The Little River WATERMARK

Friends of VOLUME 16 : ISSUE 1 DuPont Forest MEMBERSHIP NEWSLETTER Spring 2018

The American Chestnut Tree Returns with New Pairs of Genes: Part 4

By Kent Wilcox

s described in Part Two of this series, Figure 1 – Developmental stages during natural plant using traditional breeding methods A embryogenesis from zygote (fertilized egg) to mature to create a hybrid Asian/American chestnut tree embryo. Insert shows parts of a typical flower, including that is resistant to chestnut blight is a long, several ovules. Diagram courtesy of the University of tedious process. The distribution of multiple Nairobi, Kenya. genes that confer blight resistance into the sperm and eggs of hybrid trees at each step will yield some chestnuts that contain two copies in the breeding process is entirely random. of each of the desired genes, thus ensuring that Consequently, thousands of nuts from each successive generations will be blight resistant. To generation of the breeding process must be reach this goal, genetic engineers must have DNA planted and the resulting trees must be screened that contains the genes of interest, a method to for resistance and other properties. Most are transfer DNA with the genes of interest into an discarded. individual cell to create a genetically-modified cell, and a method to produce a mature, fertile The advent of molecular and cellular technolo- tree from one genetically-modified cell. A quick gies to engineer genetically-modified organisms note on terminology. The term “transgenic” is (GMOs) in the laboratory has led to the rapid cre- used for organisms that have been genetical- ation of commercially-important plants such as ly-modified by introduction of DNA from an corn, cotton, and soybean with introduced genes unrelated species (such as introducing DNA from that confer resistance to pests and disease. Can wheat into an American chestnut) and the term blight-resistant chestnut trees be created by “cisgenic” is used for organisms that have been the same technology? To address this question, genetically modified by introduction of DNA from let’s consider methods and problems associated a related species (such as introducing DNA from with genetic engineering of plants. In the case of a Chinese chestnut into an American chestnut). chestnut trees, the end goal is to create a diverse GMO is a broader term that applies to any organ- population of trees that produce both sperm ism modified by genetic engineering, regardless and eggs that contain the desired genes. Cross- of the DNA source. fertilization between first generation GMO trees Continued on Page 2 1 American Chestnut Tree Returns Continued from Page 1

Identifying the genes that confer blight resis- tance in Asian chestnut trees is a difficult process that requires comparing the genetic makeup of blight-resistant and non-resistant chestnut trees. Sequencing the DNA from the 12

different chromosomes (designated A through Figure 2 L) in Chinese chestnut trees is nearly complete (Hardwood Genomics Project, funded by the Forest Health Initiative). The corresponding proj- ect for the American chestnut tree has been ini- tiated (HudsonAlpha Institute for Biotechnology, funded by The American Chestnut Foundation) but is far from complete. When that sequencing project is finished, it might be assumed that a direct comparison of the genes in Chinese and American chestnut trees would reveal the genes

that confer blight resistance, but it’s not that Figure 3 simple. Chinese and American trees differ in growth morphology, flowering time, and many Figure 2 – A callus (disorganized cluster of somatic cells) other characteristics that are due to genetic dif- produced from fern tissue. Photo courtesy of Blahnais, Wikimedia Commons. Figure 3 – Embryogenic cells ferences. Low resolution chromosomal analyses (well-organized globular clusters of cells) produced by of blight-resistant hybrid trees created by tra- somatic cell embryogenesis from (A) American chestnut ditional breeding suggest that genes located in tree and (B) sweet orange tree. Photos courtesy of (A) specific regions on chromosomes B, F, and G in Linda Polin McGuigan, SUNY College of Environmental Chinese chestnuts confer some degree of blight Science and Forestry, (B) Randall Niedz, Agricultural resistance (Rebecca Hirsch, “Blight Resistance: Research Service, USDA. It’s in the DNA”, Journal of The American Chestnut Foundation (JTACF), March/April 2012). shoot apical meristem, the root apical meristem Thus there are at least three and probably more (radical), and two leaves (cotyledons) (Figure 1). genes that contribute to blight resistance. Once During germination, these components emerge identified, acquiring the DNA for the desired from a nut and form a seedling (Amy Miller et genes will be relatively easy, thanks to advances al., “How a Flower Becomes a Chestnut”, JTACF, in synthesizing DNA. March/April 2014).

