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Dicle Donmez, Ozhan Simsek, Tolga Izgu, Yildiz Aka Kacar, Yesim Yalcin Mendi, "Genetic Transformation in Citrus", The Scientific World Journal, vol. 2013, Article ID 491207, 8 pages, 2013. https://doi.org/10.1155/2013/491207
Genetic Transformation in Citrus
Citrus is one of the world’s important fruit crops. Recently, citrus molecular genetics and biotechnology work have been accelerated in the world. Genetic transformation, a biotechnological tool, allows the release of improved cultivars with desirable characteristics in a shorter period of time and therefore may be useful in citrus breeding programs. Citrus transformation has now been achieved in a number of laboratories by various methods. Agrobacterium tumefaciens is used mainly in citrus transformation studies. Particle bombardment, electroporation, A. rhizogenes, and a new method called RNA interference are used in citrus transformation studies in addition to A. tumefaciens. In this review, we illustrate how different gene transformation methods can be employed in different citrus species.
Citrus species are the most widely grown fruit crops. Despite substantial genetic diversity and interspecific fertility, the genus Citrus includes some of the most difficult species to breed [1, 2]. This is due to several obstacles for conventional breeding. For example, most species are highly heterozygous and produce progeny that segregate widely for many characters when crosses are made. The juvenile periods are often very long, self- and cross-incompatibility and pollen and/or ovule sterility are relatively common, and the presence of adventitious somatic embryos in the nucellus of developing ovules of the most of Citrus greatly limits hybrid production [2, 3].
The genus Citrus possesses several undesirable characteristics including salt and cold sensitivity [4, 5]; they are also susceptible to diseases caused by fungi, bacteria and viruses, such as Citrus exocortis viroid (CEV), Citrus infectious variegation virus (CIVV), Citrus cachexia viroid (CCaV) and Citrus tristeza closterovirus (CTV) [5, 6]. Classical genetic selection, gene transfer, grafting, and micrografting techniques can contribute to the improvement of Citrus and propagation of selected species. Therefore, in vitro manipulation procedures leading to a rapid, direct bud regeneration for efficient micropropagation as well as genetic transformation are needed as a first step towards Citrus improvement. Practical benefits resulting from in vitro culture methods have already been reported in Citrus [5, 7, 8]. Recent developments in gene transfer techniques via the classical regeneration method have been applied to this genus and have opened the way to induce a specific genetic change within a period of time shorter than using the classical genetic selection method [5, 9, 10].
Conventional breeding methods have demonstrated limitations with respect to citrus improvement due to some of the biological characteristics of woody plants such as nucellar polyembryony, high heterozygosity, long juvenile period, and autoincompatibility [11, 12]. The development of biotechnological tools has made it possible to overcome some of these problems. In the specific case of citrus breeding programs, somatic hybridization [12–14] and genetic transformation [12, 15, 16] have been applied in many countries [10, 12, 17, 18].
In recent years, there has been a major thrust in citrus improvement as competition from international citrus markets, disease, and pest pressure and other abiotic and biotic stress conditions stimulate worldwide interest [19, 20]. Several strategies exist for the genetic improvement of citrus including conventional breeding and genetic transformation [20, 21]. Currently, genetic transformation of citrus as a tool for citrus improvement is gaining popularity. This method is especially useful in cases where it is not possible to introduce a particular trait of interest to another elite cultivar using conventional breeding. Citrus cultivars vary in their response to in vitro organogenesis and genetic transformation. This results in the need for cultivar-specific optimization of in vitro protocols [20, 22].
Among the several methods available for the genetic transformation of citrus, the most popular method to transform a wide range of citrus cultivars is Agrobacterium-mediated transformation using epicotyl explants as target cells for incorporation of the T-DNA [20, 23]. However, this method is not suitable for the transformation of any seedless cultivar. Also, special cultivars in the mandarin group remain robust to transform using this method [20, 22, 24].
2. Transformation Studies in Citrus
Genetic transformation and somatic hybridization studies are already integrated in Citrus breeding programs in several countries. Genetic transformation of Citrus is a promising tool that enables the introduction of desirable traits without altering the genetic background . Genetic transformation of citrus has been reported, by using several methods (Table 1).
Agrobacterium has been the most frequently used genetic transformation method in Citrus with explants collected from seedlings germinated in vitro or under greenhouse conditions .
Transformation studies have been done for two decades in citrus. In the last few years, different transformation methods such as RNA silencing are used. In order to carry out successful gene transformation studies in citrus, optimized in vitro regeneration protocol is needed. Researchers should optimize efficient regeneration protocol before starting transformation studies. There are also many efficient regeneration protocols published in different citrus species.
Orbović et al.  investigated the effects of seed age on shoot regeneration potential and transformation rate of “Duncan” and “Flame” grapefruit cultivars, along with “Hamlin” sweet orange cultivar. Genetic transformation of citrus explants was carried out as previously described  using A. tumefaciens strain EHA105  containing a binary vectors derived from pD35s . In conclusion, the regeneration potential and transformability of citrus juvenile explants are different among cultivars and also change within the fruit harvest season. Because of these findings, especially the latter one, it will be extremely difficult to develop a universal protocol for genetic transformation of citrus. Optimal transformation efficiency will require flexible procedures that account for cultivar variability and timing of seed collection. In another study, a protocol was developed for regeneration of transgenic plants via A. tumefaciens-mediated transformation of leaf segments from “Valencia” sweet orange (C. sinensis L. Osbeck) using gfp (green fluorescence protein) as a vital marker . The transformation methodology described by Khan et al.  was an important finding for generating transgenic plants using leaf segments as explants.
