agglutinated through drying, 0.1 ml
of PEG is slowly added. [Preparation of PEG: 25% polyethylene glycol 6000,
0.7 M sorbitol, 0.1 M calcium chloride (CaCl 2 ), 0.1 M Tris, pH 10.0]
5. Rinse slowly with Tris-sorbitol buffer solution.
In this case, the protoplasts of laver (a red alga) will assume a reddish tinge and
the protoplasts of sea lettuce (a green alga) a green tinge due to differences in
pigments, which makes it easy to verify by microscope whether protoplast fusion
has occurred. Because the red and green alga fusion occurs between very distance
species, complete fusion of protoplasts will not be easy to achieve. It will also be
difficult to conclude that fusion has occurred for any product (Reddy et al. 2008).
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6 Seaweed Biotechnology
shown to be reproducible. Recent years have seen greater available of electrical cell
fusion devices using electrofusion, or fusion through electrical stimulation. The
pomato, created from the fusion of potato and tomato protoplasts, may be the
best-known example of cell fusion in higher plants. Many other hybrids have also
been produced through cell fusion recently, most notably with members of the
nightshade family (Cheney et al. 1986).
Examples of cell fusion use in seaweed remain rare, with only minor reports
from studies of freshwater algae. A recent successful and reproducible example in
seaweeds was obtained from a syncytium produced through the PEG and electrofusion methods. It remains difficult, however, to distinguish whether complete
fusion has occurred even within seaweeds from the same genus and species, or
between different species and families. Another issue concerns whether the new cell
resulting from fusion will be capable of developing into a new seaweed organism.
Development of new techniques for syncytium (hybrid) culturing and selection may
become an important issue in the future (Waaland 1975).
Let us consider an actual example of seaweed cell fusion using protoplasts.
Figure 6.8 shows the principles of cell fusion through the PEG method. Even with
this fusion method, cell fusion occurs randomly, and cells of different species do not
fuse at a 1:1 ratio. The fusion rate is around 10% for the same species and 15% for
different species, but because fusion occurs within the same species or with two or
more cells, target cells must be selected from these for culturing. In some cases,
traits from both types may not be expressed in the fused cell, and the target trait
may not be expressed. Many issues remain to be addressed in the selection of fused
cells and subsequent regeneration through culturing, and continued research is
expected to result in functional improvements going ahead.
As an example of the PEG method, cell fusion involving the red alga laver and
the green alga sea lettuce occurs according to the following sequence (Fig. 6.8).
1. Protoplasts are separated from laver (a red alga) and sea lettuce (a green alga).
2. Protoplasts are mixed at a 1:1 ratio in a Tris-sorbitol buffer solution and adjusted
to 10 per milliliter.
3. Two to three droplets of the mixture are transferred to an agar-coated glass slide
and allowed to naturally draw for several minutes.
4. After verification that the protoplasts have agglutinated through drying, 0.1 ml
of PEG is slowly added. [Preparation of PEG: 25% polyethylene glycol 6000,
0.7 M sorbitol, 0.1 M calcium chloride (CaCl 2 ), 0.1 M Tris, pH 10.0]
5. Rinse slowly with Tris-sorbitol buffer solution.
In this case, the protoplasts of laver (a red alga) will assume a reddish tinge and
the protoplasts of sea lettuce (a green alga) a green tinge due to differences in
pigments, which makes it easy to verify by microscope whether protoplast fusion
has occurred. Because the red and green alga fusion occurs between very distance
species, complete fusion of protoplasts will not be easy to achieve. It will also be
difficult to conclude that fusion has occurred for any product (Reddy et al. 2008).
162
6 Seaweed Biotechnology