A brief refresher on plant cell biology is impera- Because reproductive cells are haploid and have tive to understand what follows. Most plant cells the capacity to generate an entire tree after are diploid, which means they have two copies fertilization, one might assume that the best of each chromosome. The term “somatic cell” is method to create a GMO chestnut tree would be used to distinguish diploid cells from reproduc- to transfer the desired genes directly into the tive cells (sperm and eggs) which are haploid nuclei of sperm and eggs, use in vitro fertiliza- (one copy of each chromosome). Fertilization tion to make a diploid zygote, and culture the of an egg with a sperm creates a diploid cell zygote to create a mature embryo that becomes called a zygote, which undergoes multiple a seedling. However, the tough outer wall of pol- rounds of cell division to become a mature len grains is a barrier that blocks DNA transfer embryo within a nut. The mature embryo con- to the sperm inside. Sperm could be extracted sists of differentiated somatic cells that form the from pollen, but DNA transfer would probably

2 Continued on Page 3 American Chestnut Tree Returns successes reveal that young, newly-differentiated Continued from Page 2 somatic cells are receptive to reprogramming to become non-differentiated embryonic cells, which is the key step in somatic cell embryo- genesis. This step is accomplished by placing the somatic cell tissues in culture medium that contains nutrients and 2,4-dichlorophenoxyacetic acid (2,4-D), a synthetic analog of the plant hor- mone auxin that induces somatic cells to dedif- ferentiate as they grow and divide. (Ironically, 2,4-D is used as an herbicide that kills plants by inducing uncontrolled cell growth.) Under the influence of 2,4-D, the dividing cells form an aggregate that consists of disorganized clusters of cells called a callus (Figure 2) or a proembryo-

Figure 4 genic mass (PEM) that consists of embryogenic and nonembryogenic cells (Figure 3). After PEMs Figure 4 – American chestnut shoot and leaves emerg- are observed, the culture conditions are changed ing from a mature embryo created by somatic cell to stimulate embryogenic cells to transition to embryogenesis. Photo courtesy of Linda Polin McGuigan, an immature embryo. Further changes in the SUNY College of Environmental Science and Forestry. culture conditions induce the immature embryo fail because sperm DNA is highly compact and to differentiate and form a mature embryo with sperm nuclei lack mechanisms required for DNA two cotyledons and components of the apical recombination. There is an easy method to trans- meristem (the shoot that grows above ground) fer DNA into the ovules (structures that contain (Figure 4). Refrigeration (40°F) of the mature eggs, Figure 1) of some flowers, but thus far this embryo for two or three months is required floral dip method has succeeded with only a few to induce germination, which occurs when an species (not including chestnut flowers), pri- initial root called the radicle extends from the marily because most plant ovules are relatively embryo. This delicate assemblage of differenti- inaccessible to DNA transfer methods. Given ated cells can be placed in potting soil to form the difficulties of introducing DNA into haploid a plantlet (since there is no outer coat around reproductive cells from chestnut trees, investiga- the mature embryo to form a seed, scientists tors have chosen to begin with somatic cells and prefer the term plantlet rather than seedling). induce them to undergo somatic cell embryogen- Establishing and optimizing conditions for each esis, an artificial process in which a somatic cell of these steps required a lot of time and effort. from a plant is induced to form an embryo. Dr. Scott Merkle and his colleagues in the School of Forest Resources at the University of Georgia Obtaining somatic cells from mature trees would worked for six years to find conditions to induce allow genetic engineers to choose a tree with American chestnut somatic cell embryos to ger- the desired characteristics before starting the minate and another eight years to increase the process of somatic cell embryogenesis. However germination frequency from 0.4% to 40%. somatic cell embryogenesis using tissues from mature American chestnut trees has not suc- While working on methods to achieve somatic ceeded. Fortunately, since 1985, investigators cell embryogenesis, scientists were also working working with several species of chestnut trees on methods to transfer DNA into somatic chest- have reported successes in generating embryonic nut cells. The first attempts used gene guns that cells from somatic cells obtained either from shoot tiny gold particles covered with DNA into immature embryos within chestnuts or from cells. The method works, but is very inefficient cotyledons in newly-germinated seedlings. These because most of the cells are killed. Currently the preferred method is to use a bacterial

