Production of Three New Grapefruit Cybrids with Potential for Improved Citrus Canker Resistance
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In Vitro Cell.Dev.Biol.—Plant (2017) 53:256–269 DOI 10.1007/s11627-017-9816-7 PLANT TISSUE CULTURE Production of three new grapefruit cybrids with potential for improved citrus canker resistance Ahmad A. Omar1,2 & Mayara Murata1 & Qibin Yu1 & Fred G. Gmitter Jr.1 & Christine D. Chase3 & James H. Graham1 & Jude W. Grosser1 Received: 6 January 2017 /Accepted: 8 March 2017 /Published online: 27 April 2017 / Editor: Wenhao Dai # The Society for In Vitro Biology 2017 Abstract Cybrid production, combining the nucleus of one polymorphism between kumquat and grapefruit and were used species with alien cytoplasmic organelles of another, is a poten- to validate the cybrids. All the cybrids had the mt genome of tially valuable method used for improvement of various crops kumquat, and most had the cp genome of kumquat with a few including Citrus species. Furthermore, this technology is con- exceptions. EST-SSR marker analysis confirmed that the nucle- sidered a non-GMO biotechnology strategy. In citrus, cybrid ar genome in all the generated cybrids came from the grapefruit plants can be produced as a by-product of somatic fusion. parent. All the cybrid clones have been propagated and are Host resistance is the most desirable strategy for control of undergoing extensive canker assays to identify any clones that citrus canker. By using a cybridization approach, several puta- have improved canker tolerance/resistance. These cybrid pop- tive cybrids were created by protoplast fusion of embryogenic ulations provide a valuable tool for investigating the contribu- suspension culture-derived protoplasts of canker resistant tion of cytoplasmic organelles to plant disease resistance. ‘Meiwa’ kumquat (Citrus japonica Thunb), with mesophyll- derived protoplasts of three grapefruit (Citrus paradisi Keywords Disease resistance . Microsatellite marker . Macfad.) cultivars ‘Marsh,’‘Flame,’ and ‘N11-11’ somaclone Organelle inheritance . Mitochondrial introns . Chloroplast of ‘Ruby Red.’ In an effort to generate new grapefruit cultivars genome with enhanced canker resistance, putative cybrid grapefruit plants morphologically equivalent to standard grapefruit from all three combinations were produced. Four mitochondrial (mt) Introduction introns, a mt ribosomal RNA spacer region, and four chloro- plast (cp) DNA regions previously shown to have polymor- Citrus canker is one of the most devastating diseases for citrus phism among different Citrus species were tested. Four molec- industries in many regions around the world, which causes a ular markers, two mt DNA regions (intron nad7i2 and a rRNA huge economic loss. Citrus canker is caused by a gram- spacer), and two cp DNA regions (NADH dehydrogenase sub- negative bacteria, Xanthomonas citri subsp. citri, (Xcc) (syn- unit K (ndhk)geneandatrnG-trnR intergenic spacer) revealed onym, Xanthomonas axonopodis pv. citri strain A). Citrus spp. and relatives vary in terms of their susceptibility to citrus canker disease, with most of the commercially important Ahmad A. Omar and Mayara Murata contributed equally to this work. grown citrus types rated as susceptible hosts to Xcc * Jude W. Grosser (Schubert et al. 2001). For instance, grapefruit is highly sus- [email protected] ceptible to Xcc (Schubert et al. 2001,Grahamet al. 2004), while kumquats are considered to be highly resistant to citrus 1 University of Florida, IFAS, CREC, 700 Experiment Station Road, canker. Disease symptoms include canker lesions on leaves, Lake Alfred, FL 33850, USA stems, as well as fruit. Severe infections can cause both defo- 2 Biochemistry Department, College of Agriculture, Zagazig liation and fruit drop, which result in decreasing the produc- University, Zagazig 44511, Egypt tivity and profitability of affected trees. Blemished fruit is not 3 Horticultural Sciences Department, University of Florida, IFAS, accepted in the marketplace and can significantly impact ex- Gainesville, FL 32611, USA port potential. PRODUCTION OF THREE NEW GRAPEFRUIT CYBRIDS 257 Kumquats (Citrus japonica Thunb) (synonym, Fortunella Wallin et al. (1978) to produce cybrids, but found it to be spp.), closely related to citrus and sexually compatible, exhibit challenging and not applicable for citrus breeding. However, high levels of field resistance to citrus canker (Schubert et al. verified cybrid plants have been recovered spontaneously as a 2001). Khalaf et al. (2007) reported sharply contrasting phe- by-product from intraspecific, interspecific, and intergeneric notypes between ‘Duncan’ grapefruit (Citrus paradisi symmetric somatic hybridization experiments in plants, espe- Macfad.) and ‘Nagami’ kumquat (C. japonica) when both cially citrus (Grosser et al. 1996,Cabassonet