New acid citrus selections for Florida

Lemon and lime-like selections with niche market potential are being developed with biotechnology at the University of Florida.

By Jude Grosser, Zenaida Viloria and Manjul Dutt

re you ready for a purple margarita? Would you like a fragrant, juicy lime is a naturally occurring citrus lemon for your iced tea with no seeds to clog your straw or dishwasher triploid, which is why it is seedless. drain? How about some seedless lime trees that are cold-hardy enough The new seedless watermelons in the Afor Central Florida? These and many more interesting acid-citrus marketplace are also triploids. selections are now on the horizon, including some with good ornamental potential. Due to the fact that new and This article will focus on progress in using emerging biotechnologies to develop improved citrus cultivars must be interesting new citrus cultivars in the lemon and lime group. Cultivars include seedless to compete in the national some that will not have regulatory constraints, and also a genetically modified and international marketplace, the organism (GMO)-derived purple Key lime as a teaser for the future. University of Florida’s Citrus Research and Education Center (UF/CREC) LEARNING FROM they are triploids. People and most citrus improvement team (working THE BANANA citrus trees are diploid, meaning with Fred Gmitter) has formulated Have you ever wondered why you there are two sets of chromosomes in several ways to create triploids as a key never find seeds in your bananas? Did each cell. Triploid bananas have three method of developing seedless citrus you know that there are wild-type sets of chromosomes per cell. This cultivars, especially in the mandarin bananas growing in some jungles disrupts the chromosome pairing group. The team’s primary method around the world that are full of mechanism that occurs during meiosis for producing new, seedless, triploid seeds? The reason commercial banana (cell division), resulting in sterility hybrids is a method called interploid cultivars do not have seeds is because and few, if any, seeds. The Persian hybridization, which is simply the

30 Citrus Industry July 2015 conventional breeding of a diploid interploid crosses. The majority of approved for release by the IFAS parent with a tetraploid parent that has the UF/CREC team’s efforts using Cultivar Release Committee (Figure 1, four sets of chromosomes per cell. this technology target fresh citrus page 32). The first selection approved The team has pioneered a groups most important to Florida, i.e. for release is C4-5-27, produced from technique called cell fusion to mandarins, oranges and grapefruit, a cross of Key lime with a tetraploid produce most of the tetraploid but some small efforts in the acid lemon (we call this a “lemonlime”). parents used in this approach. citrus category have been made with It produces nearly seedless fruit that Tetraploid hybrids produced by this very promising results. resembles a Key lime in size and flavor, technique could be called test-tube and yields quite well. This selection babies (in reality, they are petri-dish NEW HYBRIDS WITH has ornamental potential as the new babies). This technique fuses cells POTENTIAL FOR growth flushes exhibit an intense red/ isolated from tissue cultures that COMMERCIALIZATION purple color that remains until leaves are embryogenic, meaning each cell Overall, the citrus improvement are fully expanded, and the flowers are has the capacity to develop into an team has produced more than 20,000 fairly large with a beautiful, dark-pink embryo, just like you would find triploid citrus hybrids, including a color. Fruit from this selection does not in a seed, with cells isolated from few hundred in the acid citrus fruit store well, making it more attractive for leaves of the second complementary group. Many of these have flowered dooryard use. parent. (Leaf cells cannot regenerate and fruited, and so far, 10 from this The second selection approved for new trees by themselves.) Use of an group have been selected that may have release is the Iced Tea hybrid lemon, embryogenic parent is necessary to commercial potential. All 10 of these a seedless triploid that is uniquely provide the capacity for whole-tree have been entered into the Division of shaped like a tangelo. Fruit size is larger regeneration from any fused cells. Plant Industry Parent Tree Program, than a standard lemon. Fruits are juicy Before a fusion can be performed, and pathogen-free budwood is now with a very pleasant lemon fragrance. the rigid, cellulose plant cell walls must available for trials. This hybrid is two-thirds lemon be removed to produce naked cells Two of the selections have been and one-third sweet orange, so it is (called protoplasts). These cells still have an intact membrane and internal components. A battery of enzymes under specific osmotic conditions digests away the cell walls. The Lab resulting naked cells can then be Technologies for improved wear resistance induced to fuse, using a chemical called and corrosion resistance of stainless steel polyethylene glycol. materials Following the fusion, cells regrow their walls and begin dividing to form ����� �� �� ��e ������ e��� colonies, some of which develop into ����� ��� embryos from which we can recover whole plants. A machine called a table-top flow cytometer is used to determine the ploidy (number of sets of chromosomes) level of the regenerating plants, efficiently identifying any tetraploids. Then, a genetic fingerprinting technique is WITH used to determine if the tetraploid GROW YOUR BUSINESS resulted from the fusion of two cells from the embryogenic parent CITRUS INDUSTRY MAGAZINE. (called an autotetraploid) or the targeted fusion of cells from each Proudly serving the citrus parent (called an allotetraploid). community since 1920. Allotetraploid hybrids produced by this technique are called somatic hybrids. The tetraploids are grown Contact Bob Guerriere for advertising information. in the field, and when they reach the (352) 671-1909 / [email protected] flowering stage, they can be used in Citrus Industry July 2015 31 Citrus News on