Continued on Page 8 3 A WALK ON THE WILD SIDE

Red Fox The Red Fox (Vulpes vulpes) is not endemic to North Carolina but was introduced here in the 1700’s by settlers who wanted to fox hunt here as they had done in Europe. They like to live around farms where there are lots of rodents. The male Copperhead Snakes is called a ‘dog’, the female a ‘vixen’ and the young The Copperhead Snake (Agkistrodom contortrix) are ‘kits’. The tail is called a ‘brush’ and they have is fairly common in DuPont Forest and is venom- 28 different vocalizations. ous, and though the bite is painful, no person in N.C. has ever died from such a bite. Nationwide, the total number of fatalities has been only 1-2. Still, persons bitten by one should go to an emer- gency room. Large dogs usually survive without treatment but smaller dogs should be taken to a vet. Most bites occur at night when people go out into their yards barefoot or wearing only flip-flops and they step on a snake. Maximum size for a copperhead is about 4 feet. The newborn young have bright green or yellow tails.

Photos and article by Alan Cameron, Friends of Striped Skunk DuPont Forest Board Member and 12-year volunteer Striped Skunks (Mephitis mephitis) are common with the NC Wildlife Resources Commission. He does Friends of but rarely seen as they are nocturnal. They are 90% of his work in DuPont Forest. DuPont Forest omnivores but feed mostly on insects. Skunks do not hibernate. They can spray up to 12 feet and treatment is not tomato juice as many people think but is 1 quart of hydrogen peroxide, 1/4 cup baking soda and 1-2 tsp liquid soap. Lather, rinse, repeat, and do it outside. Skunks can carry rabies so avoid any you see active during the day and call the county animal control office.

4 A NOTE FROM THE EXECUTIVE DIRECTOR

Sharing is Caring: A Guide to Sharing & Caring for the DuPont Forest Trails Article by Sara Landry, Executive Director of Friends of DuPont

As we move into the busy summer months in DuPont Forest, it is a good time for a refresher on the Forest Rules and trail etiquette. FOREST RULES In 2017 the N.C. Forest Service hired two Law Enforcement Officers for DuPont Forest with full authority to write tickets for violations to the Forest Rules. No Alcoholic Beverages - There are plenty of places a hop, skip, and a jump from the Forest where you can have a cold beer after your long hike or ride. Save yourself the trouble and don’t bring that six pack into the Forest with you. Dogs Must be on a Leash at All Times - No exceptions, no matter how cute or friendly your LEAVE NO TRACE dog is. Tickets have already been written for vio- lating this Forest Rule. When you are out on the trails, bring a plastic bag with you to hold all of your trash. That includes Stay Off Waterfalls - One slippery rock can lead banana and orange peels! It takes two to five to a tragedy near a waterfall. Tragic deaths at weeks for the peels to decompose. There are waterfalls are reported every year in Western dumpsters available at the High Falls and Hooker North Carolina. Nine people have died in fatal falls Falls Access Areas. in state parks since 2005. For your safety, please follow the guidelines below: TRAIL ETIQUETTE • Observe all posted signs leading to a water- • Hikers, runners, and bikers should fall area. always yield to horses. • Stay on marked trails and observation • Bikers should yield to hikers and runners. areas. • When approaching from behind a friendly • Do not jump off waterfalls or dive into pools greeting can go a long way. (not even at Hooker Falls). • Be kind to each other. • Do not climb on rocks above waist height. Equestrians • Do not swim or wade upstream near a Avoid bringing horses into the Forest who are not waterfall. already desensitized to the many things encoun- • Expect slick rocks around waterfalls tered within a multi-use trail system. It is always a good idea to have a “been there, done that” buddy • Watch your children at all times. horse accompany a young or inexperienced Visitor may swim at his or her own risk in DuPont’s horse. Little River, except within 300 feet upstream Some trail users are intimidated by the size of of the top of a waterfall (and other designated horses and are not sure of what to do when they non-swimming areas). Continued on Page 7 5 MEET THE N.C. FOREST SERVICE STAFF AT DUPONT FOREST

By Sarah Patton, DuPont Forest Service Administrative Assistant

DuPont State Recreational Forest and Holmes Educational State Forest have recently undergone staff additions and organization restructuring, and I am excited to introduce the 23 staff in a series of articles.

