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Using Genetically Modified Biotechnology to Improve Citrus

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Using Genetically Modified Biotechnology to Improve Citrus COVER STORY Using genetically modified biotechnology to improve citrus By Manjul Dutt and Jude Grosser lorida has a large and vibrant citrus industry, mainly producing grapefruits and sweet oranges. This industry has faced numerous challenges in the past, in the form of diseases such as citrus canker and the tristeza virus or damage due to hurricanes and freezes. Growers have lost hundreds of thousands of trees to these challenges, but have always bounced Fback by planting new groves and resets in their existing groves. The recent huanglongbing (HLB) outbreak has led the industry to a crossroads. This bacterial disease caused by a non- indigenous pathogen has resulted in substantial economic losses and threatens the survival of our industry. Resistance to HLB is not present in the commercial grapefruit and sweet orange cultivars grown in Florida. Citrus canker disease remains a problem, and a relatively new introduced fungal disease, citrus black spot, also has the potential to be a problem. Improved commercially acceptable cultivars that can effectively control these diseases are urgently needed to maintain a vibrant industry. CONVENTIONAL AND MUTATION BREEDING producing changes that can result in an improved cultivar. Citrus genetic improvement efforts utilize different Natural mutations have resulted in most of our sweet techniques to produce the same outcome. In conventional oranges and grapefruits, and artificial mutations have citrus breeding, scientists mix two sets of DNA — one from produced seedless mandarins such as N40W-6-3 (Seedless the mother and the other from the father — to produce hybrid Snack), Tango, Fairchild LS and Kinnow LS as well as the embryos through a process called meiosis. These embryos scab-tolerant mandarin US Furr ST. Several of the new sweet give rise to hybrid seeds that nature alone would be unlikely oranges recently released by University of Florida/Institute to ever produce. A large population of seeds (containing the of Food and Agricultural Sciences (UF/IFAS), including DNA from both mother and father) have to be subsequently Valquarius and OLL-8, are products of somaclonal variation. germinated and rigorously evaluated in order to identify Citrus has a long juvenile period, and most cultivars progeny exhibiting industry-favorable characteristics. take at least six years from seed germination to flower. Improved citrus cultivars have also been produced Rapid genetic improvement to combat emerging abiotic and through natural as well as induced mutations. Natural biotic conditions is usually hampered by this long juvenile mutations have occurred spontaneously over time. Tree limbs phase. Although every single improved citrus cultivar containing a natural mutant and fruits observed to be very grown in the world has arisen from the above-mentioned different from the parent have been selected, evaluated and techniques, these techniques cannot always guarantee mass-propagated. Tissue culture-induced cell mutations the genetic integrity of the new cultivar in relation to the (somaclonal variation), or zapping budwood with radiation existing parental cultivar(s). In these cases, planted trees to induce mutations, can also result in the development of may contain thousands of unknown changes. Such methods improved cultivars. Both of these are shotgun approaches have been used for almost a century. There is minimal risk, requiring large plant populations to find improved selections. nobody worries about regulating such arrangements, and These processes can cause bits and pieces of DNA to break the products are perfectly acceptable for use in organic and rearrange themselves differently, or a point mutation production. These radical transformations work, but that is a single nucleotide change in the DNA’s sequence, nobody worries too much about them. 10 Citrus Industry September 2015 WHAT IS GM PLANT IMPROVEMENT? physiological or biochemical advantage to the new plant. In In contrast to conventional plant improvement using citrus, the new plant could now have a new gene that imparts the above mentioned techniques, production of genetically tolerance to diseases without changing the DNA of the plant modified (GM) plants (also called transgenic or genetically or the integrity of the cultivar. This is often referred to as engineered plants) can result in rapid improvement of “precision breeding.” an existing variety. The most commonly used process of introducing a gene to a plant is called “Agrobacterium- WHAT ARE WE DOING? mediated genetic engineering.” With this process, a specific The citrus improvement team at UF utilizes GM gene (DNA sequence) of interest is inserted into the host technologies to