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WOCA-AM 1370 Figure 1. Clockwise from the top left: seedless triploid lemonlime C4-5-27 (approved WOCA-FM 96.7 for release); the Iced Tea triploid hybrid lemon (approved for release); seedless triploid lemorlime C4-9-33; seedless triploid lemorlime C4-5-20. n Palatka expected to be slightly more cold-hardy of the more desirable triploids produce WIYD-AM 1260 than a standard lemon. excessively thorny trees. n Also featured in Figure 1 are two Port St. Lucie seedless, triploid lime hybrids — C4-5- ANTHOCYANINS AND WPSL-AM 1590 20 and C4-9-33 — which we refer to as PURPLE CITRUS n “lemorlimes,” since they are one-third Anthocyanins are naturally Sebring lime, one-third lemon and one-third occurring compounds in plants and WWOJ-FM 99.1 sweet orange. Both of these lime-like are responsible for the coloration WWTK-AM 730 hybrids produce larger and juicier in blueberries, grapes, cherries, fruit than Persian lime. The C4-5-20 pomegranates and many other n Stuart fruit comes off the tree looking like it fruits and vegetables. In citrus, the has already been waxed in the packing blood orange contains high levels WSTU-AM 1450 house. The C4-9-33 selection has of anthocyanins, and the purple For airtimes, visit an attractive green interior and very coloration is due to its presence. www.SoutheastAgNet.com pleasant limy fragrance. These two Anthocyanins have been studied selections will be considered for release extensively for their numerous during the next year. Triploid hybrids pharmacological properties. Their of this type produced by interploid major roles include antioxidant Suppliers hybridization are not regulated benefits, ability to protect against free Are Your Customers as GMOs, and thus can be readily radical scavenging, anti-cancer activity Hearing You? commercialized. The most obvious and prevention of cardiovascular downside to this approach is that many disease, among others. Farm Broadcasting Works!

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32 Citrus Industry July 2015 Commercial blood oranges are derived from the sweet orange. They arose through mutations from a sweet orange variety in the Mediterranean region. These blood oranges cannot be commercially grown in the citrus belt in Florida due to their inability to color up in the absence of appropriate cold conditions lacking in our subtropical climate. The UF/CREC team isolated and subsequently expressed the transcription factor VvmybA1 (induces the anthocyanin biosynthesis in grape skins) and Ruby (induces the anthocyanin biosynthesis in blood oranges) in Key lime using standard genetic engineering technology (Figure 2). Expression of these transcription factors enhanced the anthocyanin production in Key limes and resulted in production of anthocyanin in most plant tissues. The transcription factor was also expressed in the fruit pulp. Figure 2. Top: A young anthocyanin These plants have both ornamental overexpressing Key lime plant (left) with a regular plant for comparison on the right. as well as potential health benefits, and Bottom: Cross-section of an anthocyanin the fruits with elevated anthocyanin expressing Key lime fruit. levels are being evaluated. These genes are also currently being incorporated More recent work has focused on into other selections in the citrus disease resistance. Disease-resistant improvement pipeline, including parents have been developed and commercial-quality sweet oranges, for incorporated into interploid crosses. The the development of a subtropical blood UF/CREC team will continue to produce orange cultivar. Having genetically new and improved high-quality citrus modified citrus with improved nutri- triploids, and many of them are expected tional value should help convince the to be more tolerant to HLB, including public that new fruits produced by this some in the lemon and lime category. technology can be beneficial to society. GMO approaches with tremendous potential are also being pursued. CONCLUDING REMARKS Emerging biotechnologies are Jude Grosser is a research professor, and creating many new tools to facilitate Manjul Dutt is an assistant research traditional plant breeding. As a result, scientist — both at the University of many new selections with commercial Florida/IFAS Citrus Research and and niche market potential are being Education Center in Lake Alfred. developed and released. High-value, Zenaida Viloria is a part-time research niche-market selections should provide assistant at the Kingsville Citrus Center new opportunities for smaller, family- in Weslaco, Texas. For more information, type citrus farmers. contact Jude Grosser at [email protected].

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