Justin Carpenter - Maintenance Supervisor see how they work.” When he is not working at Justin Carpenter took his position at DuPont DuPont, John enjoys spending time with his family State Recreational Forest as the Maintenance or refurbishing antique radios and cars. John’s Supervisor in September 2014. Justin supervises responsibilities at DuPont include keeping the DuPont’s Maintenance Branch which oversees vehicles and machines in operating order. He is facilities, dams, vehicles, equipment, and grounds. also responsible for ensuring that all fire machin- Prior to DuPont, Justin had more than 15 years of ery stays fire-ready. contractor work. He specialized in disassembling and reconstructing 140 to 180 year old Amish Buck Jones - Maintenance hand-hewn log homes. One of the projects he Buck Jones was born and raised in Brevard, N.C. worked on was the construction of what is now While in high school he won a Future Farmers of the Aleen Steinberg Visitor Center. America competition in small engines, leading Justin hopes that in the future DuPont can focus him to be hand-picked to attend the John Deere more on forest operations and avoid becoming Company school. He graduated with certifications structured like a park. “I’d like to see DuPont go in hydraulics, diesel engines, brakes systems, more in depth with the N.C. Forest Service mis- electrical systems, and two cycle engines to name sion statement; to protect, manage, and promote a few. He worked for the John Deere company for forestry resources. There are so many valuable 20 years as a mechanic, sales rep and then as the resources here; the lakes, the waterfalls, the manager of the John Deere store in Brevard. Buck forest land and the timber land. I know we’re very has been in Fire and Rescue for 27 years, is a cer- fortunate that we log, manage, burn, fish, and tified EMT, and is currently an active responder at hunt here, but I’d like to see more of those activi- a local Fire and Rescue station. In his spare time, ties and feel less like we’re a tourist attraction.” Buck loves to restore vehicles from the 1930s, 1940s and 1950s and says, “they had class, they John Morris - Mechanic had style. They didn’t have creature comforts. It’s cool to get in a vehicle and roll down the window John Morris, who began working at DuPont because you don’t have air conditioning. Enjoy the State Recreational Forest in October 2017, is road, instead of enjoying the stuff in your vehicle.” the Forest’s new Mechanic. Born and raised in Transylvania County, John grew up hiking, hunt- Buck joined DuPont as a part-time Maintenance ing, and fishing at DuPont while his father worked employee in 2014. He became a full-time for 38 years in the coding department at DuPont employee in March 2017 and works to maintain Corporation. the Forest’s facilities and grounds. Buck drives the fuel truck for the N.C. Forest Service helicopter Besides working at DuPont, John owns a car and is also a helicopter bucket crewman. garage where he does automobile repairs, metal fabrication, and machine work. He also worked When asked what he loves most about his job, as a plumber for 19 years. John enjoys working Buck said, “Being outside. And the variety that my with his hands, especially if he can fix something job gives me and the variety of things to do.” that is broken. “Ever since I was old enough to turn a screwdriver I was taking toys apart to Continued on Page 5 6 Continued from Page 4 if you are in control when you come bombing Bob Twomey - Maintenance down Ridgeline (for example). A little friendly communication can go a long way. Bob Twomey, born and raised in Asheville, N.C., graduated from N.C. State with a BS When yielding to horses, move to the downhill in Conservation in 1977. Bob worked as a side. It reduces your size and appearance and Soil Conservationist in Sampson County and makes the horse less likely to spook. Engage Rutherford County and later became the District the rider in friendly conversation as this can Conservationist for Catawba County for 5 years help identify you as a human to a horse. and then Transylvania County for 28 years. His Protect the trails and look for a safe a durable job was to develop, maintain, and enhance a spot to stop when yielding for hikers or eques- conservation program with the landowners. trians. When riding on a single track come to a Bob believed the beauty of the job was in the complete stop and then side step off the trail. good relations with landowners and land agen- This helps us avoid erosion. cies. Bob’s career spanned over 37 years before he retired as District Conservationist of both Use a controlled speed and stay single file on Transylvania and Henderson Counties. singletrack trails. Bob’s retirement lasted 32 days before he was Hikers hired as DuPont’s Visitor Center Manager in Stay on the trail! Hiking off trail damages frag- January 2013. In March 2017, Bob transferred to a ile plants and erodes our trails. maintenance position. His duties include upkeep of facilities, trails, lakes, and dams. Bob loves the Communicate with equestrians and try not variety of his job and not knowing what he will be to make any sudden movements that might doing from one moment to the next. “It isn’t the spook the horse. same thing every day. You set your work plan for Keep your dog on a leash at all times. It is a the day, but that can change in a radio call.” Bob Forest Rule and you can risk a ticket! has been involved with volunteer rescue for 44 years and has maintained his EMT certification Keep your dog close to you when passing since 1974. children, horses, or other dogs no matter how Friends of friendly your dog may be. DuPont Forest Technology on the Trail When taking pictures be aware of your sur- Sharing is Caring roundings and don’t block the trail. Continued from Page 5 Hiking or riding with earbuds in place makes it encounter a horse on a trail. Give a friendly “hello” difficult to communicate with other users and and continue to carry on a conversation so your reduces your ability to hear what’s going on horse can identify them as human not a mountain around you. lion or bear. As Friends of the Forest, following the Forest Help protect our trails and stay on the trail. Ride Rules and practicing proper trail etiquette is single file in the middle of the path and don’t take one of the best way we can protect DuPont. shortcuts or cut across switchbacks, which can Tell your friends! Share with visitors! And trample fragile plants and create erosion. thank you!