al. 2001,Guo plants were challenged with ahighconcentrationofXcc et al. 2004b). Guo et al. (2013) reviewed cybrids arising from (108 cfu mL−1). Kumquat leaves showed rapid necrotic le- more than 40 combinations by symmetric fusion. These cybrids sions that appeared to be a hypersensitive response (HR), usually have the nuclear genome of the mesophyll parent and while grapefruit leaves showed water-soaking and blister- mitochondria (mt) genome of the embryogenic callus parent, like lesions, which are typical citrus canker symptoms. while the chloroplast (cp) genome is generally randomly Furthermore, gene expression analysis revealed a differential inherited (Grosser et al. 1996,Moreiraet al. 2000,Guoet al. transcriptional response, which suggested delayed HR in 2004b, Grosser and Gmitter 2005). Citrus somatic hybridiza- C. paradisi. In addition to the phenotype contrast, Fu et al. tion by symmetric fusion of callus protoplasts and mesophyll (2012) described different transcriptional responses to Xcc in protoplasts has been a powerful tool in citrus breeding, and ‘Meiwa’ kumquat (C. japonica)and‘Newhall’ navel orange numerous somatic hybrid plants from desirable parental com- (Citrus sinensis (L.) Osbeck) using microarrays, with ‘Meiwa’ binations have been produced (Grosser et al. 2000, Grosser and exhibiting upregulated expression of genes involved in the Gmitter 2005, 2011,Grosser et al. 2010b). Usually, regenerated response to biotic stimulus and in the defense response. diploid plants with morphological features similar to their leaf- Wang et al. (2011) found structural differences and physiolog- derived protoplast parent attracted attention, because leaf pro- ical responses to citrus canker inoculation between ‘Meiwa’ toplasts have been shown not to have the capacity for plant kumquat and ‘Newhall’ navel orange. ‘Meiwa’ kumquat regeneration in currently available culture schemes (Moreira showed lower stomatal density and smaller stomatal size than et al. 2000). By examining those diploid plants produced by ‘Newhall’ navel orange. In contrast, ‘Meiwa’ had a higher symmetric fusion, it was shown that they inherited mt DNA epicuticular wax content than ‘Newhall.’ The enzymatic ac- from the corresponding embryogenic callus or suspension cul- tivity also differed after Xcc inoculation in that ‘Meiwa’ ture parent, and the nuclear DNAwas from the leaf parent (Tusa showed significantly higher activities of catalase, peroxidase, et al. 1990, Grosser et al. 2000,Moreiraet al. 2000, Cabasson and phenylalanine ammonia-lyase in comparison with et al. 2001, Guo et al. 2004b, Grosser and Gmitter 2005). ‘Newhall.’ Also, in kumquat, genes responsive to Xcc infec- Therefore, citrus symmetric somatic cybridization makes it pos- tion included genes related to oxidative stress response and sible to transfer mt DNA-controlled traits from an embryogenic encoding proteins located in the mitochondria, the cell mem- callus/suspension parent to the leaf parent without altering cul- brane, and the chloroplast (Khalaf et al. 2011). Researchers tivar integrity (Satpute et al. 2015). Recently, Aleza et al. are trying to produce citrus plants resistant to Xcc by using (2016) were able to identify one tetraploid cybrid and several transgenic approaches (Boscariol et al. 2006, Omar et al. diploid cybrids exhibiting a range of mitochondrial and chloro- 2007,Mendeset al. 2010,Liet al. 2014,Penget al. 2015) plastic genome combinations from protoplast fusion between or by in vitro mutagenesis with ethyl methane sulphonate ‘Chios’ mandarin (Citrus reticulata) callus and ‘Clementine’ (EMS) (Ge et al. 2015). mandarin (C. reticulata) leaves or ‘Chios’ mandarin callus Moving cytoplasmic organelles between protoplasts in pro- and ‘Sanguinelli’ sweet orange (Citrus sinensis (L.) Osbeck) toplast fusion experiments was first reported by Maliga et al. callus. Previous work with putative grapefruit cybrids contain- (1982). Methods utilized to successfully achieve organelle ing cytoplasm from ‘Valencia’ sweet orange (C. sinensis (L.) transfer include removal of nuclear DNA by cytochalasin B Osbeck) showed that the cybrids exhibited improved canker (Wallin et al. 1978) and gradient ultracentrifugation (Lorz tolerance more similar to that of ‘Valencia’ than to the suscep- et al. 1981). Some researchers used these methods in attempt tible grapefruit control (J.H. Graham and J.W. Grosser, unpub- to produce cybrid plants with specific agronomical traits (Sakai lished data). Thus, it was hypothesized that cytoplasm from the and Imamura 1990, Spangenberg et al. 1990). Citrus cybrids highly canker resistant kumquat should provide an even higher were first produced by Vardi et al. (1987, 1989), by fusion of level of tolerance in the presently reported grapefruit cybrids. irradiated protoplasts (to destroy nuclei) with protoplasts whose To