In addition to transformation studies via A. tumefaciens, recently, A. rhizogenes has been used. Many reports suggest the use of A. rhizogenes for expression of the rol genes and also to deliver foreign genes to susceptible plants . The hairy root harbours the T-DNA segment of Ri-plasmid within its nuclear genomes . A. rhizogenes are also capable of transferring the T-DNA of binary vectors in trans, thereby facilitating the selection of transgenic plants from screened hairy roots . A. rhizogenes-mediated transformation system was found to be very useful in genetic manipulation of plants for the production of phytochemicals , large scale secondary metabolite production , monoclonal antibody production , and phytoremediation . There are many reports that suggest the successful use of A. rhizogenes harbouring binary vectors with desired gene constructs  for plant genetic transformation . Due to low transformation efficiency of A. rhizogenes, many researchers have worked to optimize transformation methods.
Chávez-Vela et al.  used A. rhizogenes A4 agropine-type strain to develop the transformation system. A4 contains wild-type plasmid pRi A4 which confers hairy-root genotype and binary vector pESC4. In the study seventy-five-day-old sour orange seedlings were used and transgenic sour orange (C. aurantium L.) plants were regenerated from A. rhizogenes transformed roots. 91% of explants produced transformed roots with an average of 3.6 roots per explant.
In another study transgenic Mexican lime (C. aurantifolia (Christm.) Swing) plants were regenerated from tissues transformed by A. rhizogenes strain A4, containing the wild-type plasmid pRiA4 and the binary vector pESC4 with nos-npt II and cab-gus genes. More than 300 Mexican lime transgenic plants were obtained, 60 of which were adapted to growing in soil .
In addition to the indirect gene transfer methods, there are studies performed by direct gene transfer methods in citrus. Bespalhok Filho et al.  carried out to optimize the conditions for transient gene expression through particle bombardment on Carrizo citrange (C. sinensis Poncirus trifoliata) thin epicotyl sections. The best conditions for transient GUS expression were M-25 tungsten particles, 1550 psi helium pressure, 9 cm distance between specimen, and DNA/particle holder and culture of explants in a high osmolarity medium ( sorbitol) 4 h prior and 20 h after bombardment. Under these conditions, an average of 102 blue spots per bombardment (20 explants/plate) were achieved. It is stated that protocol is currently being used for transformation of Carrizo citrange and sweet orange (C. sinensis).
Electroporation is an effective direct gene transfer system used for citrus transformation. Hidaka and Omura  used electroporation methods for gene transformation in citrus. Protoplasts were prepared from embryogenic callus of “Ohta” ponkan (C. reticulata Blanco) and electroporation with exponential decay pulses was carried out in the solution containing the β-glucuronidase (GUS) chimeric gene coupled to the CaMV 35S promoter (pBI221). At 24 hr after incubation, significant GUS activity was detected in the cells by fluorometric assay. Another alternative method for direct gene transformation had been developed in sweet orange (C. sinensis (L.) Osbeck). Plasmid DNA encoding the nondestructive selectable marker enhanced green fluorescent protein gene was introduced using polyethylene glycol into protoplasts of “Itaborai” sweet orange isolated from an embryogenic nucellar-derived suspension culture. Following protoplast culture in liquid medium and transfer to solid medium, transformed calluses were identified via expression of the green fluorescent protein, physically separated from nontransformed tissue and cultured on somatic embryogenesis induction medium. Transgenic plantlets were recovered from germinating somatic embryos and by in vitro rooting of shoots .
As well as the transformation studies conducted for gene expression, several studies conducted for gene silencing. RNA interference (RNAi) are a posttranscriptional gene-silencing phenomenon induced by double-stranded RNA. It has been widely used as a knockdown technology to analyze gene function in various organisms. Although RNAi was first discovered in worms, related phenomena such as posttranscriptional gene silencing and coat protein-mediated protection from viral infection had been observed in plants prior to this. In plants, RNAi is often achieved through transgenes that produce hairpin RNA. For genetic improvement of crop plants, RNAi has advantages over antisense-mediated gene silencing and cosuppression, in terms of its efficiency and stability . Soler et al.  stated Citrus tristeza virus (CTV), the causal agent of the most devastating viral disease of citrus, has evolved three silencing suppressor proteins acting at intra- (p23 and p20) and/or intercellular level (p20 and p25) to overcome host antiviral defence. Mexican lime was transformed with an intron-hairpin vector carrying full-length, untranslatable versions of the genes p25, p20, and p23 from CTV strain T36 to silence the expression of these critical genes in CTV-infected cells. Three transgenic lines presented complete resistance to viral infection, with all their propagations remaining symptomless and virus-free after graft inoculation with CTV-T36, either in the nontransgenic rootstock or in the transgenic scion. Accumulation of transgene-derived siRNAs was necessary but not sufficient for CTV resistance. Inoculation with a divergent CTV strain led to partially breaking the resistance, thus showing the role of sequence identity in the underlying mechanism. Results are a step forward to developing transgenic resistance to CTV and also show that targeting simultaneously by RNA interference (RNAi) the three viral silencing suppressors appear critical for this purpose, although the involvement of concurrent RNAi mechanisms cannot be excluded.
