Kirk Emch

1. Transfer of an Arctic Fish Antifreeze Protein (AFP) into Strawberries to Increase Frost Resistance Kirk Emch Ocean Pout

2. 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.

3. 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.

4. 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.

5. 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.

6. 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.

7. 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

8. 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.

9. 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

10. 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. (DeVrieset 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.

11. Khammuang et. al (2005) • Researchers began bydesigning 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.

12. 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 XbaI - BamHI fragment of strawberry optimized codon of AFP gene into plasmid pBI121.

13. 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. tumefaciensby 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.

14. 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.

15. 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.

16. 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.

17. 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”

18. Arguments Against • This website explains how they disproved the blue strawberries. • http://www.hoaxorfact.com/Science/blue-strawberries-genetically-modified-by-fish-genes-facts-analysis.html

19. 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." ActaHort, n.d. Web. • Fletcher, Garth L. "Antifreeze Proteins of Teleost Fishes." Annual Review Physiology 63 (2001): 359-90. Print.