\r\n\tNearly 25% - 30% of the world population is affected by neurological diseases exerting a hard financial strain on the healthcare system. The costs are estimated at around $800 billion annualy, expected to exponentially increase as the elders, at high risk of debilitating neurological diseases, will double by 2050. A varied spectrum of neuroprotective strategies has been suggested, including combined antioxidative-anti-inflammatory treatments, ozone autohemotherapy, hypothermia, cell therapy, the administration of neurotrophic factors, hemofiltration, and others. Distressingly, none of the currently available neuroprotective approaches has so far proven to prolong either life span or the cardinal symptoms of the patients suffering from brain injury. Last but not least, translational studies are still lacking.
\r\n\r\n\tThe book aims to revisit, discuss, and compile some promising current approaches in neuroprotection along with the current goals and prospects.
",isbn:"978-1-83880-440-4",printIsbn:"978-1-83880-439-8",pdfIsbn:null,doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"10acd587ca2c942616bfc09c4b79df39",bookSignature:"Dr. Matilde Otero-Losada, Dr. Francisco Capani and Dr. Santiago Perez Lloret",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/8087.jpg",keywords:"IKKβ/NF-κB pathway, neuroendocrine studies, anti-inflammatory agents, Bipolar disorder, oxidative metabolism, metabolic syndrome, Parkinson's disease, Alzheimer's disease, neurotrophins, growth factors, ATP-mediated calcium signalling, glutathione peroxidase",numberOfDownloads:162,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"September 10th 2019",dateEndSecondStepPublish:"October 1st 2019",dateEndThirdStepPublish:"November 30th 2019",dateEndFourthStepPublish:"February 18th 2020",dateEndFifthStepPublish:"April 18th 2020",remainingDaysToSecondStep:"2 months",secondStepPassed:!0,currentStepOfPublishingProcess:4,editedByType:null,kuFlag:!1,editors:[{id:"193560",title:"Dr.",name:"Matilde",middleName:null,surname:"Otero-Losada",slug:"matilde-otero-losada",fullName:"Matilde Otero-Losada",profilePictureURL:"https://mts.intechopen.com/storage/users/193560/images/system/193560.jpeg",biography:"Dr. Matilde Otero-Losada graduated at the School of Pharmacy and Biochemistry, University of Buenos Aires (UBA) Argentina; pursued her studies in Neuropharmacology getting her Sci.D. in Neuropharmacology (UBA, Argentina); and completing her Ph.D. in Psychiatry at the Wolverhampton University, WLV, UK. \r\nHer following studies in Psychometrics and Statistical Methods, Radioisotopes and Radiochemistry, Signal Processing and Microcomputers, took her to the University of California San Diego (UCSD) for training in human Psychophysics. \r\nBack in Argentina, she carried on studying smell, taste and trigeminal perception at the Hospital de Clínicas, UBA. \r\nShe focused on the study of metabolic syndrome, soft drinks and cardiovascular-renal morbidity for the last ten years, and in the last two years she is back to her roots: Neurosciences. \r\nWith over 90 papers published in prestigious journals indexed in PubMed, Embase and Scopus and book chapters authored, as Senior Researcher of the National Research Council (Argentina), she customarily reviews manuscripts and is acknowledged for her scientific writing, and editing capacities.",institutionString:"University of Buenos Aires",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"2",totalChapterViews:"0",totalEditedBooks:"0",institution:null}],coeditorOne:{id:"120703",title:"Dr.",name:"Francisco",middleName:null,surname:"Capani",slug:"francisco-capani",fullName:"Francisco Capani",profilePictureURL:"https://mts.intechopen.com/storage/users/120703/images/system/120703.jpeg",biography:"Dr. Francisco Capani graduated at the School of Medicine, University of Buenos Aires, (UBA) Argentina and completed his doctoral studies in Neurosciences at the Institute of Cell Biology and Neuroscience Prof E. De Robertis, School of Medicine (UBA), Argentina. Then he moved abroad to perform his postdoctoral studies at the University of California San Diego (UCSD-NCMIR) and the Karolinska Institute, Department of Neuroscience. Over an