Transfer of an Arctic Fish Antifreeze Protein (AFP) into
Strawberries to Increase Frost Resistance
Kirk Emch
Ocean Pout
General Strawberry Information
 The United States is the largest producer of strawberries in
the world.
 In 2010 the U.S. accounted for 28% of the world total for
strawberries produced.
 In 2012 the total value of production was 2.4 billion dollars.
 Most are produced in southern states and California.
Why Strawberries Need Frost Resistance
 Strawberries grow in temperate climate regions which are
capable of having low temperatures and frost.
 Spring frosts cause damage to the flowers of the plant leading
to poor yields and erratic fruiting.
 Frost on average causes millions of dollars in damages and
drives up the price of the fruit for the consumer.
Why Strawberries Need Frost Resistance Cont.
 Frost or cold resistant plants would also help eliminate after
harvest losses.
 Strawberries are a soft and fragile fruit therefore they have
a very short shelf life and must be stored and shipped at low
temperatures.
 It is not uncommon for the storage and shipping
temperature to get to close to freezing and harm the
berries.
Frost Prevention Practices
 Floating row covers – easy and effective but really only for
small acreages, so not applicable to large scale producers.
 Wind machines – only provide a few degrees of protection
and work the best with plants that bloom late, so not
efficient enough to save a large scale berry farm from a heavy
frost.
Frost Prevention Practices Cont.
 Sprinkler irrigation – works well for protecting strawberries
but it is risky and an expensive irrigation system is required.
Growers must be careful not to overwater their crops or to
water during a period where evaporative cooling can take
place and harm the plants.
 Genetically engineered plants could provide an opportunity
to eliminate expensive and less efficient preventative
practices.
Discovery of AFP’s
 Antifreeze proteins or AFP’s
were discovered by Arthur
DeVries and colleagues in 1969.
 First discovered in a Antarctic
Nototheniid fish; AFP’s opened
a whole field of study into cold
resistance.
 Since the discovery of AFP’s in
fish several other AFP’s have
been discovered in other
organisms such as bacteria,
fungi, plants, insects, and
vertebrates.
Arthur DeVries and Nototheniid fish
What are AFP’s?
 AFP’s are proteins that reduce and prevent the damage
caused to an organism by freezing.
 These proteins are able to utilize their unique structures that
hinder freezing by binding to and preventing young ice
crystals from growing. (Fletcher 2001)
 The AFP we will focus on for this research is type III which
comes from the Ocean Pout.
Type III AFP
 There are four types of AFP’s that come from fish and all
have different structures, so all have different ways they bind
to ice crystals.
 The type III AFP is made from short β-strands and one helix
turn, resulting in a flat faced globular fold. (Fletcher 2001)
AFP type III
The Plants
 In one of the early papers DeVries published about AFP’s
he made a statement saying that the transfer of genes that
encode for AFP’s from fish is one means of increasing frost
resistance. (DeVries et al., 1970)
 With all of the problems strawberries encounter when
exposed to cold temperatures it seems plausible to try and
put an AFP gene into them.
 The type III AFP was chosen because even at low levels of
ice crystallization, AFP activity of type III is the most active.
 More activity means greater cold or frost resistance, which
equates to better protection and more money saved.
Khammuang et. al (2005)
 Researchers began by designing and constructing the
optimized AFP gene and the plant expression vector.
 A codon with high frequency use was selected and used to
design the primers: AFP-F1, AFP-R1, AFP-F2, AFP-R2.
 These primers constructed the strawberry optimized codon
gene encoding antifreeze protein by polymerase chain
reaction (PCR).
 The modified gene was ligated to the pT7blue2 plasmid by TA
cloning.
Khammuang et. al (2005)
 The pT7blue2 plasmids that contained the optimized gene were
digested Xba I and Bam HI.
 This fragment was then ligated into the digested pBI121 plasmids
which were then transformed to E.coli JM109.
Figure 1. Construction of plant expression vector by insertion of the Xba I - BamH I fragment
of strawberry optimized codon of AFP gene into plasmid pBI121.
Khammuang et. al (2005)
 Plasmids from the pBI121 that contained the correct gene were
called pBB and contained the NOS gene for kanamycin
resistance with its promoter, and CaMV35S promoter.
 pBB was introduced into A. tumefaciens by triparental mating.
 Once the agrobacterium contained these plasmids it was used
to infect the plants.
 Explants were transferred to MS medium containing Kanamycin
so any plant that did not contain the correct DNA would die.
Khammuang et. al (2005)
 Plants that didn’t die during the Kanamycin test were then
subjected to DNA extraction and PCR analysis to prove the
correct gene was present.
Khammuang et. al (2005)
Figure 5. Detection of the transgene by PCR. Genomic DNA from pSW1 (a) and pBB (b)
transformed individual strawberry plants of T0 generation. M, marker; N,
nontransformed strawberry plant; 1, pSW1 plasmid as positive control; 2-8,
transformants of pSW1; 9, pBB plasmid as positive control; 10-12, transformants
of pBB.
Khammuang et. al (2005)
 The transformation efficiency was not so great, out of
138 explants there were 3 that survived the kanamycin
test and those 3 were also PCR positive that the AFP
gene was of correct size.
 Until they can get more transgenic plants cold
resistance tests are on hold.
 Nevertheless they were able to put an AFP gene from a
fish into a strawberry.
 So until further testing we do not know how well this
will work if at all.
Arguments Against
 Unfortunately even if further research gets done and proves that
this technology is something we should be producing it may
never see the field due to the media and organizations against
GMO’s.
 Remember the pT7blue2 plasmids that the PCR product of the
modified gene was ligated into?
 Some person took this research and made up a story of how
engineering strawberries to have fish genes using this blue
plasmid for antifreeze properties “accidentally turned the
strawberry blue and scientists has not expected this”
Arguments Against
 This website explains how they disproved the blue
strawberries.

http://www.hoaxorfact.com/Science/blue-strawberries-genetically-modified-by-fishgenes-facts-analysis.html
References
 Khammuang, Saranyu. "Agrobacterium-mediated Transformation of
Modified Antifreeze Protein Gene in Strawberry." Science Technology
4th ser. 27 (2005): 693-703. Web.
 Firsov, A. P., and S. V. Dolgov. "AGROBACTERIAL TRANSFORMATION
AND TRANSFER OF THE ANTIFREEZE PROTEIN GENE OF WINTER
FLOUNDER TO THE STRAWBERRY." Acta Hort, n.d. Web.
 Fletcher, Garth L. "Antifreeze Proteins of Teleost Fishes." Annual
Review Physiology 63 (2001): 359-90. Print.