Genetic transformation is an attractive alternative technique for citrus genetic improvement. However, transformation efficiencies are generally low, and protocols are dependent on species, or even cultivar dependent. One of the limitations within this technology is low plant regeneration frequencies especially for many of the economically important citrus species . In addition, difficulty in rooting transgenic shoots for some citrus cultivars has been reported [10, 89, 91]. Development of effective genetic transformants therefore requires specific studies on in vitro regeneration conditions for each genotype.
The development of direct genetic manipulation techniques has provided new opportunities for plant improvement. Plant transformation has made it possible to modify just one or two traits, while retaining the unique characteristics of the original cultivar. The characters that could potentially be manipulated by genetic transformation of Citrus include pest and disease resistance, growth habit, and fruit quality. In order to use this technology, it is essential to develop efficient genetic transformation systems for Citrus. .
- F. G. Gmitter, J. W. Grosser, and G. A. Moore, “Citrus,” in Biotechnology of Perennial Fruit Crops, F. A. Hammerschlag and R. E. Litz, Eds., pp. 335–369, CAB International, Wallingford, UK, 1992.
- E. Pérez-Molphe-Balch and N. Ochoa-Alejo, “Regeneration of transgenic plants of Mexican lime from Agrobacterium rhizogenes-transformed tissues,” Plant Cell Reports, vol. 17, no. 8, pp. 591–596, 1998.
- G. A. Moore, C. C. Jacono, J. L. Neidigh, S. D. Lawrence, and K. Cline, “Transformation in Citrus,” in Plant Protoplasts and Genetic Engineering IV, Y. P. S. Bajaj, Ed., vol. 23 of Biotechnology in Agriculture and Forestry, pp. 194–208, Springer, Berlin, Germany, 1993.
- P. Garcia-Agustin and E. Primo-Millo, “Selection of a NaCl-tolerant Citrus plant,” Plant Cell Reports, vol. 14, no. 5, pp. 314–318, 1995.
- B. Van Le, N. Thanh Ha, L. T. Anh Hong, and K. Trân Thanh Vân, “High frequency shoot regeneration from trifoliate orange (Poncirus trifoliata L. Raf.) using the thin cell layer method,” Comptes Rendus de l'Academie des Sciences, vol. 322, no. 12, pp. 1105–1111, 1999.
- V. Greño, L. Navarro, and N. Duran-Vila, “Influence of virus and virus-like agents on the development of citrus buds cultured in vitro,” Plant Cell, Tissue and Organ Culture, vol. 15, no. 2, pp. 113–124, 1988.
- J. W. Grosser, F. G. Gmitter Jr., and J. L. Chandler, “Intergeneric somatic hybrid plants of Citrus sinensis cv. Hamlin and Poncirus trifoliata cv. Flying Dragon,” Plant Cell Reports, vol. 7, no. 1, pp. 5–8, 1988.
- R. Ghorbel, L. Navarro, and N. Duran-Vila, “Morphogenesis and regeneration of whole plants of grapefruit (Citrus paradisi), sour orange (C. aurantium) and alemow (C. macrophylla),” Journal of Horticultural Science and Biotechnology, vol. 73, no. 3, pp. 323–327, 1998.
- J. Kaneyoshi, S. Kobayashi, Y. Nakamura, N. Shigemoto, and Y. Doi, “A simple and efficient gene transfer system of trifoliate orange (Poncirus trifoliata Raf.),” Plant Cell Reports, vol. 13, no. 10, pp. 541–545, 1994.
- M. A. Gutiérrez-E, D. Luth, and G. A. Moore, “Factors affecting Agrobacterium-mediated transformation in Citrus and production of sour orange (Citrus aurantium L.) plants expressing the coat protein gene of Citrus tristeza virus,” Plant Cell Reports, vol. 16, no. 11, pp. 745–753, 1997.
- R. Ghorbel, J. Juárez, L. Navarro, and L. Peña, “Green fluorescent protein as a screenable marker to increase the efficiency of generating transgenic woody fruit plants,” Theoretical and Applied Genetics, vol. 99, no. 1-2, pp. 350–358, 1999.
- R. L. Boscariol, W. A. B. Almeida, M. T. V. C. Derbyshire, F. A. A. Mourão Filho, and B. M. J. Mendes, “The use of the PMI/mannose selection system to recover transgenic sweet orange plants (Citrus sinensis L. Osbeck),” Plant Cell Reports, vol. 22, no. 2, pp. 122–128, 2003.
- J. W. Grosser and F. G. Gmitter, “Protoplast fusion and citrus improvement,” Plant Breeding Reviews, vol. 8, pp. 339–374, 1990.