eight-year period, his research focused on synaptic organization, combining electron tomography, 3-D reconstruction, and correlative light and electron microscopy techniques. Upon his return to Argentina in 2006, he devoted to study the mechanisms involved in the pathophysiology of the perinatal asphyxia supported by his broad experience in electron microscopy. He has published 101 papers in recognized journals and has been invited assisting in a speaker capacity to several international conferences.",institutionString:"University of Buenos Aires",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"4",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"University of Buenos Aires",institutionURL:null,country:{name:"Argentina"}}},coeditorTwo:{id:"168475",title:"Dr.",name:"Santiago Perez",middleName:null,surname:"Lloret",slug:"santiago-perez-lloret",fullName:"Santiago Perez Lloret",profilePictureURL:"https://mts.intechopen.com/storage/users/168475/images/system/168475.jpeg",biography:"Dr. Santiago Perez Lloret is a leading expert in neurophysiology and neuropharmacology with more 90 papers published in international medical journals (H-index= 29). He has been recently awarded Top 1% reviewer in neurosciences (Publons.com). After obtaining his MD and PhD, he pursued master courses in pharmacoepidemiology, clinical pharmacology and biostatistics at the Universities of Bordeaux and Paris. Dr. Perez Lloret is Assistant professor of Neurophysiology at the Medicine School of the Buenos Aires University and Associate Researcher at the Cardiology Research Institute, University of Buenos Aires, National Research Council. He is member of the International Parkinson’s Disease and Movement Disorder Society (MDS), where he is Co-editor of the Webpage and collaborates in several committees, including the Educational and the Evidence-based Medicine Committees.",institutionString:"University of Buenos Aires",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"0",totalChapterViews:"0",totalEditedBooks:"0",institution:{name:"University of Buenos Aires",institutionURL:null,country:{name:"Argentina"}}},coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"16",title:"Medicine",slug:"medicine"}],chapters:[{id:"69122",title:"Lifestyle Factors, Mitochondrial Dynamics, and Neuroprotection",slug:"lifestyle-factors-mitochondrial-dynamics-and-neuroprotection",totalDownloads:83,totalCrossrefCites:0,authors:[null]},{id:"69463",title:"Polyphenols as Potential Therapeutic Drugs in Neurodegeneration",slug:"polyphenols-as-potential-therapeutic-drugs-in-neurodegeneration",totalDownloads:36,totalCrossrefCites:0,authors:[null]},{id:"69376",title:"Trends in Neuroprotective Strategies after Spinal Cord Injury: State of the Art",slug:"trends-in-neuroprotective-strategies-after-spinal-cord-injury-state-of-the-art",totalDownloads:35,totalCrossrefCites:0,authors:[null]},{id:"70228",title:"Aptamers and Possible Effects on Neurodegeneration",slug:"aptamers-and-possible-effects-on-neurodegeneration",totalDownloads:10,totalCrossrefCites:0,authors:[null]}],productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:{id:"301331",firstName:"Mia",lastName:"Vulovic",middleName:null,title:"Mrs.",imageUrl:"https://mts.intechopen.com/storage/users/301331/images/8498_n.jpg",email:"mia.v@intechopen.com",biography:null}},relatedBooks:[{type:"book",id:"6550",title:"Cohort Studies in Health Sciences",subtitle:null,isOpenForSubmission:!1,hash:"01df5aba4fff1a84b37a2fdafa809660",slug:"cohort-studies-in-health-sciences",bookSignature:"R. 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Chan and Manoj Kumar Tiwari",coverURL:"https://cdn.intechopen.com/books/images_new/3794.jpg",editedByType:"Edited by",editors:[{id:"252210",title:"Dr.",name:"Felix",surname:"Chan",slug:"felix-chan",fullName:"Felix Chan"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3621",title:"Silver Nanoparticles",subtitle:null,isOpenForSubmission:!1,hash:null,slug:"silver-nanoparticles",bookSignature:"David Pozo Perez",coverURL:"https://cdn.intechopen.com/books/images_new/3621.jpg",editedByType:"Edited by",editors:[{id:"6667",title:"Dr.",name:"David",surname:"Pozo",slug:"david-pozo",fullName:"David