Plants like sunshine. Plants don't like freezing cold. Here
in Alaska we have lots of both. The summers, though, are ideal for plant growth;
they actually grow faster during Alaska summers than in more southern latitudes.
This produces the Alaskan gigantic vegetables. Alaska's growing season is
severely limited in both spring and autumn by freezing temperatures; extending
the season by just a few days would allow plants to grow significantly bigger.
Of course we can't change the length of our summer, but maybe we can change the
plant's sensitivity to freezing.
Some animals have their own anti-freeze, enabling them to
live in below-freezing conditions. Arctic fish survive by producing a protein
keeping them alive down to -1.5°C. This anti-freeze protein, or AFP, is
produced in their liver from a gene in their DNA. AFP works by binding to ice
crystals and preventing further development. If we could copy this flounder
gene, then insert it into a plant, then the plant might produce enough AFP to
survive low temperatures. In 1990, Dr. Fawzy Georges and his staff at the
National Research Council of Canada have done just that. They built a synthetic
version of the AFP gene and inserted it into Black Mexican Sweet Corn in hopes
of improving corn's cold hardiness. This procedure involves several complex
steps: synthesizing the gene, construction of a suitable transfer vehicle, and
introduction of the vehicle into corn plants.
To synthesize the gene, Dr. Georges used a new strategy. DNA
is double stranded; normally, both strands must be synthesized. Dr. Georges
synthesized just one strand; this method costs less than producing a
double-stranded gene. To produce the AFP gene's complementary strand, he used
the cell's own machinery. He simply used chemical enzymes taken from cells to
synthetically replicate the gene in the same way done naturally.
The synthetic AFP gene was then joined with other segments of
DNA to form a transfer vehicle, or plasmid. The plasmid also contained a
promoter segment that told the corn cells to continually manufacture AFP.
Interestingly, this promoter comes from a virus infecting cauliflower. Just
after the AFP gene, Dr. Georges inserted a reporter gene called CAT. The CAT
gene produces a protein that, when mixed with another chemical, turns blue. In
this way, Dr. Georges could visually see when AFP was produced in the corn
cells.
The plasmid containing the AFP gene was next introduced into
corn. Separated plant cells, or protoplasts, however, have cell membranes not
penetrable by DNA. So, Dr. Georges made holes in the protoplasts by a process
called electroporation--the making of pores with electricity. He shocked the
protoplasts twice with 250 volts. This killed many corn protoplasts, but in
others, made holes just large enough for the plasmid to enter.
Determining if the corn protoplasts did indeed take up the
plasmid and produced AFP is the CAT gene's job. Since the CAT gene immediately
followed the AFP gene, then the presence of CAT protein indicates the presence
of AFP. Dr. Georges broke open the corn protoplasts and mixed them with the
CAT-indicating chemical. The chemical reaction showed presence of AFP.
Production of AFP within its cells is not, however,
sufficient to give corn freeze tolerance. Cells must export AFP to the spaces
between cells. The AFP protein was not exported because it apparently lacked the
correct cell signaling sequence.
Though Dr. Georges's experiment with the flounder anti-freeze
gene did not produce freeze-resistant corn, it did prove plants can produce
anti-freeze proteins. This powerful technique is a first step to production of
plants able to survive several days longer when cold temperatures return in
autumn. In Alaska, this means plants may continue to grow when long daylight
hours still persist, but temperatures have dropped to freezing. Alaska may
someday produce significant agricultural crops using techniques of genetic
engineering.