- B. M. J. Mendes, F. D. A. A. Mourão Filho, P. C. D. M. Farias, and V. A. Benedito, “Citrus somatic hybridization with potential for improved blight and CTV resistance,” In Vitro Cellular and Developmental Biology—Plant, vol. 37, no. 4, pp. 490–495, 2001.
- L. Peña and L. Navarro, “Transgenic citrus,” in Transgenic Trees, Y. P. S. Bajaj, Ed., pp. 39–54, Springer, Berlin, Germany, 1999.
- M. G. C. Costa, W. C. Otoni, and G. A. Moore, “An evaluation of factors affecting the efficiency of Agrobacterium-mediated transformation of Citrus paradisi (Macf.) and production of transgenic plants containing carotenoid biosynthetic genes,” Plant Cell Reports, vol. 21, no. 4, pp. 365–373, 2002.
- M. Cervera, C. Ortega, A. Navarro, L. Navarro, and L. Peña, “Generation of transgenic citrus plants with the tolerance-to-salinity gene HAL2 from yeast,” Journal of Horticultural Science and Biotechnology, vol. 75, no. 1, pp. 26–30, 2000.
- B. M. Januzzi Mendes, R. Luciana Boscariol, F. D. A. A. Mourão Filho, and W. A. Bastos de Almeida, “Agrobacterium-mediated genetic transformation of “Hamlin” sweet orange,” Pesquisa Agropecuaria Brasileira, vol. 37, no. 7, pp. 955–961, 2002.
- J. W. Grosser, P. Ollitrault, and O. Olivares-Fuster, “Somatic hybridization in citrus: an effective tool to facilitate variety improvement,” In Vitro Cellular and Developmental Biology—Plant, vol. 36, no. 6, pp. 434–449, 2000.
- M. Dutt and J. W. Grosser, “An embryogenic suspension cell culture system for Agrobacterium-mediated transformation of citrus,” Plant Cell Reports, vol. 29, no. 11, pp. 1251–1260, 2010.
- L. Peña, M. Cervera, R. Ghorbel et al., “Genetic transformation,” in Citrus Genetics, Breeding, and Biotechnology, I. A. Khan, Ed., pp. 329–344, CABI International, Wallingford, UK, 2007.
- M. Dutt, D. H. Lee, and J. W. Grosser, “Bifunctional selection-reporter systems for genetic transformation of citrus: mannose- and kanamycin-based systems,” In Vitro Cellular and Developmental Biology—Plant, vol. 46, no. 6, pp. 467–476, 2010.
- M. Dutt and J. W. Grosser, “Evaluation of parameters affecting Agrobacterium-mediated transformation of citrus,” Plant Cell, Tissue and Organ Culture, vol. 98, no. 3, pp. 331–340, 2009.
- R. N. Khawale, S. K. Singh, G. Garg, V. K. Baranwal, and S. A. Ajirlo, “Agrobacterium-mediated genetic transformation of Nagpur mandarin (Citrus reticulata Blanco),” Current Science, vol. 91, no. 12, pp. 1700–1705, 2006.
- N. T. Marques, G. B. Nolasco, and J. P. Leitão, “Factors affecting in vitro adventitious shoot formation on internode explants of Citrus aurantium L. cv. Brazilian,” Scientia Horticulturae, vol. 129, pp. 176–182, 2011.
- L. Y. Miyata, R. Harakava, L. C. L. Stipp, B. M. J. Mendes, B. Appezzato-da-Glória, and F. A. A. Mourao-Filho, “GUS expression in sweet oranges (Citrus sinensis L. Osbeck) driven by three different phloem-specific promoters,” Plant Cell Reports, vol. 31, no. 11, pp. 2005–2013, 2012.
- E. U. Khan, X.-Z. Fu, and J.-H. Liu, “Agrobacterium-mediated genetic transformation and regeneration of transgenic plants using leaf segments as explants in Valencia sweet orange,” Plant Cell, Tissue and Organ Culture, vol. 109, no. 2, pp. 383–390, 2012.
- P. Fávero, F. A. de Alves Mourão Filho, L. C. L. Stipp, and B. M. J. Mendes, “Genetic transformation of three sweet orange cultivars from explants of adult plants,” Acta Physiologiae Plantarum, vol. 34, no. 2, pp. 471–477, 2012.
- E. Pons, J. E. Peris, and L. Peña, “Field performance of transgenic citrus trees: assessment of the long-term expression of uidA and nptII transgenes and its impact on relevant agronomic and phenotypic characteristics,” BMC Biotechnology, vol. 12, no. 12, p. 41, 2012.
- B. Çevik, R. F. Lee, and C. L. Niblett, “Agrobacterium-mediated transformation of grapefruit with the wild-type and mutant RNA-dependent RNA polymerase genes of Citrus tristeza virus,” Turkish Journal of Agriculture and Forestry, vol. 36, no. 2, pp. 195–206, 2012.
- F. R. Muniz, A. J. De Souza, L. C. L. Stipp et al., “Genetic transformation of Citrus sinensis with Citrus tristeza virus (CTV) derived sequences and reaction of transgenic lines to CTV infection,” Biologia Plantarum, vol. 56, no. 1, pp. 162–166, 2012.