Pozo"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"18815",title:"Citrus Transformation: Challenges and Prospects",doi:"10.5772/24526",slug:"citrus-transformation-challenges-and-prospects",body:'Citrus is an important commodity worldwide and is produced in tropical and subtropical regions around the world. Annually, the total citrus fruit production is estimated to be more than 124.5 million tonnes worldwide, with China, Brazil, the United States, Mexico and India the main producers (FAO, 2011). Oranges, lemons, tangerines and grapefruits are among the most commonly grown citrus types and they are traded as fresh fruit, juice, or as concentrate. Growers, however, face important challenges for maintaining or improving yield: disease, drought, cold and soil salinity are some of the factors that can limit production and can have an important economic impact on growers. Traditional breeding methods have been used successfully over the years to improve citrus; however this is done with difficulty due to the slow growth and maturation of this crop, incompatibility, polyembryony, parthenocarpy, etc. Because traditional breeding takes such a long time the fast incorporation of desirable traits is not possible. In other instances, certain desirable traits are not present in cultivated citrus types. This has been made more evident in the battle against diseases. Diseases can appear in a region and within a few years spread and become limiting factors for production and have a major economical impact because of yield reduction and/or increased production costs. Therefore, genetic engineering via citrus transformation is an alternative method used to incorporate desirable traits into citrus genotypes.
The genetic transformation procedure involves two major processes. The first is the incorporation of the foreign gene of interest into the plant genome while the second entails the regeneration of the transformed cells into whole transgenic plants (Singh & Rajam, 2009). The success of the genetic transformation technique depends on an effective and reliable procedure as efficiencies are often low. Several techniques such as polyethylene glycol (PEG)-mediated direct uptake of DNA by protoplast (Kobayashi & Uchimaya, 1989), particle bombardment (Yao et al., 1996) and Agrobacterium-mediated transformations (Hidaka & Omura, 1993) have been developed and used with various Citrus spp. However, the latter transformation system is now the most commonly used method because it has been proven most successful with higher transformation efficiencies resulting in the production of transgenic plants (Peña et al., 2007; Singh & Rajam, 2009; Yu et al., 2002).
Although, Agrobacterium-mediated transformation is considered the best overall method, direct uptake of DNA by protoplasts and particle bombardment have their advantages over the former method. Protoplast transformation is mostly used with commercially important citrus genotypes that are either seedless or contain very few seeds, which is required in most Agrobacterium-mediated transformation procedures (Fleming et al., 2000). Here, the citrus plant is regenerated from the protoplast via somatic embryogenesis and additionally it can eliminate the need for the use of antibiotics either for plant selection or bacterial inhibition (Fleming et al., 2000). This method also allows the improvement of citrus genotypes that are sexually incompatible by producing superior scion or rootstock somatic hybrids (Fleming et al., 2000; Grosser et al., 1998a; Grosser et al., 1998b). Regeneration using this system has been used with many citrus species, including lemons [C. limon (L.) Burm. F.], limes [C. aurantifolia (Cristm.) Swingle], mandarins (C. reticulata Blanco), grapefruits (C. paradisi Macf.), sweet orange (C. sinensis Osbeck) and sour orange (C. aurantium L.). Although, limited success has previously been reported using protoplast transformation with sweet orange, rough lemon (C. jambhiri Lush.) and ‘Ponkan’ mandarin (Hidaka & Omura, 1993; Kobayashi & Uchimaya, 1989; Vardi et al., 1990). Fleming et al. (2000) have reported success in recovering transgenic sweet orange plantlets by an optimized version of this method.