create new tools to facilitate conventional plant’s genome with the help of a common pathogenic soil plant breeding. The major problem facing the industry is the bacterium called Agrobacterium, often referred to as “nature’s various diseases affecting citrus. Biotechnology-mediated own genetic engineer.” This bacterium can enter and infect strategies are being developed to combat these diseases. Just the plant cell through a wound. The bacterium inserts a as animals contain white blood cells that are involved in special sequence of its own DNA into the plant cell’s DNA. protecting the body against infections, plants also have the This original sequence of inserted wild-type DNA can be ability to produce an immune response following infection. altered, removing bad DNA (deleterious genes) and replacing This immune response [known as systemic acquired it with disease-resistant genes scientists want inserted. resistance (SAR)] occurs after plants are infected and can After the new beneficial DNA is delivered by the provide strong resistance to subsequent infections. Agrobacterium, scientists use an antibiotic to kill the An SAR gene called NPR1 isolated from Arabidopsis, a Agrobacterium, so the engineered cells are no longer infected. mustard plant relative, is being evaluated for its ability to Any engineered cell containing the small extra piece of control HLB. Inserting this gene in citrus using Agrobacterium inserted beneficial DNA can then be made to produce a keeps it switched on at all times and results in the tree’s whole plant using tissue culture techniques. This plant will immune system being constantly turned on and ready to fight therefore contain all the original DNA of the cultivar, with against infection. All genes are controlled by small sequences the addition of a small, “foreign” DNA imparting some of DNA called promoters. Simply put, a promoter is a switch Need New Citrus Varieties? Sugar Belle® Tango U.S. Early Pride Mandarin Hybrid • Low-Seeded easy • Low-Seeded tangerine • Unique specialty hybrid of peel mandarin • Same maturity period Clementine and Honeybell • Maturity range: as Fallglo • Dec.–Jan. maturity Thanksgiving–January • Now available for • High flavor — strong commercial production consumer appeal NVDMC New Varieties Development & Management Corporation s UF/IFAS-developed “UFR” rootstocks and sweet NVDMC supports new variety development oranges are available for licensing to nurseries research in Florida through Florida Foundation Seed Producers, Inc. Oranges include: s NVDMC provides access to unique and high value • Valquarius brand ‘SF14W-62’ • ‘N13-32’ (improved Hamlin (early maturing valencia) type) varieties for growers, nurseries and packers. • ‘OLL-4’, ‘OLL-8’, ‘B9-65’ • ‘UF11-1-24’ (nearly seedless Fresh and processed varieties. (improved Valencia) improved Mid-Sweet type) • ‘N7-3’ (seedless late Valencia) s NVDMC serves as licensing agent for public and Rootstocks include: Interested growers and private varieties. Working to keep citrus growers UFR-1; UFR-2; UFR-3; UFR-4; packers should contact UFR-5; UFR-6; UFR-15; UFR-16 John Watson at (352) 317- on the leading edge. and UFR-17 0703 or [email protected] Contact NVDMC for other varieties available for trial planting Staff: Peter Chaires • E-mail: [email protected] • Website: NVDMC.org Citrus Industry September 2015 11 that tells the gene to express needed for Agrobacterium-mediated engineering — even if at specific locations in the introduced DNA helps impart durable disease resistance the plant. A constitutive and has been proven to be safe. Development of GM citrus promoter or switch turns where all the introduced DNA comes from edible plants the gene on throughout the should eliminate this concern. entire plant. We have developed a unique protoplast (a naked cell We have produced several that had its cell wall completely removed using digestive transgenic sweet orange enzymes) genetic engineering (transformation) system that trees containing this gene can potentially allow the addition of only edible plant-derived — being driven by either a transgenes without any additional bacterial/viral DNA constitutive promoter or a sequences. In this system, a plant-derived visual reporter gene phloem-targeted promoter is inserted along with the gene of interest directly into an (HLB resides in the phloem) isolated citrus protoplast with the help of polyethylene glycol — that have demonstrated or electroporation. enhanced tolerance to HLB. Why is the visual reporter gene needed? In the process of Engineered sweet oranges protoplast transformation, millions of cells are exposed to the with this gene have remained foreign DNA, with many of those cells taking up the DNA. HLB resistant after two and However, only a few cells are able to successfully incorporate Figure 1: University of Florida a half years in a greenhouse
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