- N. Soler, M. Plomer, C. Fagoaga et al., “Transformation of Mexican lime with an intron-hairpin construct expressing untranslatable versions of the genes coding for the three silencing suppressors of Citrus tristeza virus confers complete resistance to the virus,” Plant Biotechnology Journal, vol. 10, pp. 597–608, 2012.
- M. Dutt, G. Ananthakrishnan, M. K. Jaromin, R. H. Brlansky, and J. W. Grosser, “Evaluation of four phloem-specific promoters in vegetative tissues of transgenic citrus plants,” The Tree Physiology, vol. 32, no. 1, pp. 83–93, 2012.
- S. N. Mondal, M. Dutt, J. W. Grosser, and M. M. Dewdney, “Transgenic citrus expressing the antimicrobial gene Attacin E (attE) reduces the susceptibility of “Duncan” grapefruit to the citrus scab caused by Elsinoë fawcettii,” European Journal of Plant Pathology, vol. 133, no. 2, pp. 391–404, 2012.
- A. Al-Bachchu, S. B. Jin, J. W. Park et al., “Agrobacterium-mediated transformation using embryogenic calli in satsuma mandarin (Citrus unshiu Marc.) cv. Miyagawa Wase,” Horticulture Environment Biotechnology, vol. 52, no. 2, pp. 170–175, 2011.
- V. Orbović, M. Dutt, and J. W. Grosser, “Seasonal effects of seed age on regeneration potential and transformation success rate in three citrus cultivars,” Scientia Horticulturae, vol. 127, no. 3, pp. 262–266, 2011.
- V. Orbović, P. Soria, G. A. Moore, and J. W. Grosser, “The use of Citrus tristeza virus (CTV) containing a green fluorescent protein gene as a tool to evaluate resistance/tolerance of transgenic citrus plants,” Crop Protection, vol. 30, no. 5, pp. 572–576, 2011.
- Y. He, S. Chen, A. Peng et al., “Production and evaluation of transgenic sweet orange (Citrus sinensis Osbeck) containing bivalent antibacterial peptide genes (Shiva A and Cecropin B) via a novel Agrobacterium-mediated transformation of mature axillary buds,” Scientia Horticulturae, vol. 128, no. 2, pp. 99–107, 2011.
- C. A. Reyes, A. De Francesco, E. J. Peña et al., “Resistance to Citrus psorosis virus in transgenic sweet orange plants is triggered by coat protein-RNA silencing,” Journal of Biotechnology, vol. 151, no. 1, pp. 151–158, 2011.
- J. Fan, X. Liu, S.-X. Xu, Q. Xu, and W.-W. Guo, “T-DNA direct repeat and 35S promoter methylation affect transgene expression but do not cause silencing in transgenic sweet orange,” Plant Cell, Tissue and Organ Culture, vol. 107, no. 2, pp. 225–232, 2011.
- L. Yang, C. Hu, N. Li, J. Zhang, J. Yan, and Z. Deng, “Transformation of sweet orange [Citrus sinensis (L.) Osbeck] with pthA-nls for acquiring resistance to citrus canker disease,” Plant Molecular Biology, vol. 75, no. 1, pp. 11–23, 2011.
- X.-Z. Fu, E. U. Khan, S.-S. Hu, Q.-J. Fan, and J.-H. Liu, “Overexpression of the betaine aldehyde dehydrogenase gene from Atriplex hortensis enhances salt tolerance in the transgenic trifoliate orange (Poncirus trifoliata L. Raf.),” Environmental and Experimental Botany, vol. 74, no. 1, pp. 106–113, 2011.
- S.-X. Xu, X.-D. Cai, B. Tan, and W.-W. Guo, “Comparison of expression of three different sub-cellular targeted GFPs in transgenic Valencia sweet orange by confocal laser scanning microscopy,” Plant Cell, Tissue and Organ Culture, vol. 104, no. 2, pp. 199–207, 2011.
- M. Dutt, M. Vasconcellos, and J. W. Grosser, “Effects of antioxidants on Agrobacterium-mediated transformation and accelerated production of transgenic plants of Mexican lime (Citrus aurantifolia Swingle),” Plant Cell, Tissue and Organ Culture, vol. 107, no. 1, pp. 79–89, 2011.
- M. Dutt, J. Madhavaraj, and J. W. Grosser, “Agrobacterium tumefaciens-mediated genetic transformation and plant regeneration from a complex tetraploid hybrid citrus rootstock,” Scientia Horticulturae, vol. 123, no. 4, pp. 454–458, 2010.
- A. Ballester, M. Cervera, and L. Peña, “Selectable marker-free transgenic orange plants recovered under non-selective conditions and through PCR analysis of all regenerants,” Plant Cell, Tissue and Organ Culture, vol. 102, no. 3, pp. 329–336, 2010.
- M. L. P. De Oliveira, V. J. Febres, M. G. C. Costa, G. A. Moore, and W. C. Otoni, “High-efficiency Agrobacterium-mediated transformation of citrus via sonication and vacuum infiltration,” Plant Cell Reports, vol. 28, no. 3, pp. 387–395, 2009.
- J. M. Barbosa-Mendes, F. D. A. A. M. Filho, A. B. Filho, R. Harakava, S. V. Beer, and B. M. J. Mendes, “Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker,” Scientia Horticulturae, vol. 122, no. 1, pp. 109–115, 2009.