Particle bombardment involves the direct delivery of DNA coated onto microprojectiles into intact cells or organized tissue via a gene gun or a biolistic particle delivery system (Yao et al., 1996). This method is used alternatively in cases where citrus genotypes are recalcitrant to Agrobacterium infection. A reason for this is that citrus is not a natural host for the bacteria (Khan, 2007). A problem that arises from this method is the low regeneration frequency of stably transformed cells from calli as was observed with tangelo (C. reticulata x C. paradisi) (Yao et al., 1996). Nevertheless, transformation efficiencies of 93%, based on transient expression experiments, have been reported with citrange (C. sinensis x P. trifoliata) when particle bombardment is carried out using thin epicotyl segments (Bespalhok et al., 2003).
This system uses the ability of the Agrobacterium-plant interaction to transfer and integrate genetic information into the plant’s genome. The bacteria, depending on the species, contain either a rhizogenic (Ri) or a tumor-inducing (Ti) plasmid which includes a T-region or transferred DNA region (T-DNA). This T-DNA region is manipulated by genetic engineering to include the gene of interest for transfer in the transformation process. The T-DNA movement from Agrobacterium occurs only onto wounded plant cells (Gelvin, 2003; Messens et al., 1990). The initiation of this transfer depends on the induction of the virulence (vir) region located in the Ti plasmid. There are 6 vir genes virA-virE and virG that make up this 35 kilobase pairs (kb) region between the left and right borders of the T-DNA. Wounded plant cells produce vir inducing compounds such as acetosyringone and α-hydroxyacetosyringone that induce the expression of these vir genes initiating the T-DNA transfer and thus transformation of the plant cells (Gelvin, 2003; Messens et al., 1990). Agrobacterium-mediated transformation experiments have been carried out with numerous hybrids and species of citrus, such as grapefruit, sour orange, sweet orange, trifoliate orange (Poncirus trifoliata Raf.), ‘Carrizo’ citrange, ‘Mexican’ lime, ‘Swingle’ citrumelo (C. paradisi x P. trifoliata), ‘Cleopatra’ mandarin, and alemow (C. macrophylla Wester) (Dominguez et al., 2000; Ghorbel et al., 2000; Gutierrez-E et al., 1997; Luth & Moore, 1999; Molinari et al., 2004; Moore et al., 1992; Peña et al., 2004, 2007). Transformation of other economically important citrus cultivars with the existing protocols has not yet been successful.
Generally, transformation efficiencies obtained by using Agrobacterium with most citrus cultivars can range from 0 to 45%. This is due to a number of limiting factors that can affect the transformation process. These include: species or cultivar specificity, age and type of explant used, competence of the citrus cells or tissues, Agrobacterium strains used and inoculation procedure, co-cultivation and pre-culturing conditions, adequate selection conditions and recovery of transgenic shoots (Bond & Roose, 1998; Costa et al., 2002; Peña et al., 2007; Yu et al., 2002).
Data from early studies indicated that the type of citrus species and cultivar used in transformation experiments affect transformation efficiencies. Bond & Roose (1998) showed that when 7 citrus cultivars, ‘Washington navel’ and ‘Olinda Valencia’ oranges, ‘Lisbon’ lemon, ‘Rio Red’ grapefruit, ‘Carrizo’ citrange, mandarin and ‘Mexican’ lime were transformed with Agrobacterium only ‘Washington navel’ and ‘Carrizo’, resulted in GUS-positive shoots. These results were indicative of the receptiveness of these cultivars to this type of transformation protocol compared to the others. Although very little diversity exists between the sweet orange cultivars, ‘Washington navel’ and ‘Olinda Valencia’, the difference that exists was sufficient to affect the transformation efficiency. As a result, different protocols have been developed for different citrus species and cultivars (Bond & Roose, 1998; Costa et al., 2002; Peña et al., 1997).