Friends of Mountain Bikers DuPont Forest Call out as you come down steep slopes or blind switchbacks and let people know if there are other bikers behind you. Bikers can be intimidat- ing to other users and sometimes it is hard to tell

7 American Chestnut Tree Returns Continued from Page 3 species called Agrobacterium tumefaciens that naturally mates with some species of plant cells. The term “mate” is used rather than “infect” because Agrobacterium tumefaciens itself does not enter the plant cell. What actually happens is that a bacterial cell makes contact with a plant cell, creates a pore in the plant cell wall, and Figure 5 injects a very specific complex of bacterial DNA and proteins through the pore into the cyto- plasm. The proteins associated with the bacterial DNA guide the DNA into the cell nucleus where the DNA is recombined with (integrated into) a plant chromosome at a random site.

Agrobacterium tumefaciens (Figure 5) exists naturally in soil and has the capacity to mate with more than 140 species of plant cells and induce a tumor-like disease commonly referred to as crown gall. The integrated bacterial DNA Figure 6 encodes enzymes for the production of hor- Figure 5 – Image taken with a scanning electron micro- mones (including auxin) that stimulate plant cells scope of Agrobacterium tumefaciens. The rod-shaped to grow and divide profusely as a benign tumor bacterial cells are about 0.003 mm long. Photo courtesy and also induce plant cells to produce nutrients of Shirley Owens, Microbe Zoo Project, Comm Tech Lab, needed by the bacteria to propagate. The net Michigan State University. Figure 6 – Gall at the base of result is a large growth on the plant called a an oak tree. Photo courtesy of Kent Wilcox. gall (Figure 6) that provides a nutritious habitat “genes of interest” at a site that causes inactiva- for Agrobacterium tumefaciens. Scientists have tion of a chestnut gene required for flowering or cleverly modified (some use the term “disarmed”) drought resistance would create an undesirable the DNA injected by Agrobacterium tumefaciens GMO chestnut tree. The other bad news is that so that it lacks the genes for production of hor- the injected DNA contains sequences required mones and nutrients, but retains the sequences for the integration process and marker genes needed to integrate a DNA fragment that con- for selecting modified cells that some GMO tains “genes of interest” into plant chromosomes. opponents consider to be undesirable foreign In the case of GMO corn and soybeans, those material. “genes of interest” include a gene that confers resistance to a pesticide or herbicide plus one or In spite of these difficulties, in 2006 Dr. William more marker genes to identify and/or select the Powell and his colleagues in the College of cells with integrated DNA. Because gene transfer Environmental Science and Forestry at the is successful in less than 10% of the cells, these State University of New York in Syracuse suc- marker genes provide a method to eliminate the ceeded in using somatic cell embryogenesis cells that lack the genes of interest. Scientists and Agrobacterium-mediated gene transfer to have tweaked the process to expand the range of create American chestnut trees with increased plant species that will “mate” with Agrobacterium resistance to blight. But since the Chinese chest- tumefaciens. That’s the good news. The bad nut genes that confer resistance to chestnut news – and this can be a deal breaker in some blight were not known in 2006, what gene did cases – is that the injected DNA integrates into Dr. Powell’s group use? To answer this ques- random sites in plant chromosomes. Insertion of tion, we need to back up a bit. The detrimental