- Z. Tong, B. Tan, J. Zhang, Z. Hu, W. Guo, and X. Deng, “Using precocious trifoliate orange (Poncirus trifoliata [L.] Raf.) to establish a short juvenile transformation platform for citrus,” Scientia Horticulturae, vol. 119, no. 3, pp. 335–338, 2009.
- B. Tan, D.-L. Li, S.-X. Xu, G.-E. Fan, J. Fan, and W.-W. Guo, “Highly efficient transformation of the GFP and MAC12.2 genes into precocious trifoliate orange (Poncirus trifoliata [L.] Raf), a potential model genotype for functional genomics studies in Citrus,” Tree Genetics and Genomes, vol. 5, no. 3, pp. 529–537, 2009.
- A. Ballester, M. Cervera, and L. Peña, “Evaluation of selection strategies alternative to nptII in genetic transformation of citrus,” Plant Cell Reports, vol. 27, no. 6, pp. 1005–1015, 2008.
- M. C. Zanek, C. A. Reyes, M. Cervera et al., “Genetic transformation of sweet orange with the coat protein gene of Citrus psorosis virus and evaluation of resistance against the virus,” Plant Cell Reports, vol. 27, no. 1, pp. 57–66, 2008.
- A. Rodríguez, M. Cervera, J. E. Peris, and L. Peña, “The same treatment for transgenic shoot regeneration elicits the opposite effect in mature explants from two closely related sweet orange (Citrus sinensis (L.) Osb.) genotypes,” Plant Cell, Tissue and Organ Culture, vol. 93, no. 1, pp. 97–106, 2008.
- A. Ballester, M. Cervera, and L. Peña, “Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination,” Plant Cell Reports, vol. 26, no. 1, pp. 39–45, 2007.
- G. Ananthakrishnan, V. Orbović, G. Pasquali, M. Ćalović, and J. W. Grosser, “Transfer of Citrus tristeza virus (CTV)-derived resistance candidate sequences to four grapefruit cultivars through Agrobacterium-mediated genetic transformation,” In Vitro Cellular and Developmental Biology—Plant, vol. 43, no. 6, pp. 593–601, 2007.
- C. Chen, Q. Zheng, X. Xiang et al., “Development of pGreen-derived GFP binary vectors for Citrus transformation,” HortScience, vol. 42, no. 1, pp. 7–10, 2007.
- C. Fagoaga, F. R. Tadeo, D. J. Iglesias et al., “Engineering of gibberellin levels in citrus by sense and antisense overexpression of a GA 20-oxidase gene modifies plant architecture,” Journal of Experimental Botany, vol. 58, no. 6, pp. 1407–1420, 2007.
- O. Batuman, Transformation of Citrus plants with constructs related to citrus tristesa virus (ctv) sequences [Ph.D. thesis], Hebrew University, 2006.
- R. L. Boscariol, M. Monteiro, E. K. Takahashi et al., “Attacin A gene from Tricloplusia ni reduces susceptibility to Xanthomonas axonopodis pv. citri in transgenic Citrus sinesis “Hamlin”,” Journal of the American Society for Horticultural Science, vol. 131, no. 4, pp. 530–536, 2006.
- F. A. Azevedo, F. A. A. Mourão Filho, B. M. J. Mendes et al., “Genetic transformation of rangpur lime (Citrus limonia Osbeck) with the bO (bacterio-opsin) gene and its initial evaluation for Phytophthora nicotianae resistance,” Plant Molecular Biology Reporter, vol. 24, no. 2, pp. 185–196, 2006.
- B. Çevik, R. F. Lee, and C. L. Niblett, “Genetic transformation of Citrus paradisi with antisense and untranslatable RNA-dependent RNA polymerase genes of Citrus tristeza closterovirus,” Turkish Journal of Agriculture and Forestry, vol. 30, no. 3, pp. 173–182, 2006.
- M. Ahmad and B. Mirza, “An efficient protocol for transient transformation of intact fruit and transgene expression in Citrus,” Plant Molecular Biology Reporter, no. 23, pp. 419–420, 2005.
- W. Guo, Y. Duan, O. Olivares-Fuster et al., “Protoplast transformation and regeneration of transgenic Valencia sweet orange plants containing a juice quality-related pectin methylesterase gene,” Plant Cell Reports, vol. 24, no. 8, pp. 482–486, 2005.
- H. B. C. Molinari, J. C. Bespalhok, A. K. Kobayashi, L. F. P. Pereira, and L. G. E. Vieira, “Agrobacterium tumefaciens-mediated transformation of Swingle citrumelo (Citrus paradisi Macf. x Poncirus trifoliata L. Raf.) using thin epicotyl sections,” Scientia Horticulturae, vol. 99, no. 3-4, pp. 379–385, 2004.
- L. Peña, R. M. Pérez, M. Cervera, J. A. Juárez, and L. Navarro, “Early events in Agrobacterium-mediated genetic transformation of Citrus explants,” Annals of Botany, vol. 94, no. 1, pp. 67–74, 2004.