Studies have also shown that lower transformation efficiencies are obtained with older segments (Moore et al., 1992; Peña et al., 1995a). Transformations of three week old ‘Washington navel’ orange epicotyl segments resulted in efficiencies of up to 87%, while 5 to 8 week old epicotyl segments gave lower efficiencies of 5 to 40% (Bond & Roose, 1998). This reduction in transformation efficiency is presumed to be the result of older epicotyl segments having a lower number of actively dividing cells and consequently less susceptible to T-DNA integration and the regeneration of shoots (Bond & Roose, 1998; Villemont et al., 1997). In addition, it is regarded that older epicotyl segments have different wound exudates or cell wall components that result in a reduction in bacterial binding or the activation of the virulence genes (Bond & Roose, 1998).
Various types of explants such as, callus, leaf sections, seeds, epicotyl nodal and inter-nodal stem segments are often used, with varying results (Hidaka & Omura, 1993; Kaneyoshi et al., 1994; Moore et al., 1992). For instance, higher transformation efficiencies are obtained from citrus callus of ‘Ponkan’ mandarin. The advantages of using callus as explants are that a larger number of transgenic plants are produced, there is rapid proliferation and chimeras are rarely observed during the regeneration process (Li et al., 2002). However, drawbacks to using this system are that some citrus varieties do not possess embryogenic potential and the regenerated plants are juvenile, resulting in a long waiting period for the evaluation of the traits of interest and, additionally, it increases the risk of somaclonal variation which results in abnormal plant morphologies (Cervera et al., 2000; Li et al., 2002). Agrobacterium-mediated transformation involving epicotyl and internodal stem segments are the predominantly used explants for regeneration of transgenic citrus plants. These types of explant are the most widely used in citrus transformation experiments and appear to be the most responsive. The disadvantage of using these types of explants is that the process is very laborious and takes a long time. Alternatively, another efficient system uses cotyledons from ungerminated mature seeds, followed by shoot regeneration via direct organogenesis (Khawale et al., 2006; de Oliveira, Fisher and Moore unpublished). The advantage of this method is that it is less time consuming and laborious. It involves the use of mature seeds that are sterilized, subsequently the seed coat is removed and the cotyledons are directly inoculated with the Agrobacterium suspension and later transferred to the appropriate selection media. The use of this type of explant eliminates the time required for germination of seedlings to produce epicotyl segments and we have obtained higher transformation and regeneration frequencies with grapefruit and sweet orange. Although GUS expression was observed, we have not yet carried out the evaluation for stable integration of the transgene in the putative transgenic plants generated by this method. However, Khawale et al. (2006) proved the stability of this transformation method in ‘Nagpur’ mandarin.
Cell division and dedifferentiation of plant cells are responsible for the explants’ competent state and result in callus proliferation (Peña et al., 1997, 2004). Observations of transformed citrus inter-nodal and epicotyl segments showed that resulting transgenic cells were localized in callus tissue and are of cambial origin. It is also suggested that certain treatments such as the inclusion of auxins, which promote active cell division and dedifferentiation of plant cells, correlated with higher transformation efficiencies (Peña et al., 2004).
A study involving the use of three different strains of Agrobacterium (C58 C1, EHA101-5 and LB4404) to transform seven citrus cultivars showed varying transformation efficiencies (Bond & Roose, 1998). In four separate experiments, strain C58 C1 had the highest transformation efficiency of 45%, while strains EHA101-5 and LB4404 resulted in transformation efficiencies of 29% and 0%, respectively (Bond & Roose, 1998).
The inoculation of the citrus explants with the Agrobacterium culture typically requires incubation periods of 1 to 30 minutes (Bond & Roose, 1998; Costa et al., 2002; Luth & Moore, 1999; Peña et al., 1997). However, incubation periods greater than 10 minutes have led to the increase in regeneration of escape shoots and a reduction in transformation efficiency (Costa et al., 2002).