8 Continued on Page 9 American Chestnut Tree Returns answer requires a separate article, but in a nutshell, the first generation transgenic trees Continued from Page 8 exhibit only moderate resistance to the blight. Figure 7 – Wild-type Resistance equivalent to that exhibited by Asian (left) and ‘Darling Chinese chestnuts will probably require elevating 54’ transgenic (right) the amount of oxo produced and/or adding addi- American chest- tional genes that contribute to resistance. Even nut trees 30 days with such improvements, first generation GMO after infection with trees are essentially clones that lack the genetic the blight fungus diversity required for optimal growth in different Cryphonectria para- ecological habitats from Alabama to Maine. The sitica. Photo courtesy American Chestnut Foundation is cross-breeding of Andrew Newhouse, SUNY College of transgenic trees with wild-type American and Environmental Science hybrid Asia/American chestnuts to create trees and Forestry. with increased diversity and resistance (Jared Figure 7 Westbrook, “Merging Genomics and Biotechology with Breeding” JTACF, fall 2016). effects of some fungi (including Cryphonectria parasitica) are due in part to the production by However, until sufficient studies establish other- fungi of oxalic acid (an organic acid originally wise, there are concerns that integration of the isolated from the wood sorrel plant Oxalis and a oxo and other foreign genes at random sites in toxic component of rhubarb leaves). Oxalic acid the twelve chromosomes of American chestnut promotes fungal invasion by causing degradation trees could have adverse effects on the long of the lignin and cellulose in the rigid walls that term survival and reproduction of GMO chestnut surround plant cells. To counteract the destruc- trees in the wild. Research is ongoing to deter- tive effects of oxalic acid, some plants (but not mine whether high levels of oxalate oxidase the American chestnut) produce an enzyme production in the roots of chestnut trees might called oxalate oxidase (oxo), which converts inhibit some mycorrhizal fungi that establish oxalic acid into carbon dioxide and hydrogen symbiotic relationships with and promote growth peroxide. An extensive investigation in the of chestnut trees. There are concerns about the 1990s of the oxalate oxidase enzyme produced potentially harmful effects of high levels of oxa- by wheat convinced scientists that this enzyme late oxidase on organisms that collect pollen or plays an important role in conferring resistance consume the nuts or leaves from chestnut trees. against fungal infections in wheat. The rush was One interesting study revealed that gypsy moth on to see if the same might be true for other caterpillars that fed for three days on leaves plants. Dr. Powell’s group used the wheat oxo from first generation transgenic American chest- gene to create GMO poplar trees and American nut trees grew 16% faster than caterpillars that chestnut trees while other groups used the same fed on leaves from wild-type American chest- gene to create GMO soybean, tomato, and pea- nut trees, but there was no follow-up study to nut plants that exhibit increased resistance to determine whether bigger caterpillars turn into fungal infection. more prolific moths. Because white rot fungi secrete oxalic acid to promote the degradation It’s been more than ten years since the first of leaves and woody tissues, scientists are test- transgenic, partially blight-resistant American ing whether decomposition of leaves with high chestnut trees were created (Figure 7). Several oxo levels takes more time. To alleviate some of thousand transgenic American chestnut trees these potentially detrimental effects, scientists are now growing on test plots in New York. Do are exploring options to replace the first version these first generation transgenic trees make the of the oxo gene that produces the enzyme in sixth generation of Chinese-American hybrids all tissues of chestnut trees with a version that obsolete? The short answer is “no”. A thorough produces oxalate oxidase only in the trunk and