- A. Domínguez, M. Cervera, R. M. Pérez et al., “Characterisation of regenerants obtained under selective conditions after Agrobacterium-mediated transformation of citrus explants reveals production of silenced and chimeric plants at unexpected high frequencies,” Molecular Breeding, vol. 14, no. 2, pp. 171–183, 2004.
- M. Kayim, T. L. Ceccardi, M. J. G. Berretta, G. A. Barthe, and K. S. Derrick, “Introduction of a citrus blight-associated gene into Carrizo citrange [Citrus sinensis (L.) Osbc. x Poncirus trifoliata (L.) Raf.] by Agrobacterium-mediated transformation,” Plant Cell Reports, vol. 23, no. 6, pp. 377–385, 2004.
- W. A. B. Almeida, F. A. A. Mourao Filho, B. M. J. Mendes, A. Pavan, and A. P. M. Rodriquez, “Agrobacterium-mediated transformation of Citrus sinensis and Citrus limonia epicotyl segments,” Scientia Agricola, vol. 60, no. 1, pp. 23–29, 2003.
- J. C. Bespalhok Filho, A. K. Kobayashi, L. F. P. Pereira, R. M. Galvão, and L. G. E. Vieira, “Transient gene expression of β-glucuronidase in Citrus thin epicotyl transversal sections using particle bombardment,” Brazilian Archives of Biology and Technology, vol. 46, no. 1, pp. 1–6, 2003.
- D. D. Li, W. Shi, and X. X. Deng, “Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene,” Tree Physiology, vol. 23, no. 17, pp. 1209–1215, 2003.
- W. A. B. Almeida, F. A. A. Mourão Filho, L. E. Pino, R. L. Boscariol, A. P. M. Rodriguez, and B. M. J. Mendes, “Genetic transformation and plant recovery from mature tissues of Citrus sinensis L. Osbeck,” Plant Science, vol. 164, no. 2, pp. 203–211, 2003.
- N. A. Chávez-Vela, L. I. Chávez-Ortiz, and E. P.-M. Balch, “Genetic transformation of sour orange using Agrobacterium rhizogenes,” Agrocencia, vol. 37, no. 6, pp. 629–639, 2003.
- V. J. Febres, C. L. Niblett, R. F. Lee, and G. A. Moore, “Characterization of grapefruit plants (Citrus paradisi Macf.) transformed with Citrus tristeza closterovirus genes,” Plant Cell Reports, vol. 21, no. 5, pp. 421–428, 2003.
- R. P. Niedz, W. L. McKendree, and R. G. Shatters Jr., “Electroporation of embryogenic protoplasts of sweet orange (Citrus sinensis (L.) Osbeck) and regeneration of transformed plants,” In Vitro Cellular and Developmental Biology—Plant, vol. 39, no. 6, pp. 586–594, 2003.
- C. Yu, S. Huang, C. Chen, Z. Deng, P. Ling, and F. G. Gmitter Jr., “Factors affecting Agrobacterium-mediated transformation and regeneration of sweet orange and citrange,” Plant Cell, Tissue and Organ Culture, vol. 71, no. 2, pp. 147–155, 2002.
- D. D. Li, W. Shi, and X. X. Deng, “Agrobacterium-mediated transformation of embryogenic calluses of Ponkan mandarin and the regeneration of plants containing the chimeric ribonuclease gene,” Plant Cell Reports, vol. 21, no. 2, pp. 153–156, 2002.
- L. Peña, M. Martín-Trillo, J. Juárez, J. A. Pina, L. Navarro, and J. M. Martínez-Zapater, “Constitutive expression of Arabidopsis LEAFY or APETALA1 genes in citrus reduces their generation time,” Nature Biotechnology, vol. 19, no. 3, pp. 263–267, 2001.
- R. Ghorbel, A. Dominguez, L. Navarro, and L. Peña, “High efficiency genetic transformation of sour orange (Citrus aurantium) and production of transgenic trees containing the coat protein gene of Citrus tristeza virus,” Tree Physiology, vol. 20, no. 17, pp. 1183–1189, 2000.
- G. A. Moore, D. Luth, M. McCaffery, V. J. Febres, S. M. Garnsey, and C. L. Niblett, “Agrobacterium-mediated transformation of grapefruit (Citrus paradisi Macf.) with genes from citrus tristeza virus,” in Proceedings of the 1st International Citrus Biotechnology Symposium, ISHS Acta Horticulturae 535, 2000.
- D. Piestun, O. Batuman, X. Che et al., “Truncated versions of the Citrus tristeza virus (CTV) replicase and basta resistance genes incorporated in transgenic Troyer citrange,” in Proceedings of the 1st International Citrus Biotechnology Symposium, ISHS Acta Horticulturae 535, 2000.
- A. Domínguez, J. Guerri, M. Cambra, L. Navarro, P. Moreno, and L. Peña, “Efficient production of transgenic citrus plants expressing the coat protein gene of Citrus tristeza virus,” Plant Cell Reports, vol. 19, no. 4, pp. 427–433, 2000.
- G. H. Fleming, O. Olivares-Fuster, S. Fatta Del-Bosco, and J. W. Grosser, “An alternative method for the genetic transformation of sweet orange,” In Vitro Cellular and Developmental Biology—Plant, vol. 36, no. 6, pp. 450–455, 2000.