The optimal Agrobacterium culture concentrations that have been determined for the effective inoculation and transformation of citrus are 5x108 and 4x107 cfu/ml, and are dependent on the citrus cultivar being transformed (Bond & Roose, 1998; Cervera et al., 1998b; Costa et al., 2002; Dominguez et al., 2000; Luth & Moore, 1999; Peña et al., 1995a; Yu et al., 2002). A limited source of bacterial cells reduces the frequency of T-DNA transfer while excess bacteria stress the plant cells (Costa et al., 2002; Yu et al., 2002).
Co-cultivation involves incubating both the explants and Agrobacterium on media containing no selective agent for the transformed cells or against the bacteria, for a period of time. An increase in the co-cultivation period has been associated with a higher number of regenerated and transformed shoots (Costa et al., 2002). Transformation frequency increased when the co-cultivation period was increased from 1 to 5 days at which it reached a maximum (Cervera et al., 1998a). However, prolonged co-cultivation periods often lead to an overgrowth of Agrobacterium which reduces the regeneration frequency of transformed shoots (Cervera et al., 1998b; Costa et al., 2002). As a result, most transformation protocols routinely use a 2 to 3 days co-cultivation period (Cervera et al., 1998b; Costa et al., 2002; Luth & Moore, 1999; Peña et al., 1997).
The composition of the co-cultivation medium also affects the transformation process. The presence of auxins such as 2,4 dichlorophenoxyacetic acid (2,4-D), in co-cultivation medium has resulted in higher transformation frequencies in comparison to co-cultivation medium containing a filter paper layer, tomato cell suspension or a cell feed layer alone (Cervera et al., 1998b; Costa et al., 2002). The use of tomato cell feeder layers with high auxin concentrations has also improved citrus transformation (Costa et al., 2002).
The principle of pre-culturing the explants on co-cultivation medium before inoculation with Agrobacterium is to promote the production of vir-inducing cell components by metabolically active cells, which enhances the transformation process (Costa et al., 2002; Spencer & Towers, 1991). However, some studies have shown that pre-culturing citrus explants has a negative effect on the transformation efficiency (Cervera et al., 1998b; Costa et al., 2002). Explants without pre-culture gave a reported 8.4-fold higher level in transformation efficiency compared to those that were pre-cultured (Costa et al., 2002). Most transformation experiments have bypassed this pre-culturing stage and have instead used acetosyringone (Cervera et al., 1998b). In nature, this phenolic compound is produced in wounded plant cells and is responsible for the activation of the vir genes. This has been shown to increase transformation efficiencies when added to the Agrobacterium inoculum and the co-cultivation medium by promoting transcription of A. tumefaciens virulence genes (Cervera et al., 1998b; Kaneyoshi et al., 1994); however, in our personal experience working with grapefruit the addition of acetosyringone does not have much of an effect on the transformation efficiencies.
Finding suitable selective agents to recover transformed cells is critical in citrus transformation in order to eliminate the high numbers of chimeras and escapes that can be obtained during the process (Gutierrez-E et al., 1997; Moore et al., 1992; Peña et al., 1995a). Hence, an effective selective agent is required to improve transformation recovery. Selection is usually based on antibiotic or herbicide resistance. Kanamycin is one of the most widely used selective antibiotics in transformation processes and is most effective when used in concentrations of up to 100 mg/L. However, shoot regeneration may be inhibited at this concentration. Other antibiotics such as geneticin and hygromycin have also been used, but are not as effective as kanamycin (Costa et al., 2002; Peña et al., 1997). The selective antibiotic can be ineffective in situations where residual Agrobacterium cells are present or neighboring transformed cells result in the break down or neutralization of the antibiotic. Invariably, non-transformed plant cells, i.e. escapes, strive in the absence of selective pressure (Cervera et al., 1998b). Other non-toxic selective genes, for instance manA, which encodes for the enzyme phosphomannose-isomerase (PMI), have been successfully used in the transformation of sweet orange (Boscariol et al., 2003). The principle is based on the ability of the transformed cells to metabolize mannose as a carbon source present in the selective medium. Additionally, the use of non-metabolizable genes instead of antibiotic and herbicide resistance genes as selective agents provides a suitable alternative and would satisfy public concerns about their dissemination into the environment and potential effect to consumers. This PMI positive selection system has been shown to be more effective than using kanamycin in many plant transformation protocols (Sundar & Sakthivel, 2008) but this did not seem to be the case in citrus.