Continued on Page 10 9 American Chestnut Tree Returns to interbreed GMO trees to create a population of trees that contain two copies of the trans- Continued from Page 9 gene. Crossing cisgenic trees with three different limbs, which are the portions of the tree that genes for blight resistance will require screening are most susceptible to infection by the blight several hundred progeny trees to find a tree fungus. with two copies of all three genes. Fortunately, Will genetic engineering replace traditional testing tissues for specific DNA sequences can breeding as the method to create blight-resistant be done in two days (Jared Westbrook, “Genomic trees? The short answer is “yes, but not entirely”, Selection for Disease Resistance” JTACF, fall with some caveats. If regulatory agencies or 2017), compared to the six to nine months members of the public are reluctant to endorse required to test trees for blight resistance. transgenic trees that contain a gene(s) from an Since the first attempts to create blight-resistant unrelated organism, repopulation of Appalachian American chestnut trees began nearly 100 years forests with American chestnut trees may not ago, there has been a lot of progress towards occur until cisgenic GMO trees with the three or the goal of repopulating Appalachian forests more Chinese chestnut genes that confer blight with American chestnut trees. Much has been resistance are created. The fact that DNA trans- learned from the traditional breeding programs ferred by Agrobacterium is integrated at random about methods to pollinate, propagate, and eval- sites in plant chromosomes is problematic, uate chestnut trees. More emphasis is now being because every new transgenic tree made by this put on the importance of diverse strains that are procedure will have the blight-resistant gene(s) adapted to specific habitats. This information inserted at a different site. Deleterious effects of is essential for current projects that combine some integration events may not become appar- traditional breeding methods with advances in ent until years after GMO nuts are planted. This genetic engineering and cell biology to create random-site-integration problem may be solved a diverse population of trees that, other than using a new technology called CRISPR, which increased resistance to fungal diseases, are allows investigators to precisely insert a gene American chestnut trees in all respects. at a predetermined chromosomal site that does not disrupt nearby genes. In terms of production The author wishes to acknowledge North time, genetic engineering has a huge advantage Carolina State University for providing access to over the thirty or more years required for six many outstanding research papers on this topic, generations of traditional breeding. The current especially those authored by Dr. William Powell method used to create a transgenic plantlet from and Dr. Scott Merkle and their colleagues. For one somatic cell takes approximately 14 months more information, readers are encouraged to and can be scaled up to produce hundreds of explore the resources available at plantlets. Diversity can be achieved by creating www.esf.edu/chestnut. somatic embryos using wild-type American chest- Kent Wilcox is a retired scientist who lives in Cedar nuts collected from a variety of geographic sites. Mountain, NC. He has been a member of Friends Because first generation transgenic trees contain of DuPont Forest since 2002 and has served on the Friends of only one copy of a transgene, it will be necessary board for six years. DuPont Forest

NEWSLETTER CONTRIBUTORS Are you interested in getting more Editor: Sara Landry involved with Friends of Dupont? If you Designer: Garnet Fisher are passionate about conserving DuPont Masthead Photo: © RobTravis.com Forest, you should consider serving on Newsletter Committee: Aleen Steinberg, our Board of Directors. Please email Jeff Alan Cameron, and Kent Wilcox Jennings at [email protected]. 10 CALENDAR OF EVENTS HIKES, TOURS & PRESENTATIONS *Members Only Events

June 2 Litter Pick Up Dupont Trails Day Saturday, June 16th Help keep DuPont beautiful! June 16 Litter haters meet four times a year Cherokee Lecture and Walk* on selected Saturdays. Sign up to be a Litter Hater at : Litter Pick Up www.dupontforest.com/volunteer/

July 14 Moonshine in These Hills

July 21 Snake Lecture* DuPont Forest Festival Saturday, August 7 September 22nd Mushroom Hike*

August 14

Medicinal Plants of the Mountains Details about each event will be shared with Friends of DuPont Forest members via email and Facebook.

JOIN FRIENDS OF DUPONT FOREST AT WWW.DUPONTFOREST.COM

MANY THANKS TO OUR FOREST FESTIVAL SPONSORS!

11 JOIN OR RENEW YOUR MEMBERSHIP

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