- A. M. P. Koltunow, S. B. Protopsaltis, and N. Nito, “Regeneration of west indian limes (Citrus aurantifolia) containing genes for decreased seed,” in Proceedings of the 1st International Citrus Biotechnology Symposium, ISHS Acta Horticulturae 535, 2000.
- Z. N. Yang, I. L. Ingelbrecht, E. Louzada, M. Skaria, and T. E. Mirkov, “Agrobacterium-mediated transformation of the commercially important grapefruit cultivar Rio Red (Citrus paradisi Macf.),” Plant Cell Reports, vol. 19, no. 12, pp. 1203–1211, 2000.
- M. Cervera, J. A. Pina, J. Juárez, L. Navarro, and L. Peña, “Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration,” Plant Cell Reports, vol. 18, no. 3-4, pp. 271–278, 1998.
- J. E. Bond and M. L. Roose, “Agrobacterium-mediated transformation of the commercially important citrus cultivar Washington navel orange,” Plant Cell Reports, vol. 18, no. 3-4, pp. 229–234, 1998.
- J.-L. Yao, J.-H. Wu, A. P. Gleave, and B. A. M. Morris, “Transformation of citrus embryogenic cells using particle bombardment and production of transgenic embryos,” Plant Science, vol. 113, no. 2, pp. 175–183, 1996.
- L. Peña, M. Cervera, J. Juárez et al., “High efficiency Agrobacterium-mediated transformation and regeneration of citrus,” Plant Science, vol. 104, no. 2, pp. 183–191, 1995.
- L. Peña, M. Cervera, J. Juárez et al., “Agrobacterium-mediated transformation of sweet orange and regeneration of transgenic plants,” Plant Cell Reports, vol. 14, no. 10, pp. 616–619, 1995.
- T. Hidaka and M. Omura, “Transformation of Citrus protoplasts by electroporation,” Journal of the Japanese Society Horticulture Science, vol. 62, no. 2, pp. 371–376, 1993.
- G. A. Moore, C. C. Jacono, J. L. Neidigh, S. D. Lawrence, and K. Cline, “Agrobacterium-mediated transformation of Citrus stem segments and regeneration of transgenic plants,” Plant Cell Reports, vol. 11, no. 5-6, pp. 238–242, 1992.
- A. Vardi, S. Bleichman, and D. Aviv, “Genetic transformation of citrus protoplasts and regeneration of transgenic plants,” Plant Science, vol. 69, no. 2, pp. 199–206, 1990.
- V. Orbovic and J. W. Grosser, “Citrus: sweet orange (Citrus sinensis L. Osbeck “Valencia”) and Carrizo citrange [Citrus sinensis (L.) Osbeck X Poncirus trifoliate (L.) Raf.],” in Agrobacterium Protocols-Methods in Molecular Biology, K. Wang, Ed., pp. 177–189, Humana Press, New York, NY, USA, 2006.
- E. E. Hood, S. B. Gelvin, L. S. Melchers, and A. Hoekema, “New Agrobacterium helper plasmids for gene transfer to plants,” Transgenic Research, vol. 2, no. 4, pp. 208–218, 1993.
- M. C. Christey, “Use of Ri-mediated transformation for production of transgenic plants,” In Vitro Cellular and Developmental Biology—Plant, vol. 37, no. 6, pp. 687–700, 2001.
- M. D. Chilton, D. A. Tepfer, A. Petit, C. David, F. Casse-Delbart, and J. Tempé, “Agrobacterium rhizogenes inserts T-DNA into the genomes of the host plant root cells,” Nature, vol. 295, no. 5848, pp. 432–434, 1982.
- J. V. Shanks and J. Morgan, “Plant “hairy root” culture,” Current Opinion in Biotechnology, vol. 10, no. 2, pp. 151–155, 1999.
- S. M. Choi, S. H. Son, S. R. Yun, O. W. Kwon, J. H. Seon, and K. Y. Paek, “Pilot-scale culture of adventitious roots of ginseng in a bioreactor system,” Plant Cell, Tissue and Organ Culture, vol. 62, no. 3, pp. 187–193, 2000.
- R. Wongsamuth and P. M. Doran, “Hairy root as an experimental system for production of antibodies,” in Hairy Roots, Culture and Applications, M. P. Doran, Ed., pp. 89–97, Harwood, Amsterdam, The Netherlands, 1997.
- T. V. Nedelkoska and P. M. Doran, “Hyperaccumulation of cadmium by hairy roots of Thlapsi caerulescens,” Biotechnology and Bioengineering, vol. 67, no. 5, pp. 607–615, 2000.
- V. Kumar, A. Sharma, B. C. N. Prasad, H. B. Gururaj, and G. A. Ravishankar, “Agrobacterium rhizogenes mediated genetic transformation resulting in hairy root formation is enhanced by ultrasonication and acetosyringone treatment,” Electronic Journal of Biotechnology, vol. 9, no. 4, pp. 1–9, 2006.
- M. Kusaba, “RNA interference in crop plants,” Current Opinion in Biotechnology, vol. 15, no. 2, pp. 139–143, 2004.
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