Recovering whole transgenic plants from transformation experiments is often difficult. Typically, most regenerated transformed shoots are either placed directly in soil containing rooting hormone or on rooting media containing varying levels (0 to 1.0 mg/l) of the auxin naphthaleneacetic acid (NAA) which promotes root development (de Oliveira et al., 2009; Gutierrez-E et al., 1997; Luth & Moore, 1999; Moore et al., 1992). Some researchers have gotten better results by first transferring the shoots to hormone-free media to eliminate the cytokinin benzyl aminopurine (BA) from the regeneration media before placing on NAA containing media. Different combinations of BA, NAA and another auxin, indole 3-butyric acid (IBA), NAA and IBA only or just IBA and BA in the rooting medium have been tested so as to improve rooting efficiency in citrus cultivars such as mandarin, lemon, ‘Troyer’ citrange and lime (Al-Bahrany, 2002; Jajoo, 2010; Moreira-Dias et al., 2000; Singh et al., 1994). Again, the concentrations of these phytohormones vary depending on the citrus genotype. High rooting efficiencies of transgenic shoots have been obtained with citrus types, such as grapefruit, ‘Carrizo’ citrange and P. trifoliata (Peña et al., 2007), but with other citrus types, the rooting efficiency is very low. This problem is overcome by shoot-tip micrografting the transgenic shoot onto a decapitated rootstock seedling (Peña et al., 1995a, 1995b).
Recent advances in genomics, both in citrus and other species, have made available an abundance of genes that can be easily cloned and used in transformation. This is particularly useful in the genetic engineering process as characterized gene(s) derived from known sources can be incorporated into the genome of a recipient plant to obtain desirable traits. Because of its economic impact, disease control is often the objective of plant improvement programs. Hence, resistance and defense genes isolated from well studied plant species have been successfully incorporated into other species to generate pathogen-resistant plants. Another successful strategy in the control of diseases has been the transformation of genes derived from pathogens which can also result in resistant plants.
According to the USDA economic research service, genetically engineered (GE) crops have been widely adopted since their introduction in 1996 (USDA, 2010). Herbicide-tolerant genetically engineered soybeans and cotton have been the most extensively and rapidly adopted GE crops in the U.S., followed by insect-resistant cotton and corn (Cao et al., 2010). The positive impact of these GE crops was due to lower labor and production costs, and gains in profitability, in addition to their increased environmental benefits. In the particular case of citrus, although a variety of transgenic types have been reported in the literature, none has reached commercialization. However, field trials, including our own, are underway. Below we describe some recent and relevant cases of transgenics in citrus.
Some of the earliest success stories in the control of diseases by genetic engineering were using pathogen-derived genes from viruses (Abel et al., 1986). When certain viral genes, particularly the capsid protein (CP), were transformed into plants they showed resistance or immunity against closely related viral strains. A well-known case in a perennial species is the control of Papaya ringspot virus by the insertion of its CP into the papaya genome. This effort virtually saved this industry in Hawaii (Gonsalves, 1998). The control mechanism that prevents viral replication in the transgenic plants was initially denominated co-suppression but it is currently referred to as RNA interference or RNA silencing.
Several studies have transformed sequences from a variety of economically important viruses into different citrus types to attempt to produce resistant plants. One of such viral diseases is caused by Citrus tristeza virus (CTV). Severe strains of CTV can dramatically reduce production and in some instances lead to tree death in a relatively short period of time (Moreno et al., 2008). In some areas of the world CTV is an important or the most important limiting factor in citrus production and incorporation of resistance by traditional breeding techniques is not possible. For this reason many laboratories have tried to genetically engineer different CTV sequences into citrus as a way to control this important pathogen. However, these attempts have never been completely successful. For example, transforming the major CP (p25) into ‘Mexican‘ lime had two types of response to viral challenge. In replicate plants, propagated from the same line (i.e. genetically identical), 10 to 33% were resistant to CTV while the rest developed typical symptoms, despite a significant delay in virus accumulation (Domínguez et al., 2002). Similar results were obtained in ‘Duncan‘ grapefruit when translatable and untranslatable versions of the major CP were transformed (Febres et al., 2003, 2008). Various forms (full length, hairpins) of the p23 gene, located in the 3‘ end of the viral genome, have also been transformed into citrus genotypes. In ‘Mexican‘ lime expression of the p23 protein produced viral symptoms in some plants (Fagoaga et al., 2005). Lines with normal phenotype (no symptoms) were further propagated and tested for CTV resistance and again the results were mixed with some plants completely immune to the virus while others from the same line had delayed symptom development and virus accumulation (Fagoaga et al., 2006; López et al., 2010). The use of the 3‘ region of the p23 and the contiguous 3‘-untranslated region (UTR), either as a hairpin or as single copy, has also been transformed into \'Duncan\', \'Flame\', \'Marsh\', and \'Ruby Red\' grapefruit and alemow plants with similar results as described above in which some plants derived from a particular line were fully resistant and others were not (Ananthakrishnan et al., 2007; Batuman et al., 2006; Febres et al., 2008). Only in one line full resistance was observed (Febres et al., 2008). This line is currently being evaluated in the field for its horticultural value and durability of the resistance under natural conditions. Other CTV genes have been used but either no transgenic plants were regenerated (p20 and minor CP/p27) or they did not show resistance (RdRp gene) (Febres et al., 2003, 2008).
Resistance to another important viral disease, Citrus psorosis virus (CPsV), has been reported in transgenic sweet orange plants transformed with intron-hairpin constructs (ihp) corresponding to the viral CP, the 54K or the 24K genes (Reyes et al., 2011). After challenge with the virus, the CP transgenic plants were more effective in controlling the CPsV and consistently showed lower virus levels and no symptom development compared to 54K and 24K transgenic plants. The study reported that the observed CPsV resistance was due to pre-activated RNA silencing rather than the siRNA accumulation levels in the ihp-CP transgenic sweet orange plants prior to virus challenge (Reyes et al., 2011).
Pathogen-derived genes have also been used to control bacterial diseases. Citrus canker, caused by Xanthomonas axonopodis pv. citri is an economically important disease, especially for the fresh fruit market. The pthA protein is involved in the pathogenesis and symptom development of this bacterial pathogen and the C-terminus contains three nuclear localizing signals (NLS) critical for the interaction with a host protein, translocation to the nucleus and function (Yang et al., 2011). By using a truncated version of the pthA gene, coding only for the C-terminus portion of the protein, it was theorized that the resulting protein would interrupt binding and function of the native bacterial pthA during infection and prevent symptom development and pathogen growth. Indeed transgenic sweet orange plants that expressed the truncated protein showed lower disease incidence and symptom development compared to wild type plants, demonstrating a certain degree of resistance (Yang et al., 2011). The authors are currently conducting field experiments to determine the effectiveness of this strategy under natural conditions.
In another strategy, also to control citrus canker, a hrpN gene derived from Erwinia amylovora was transformed into ‘Hamlin’ sweet orange plants. The hrpN encodes a harpin protein that elicits the hypersensitive response (HR) and systemic acquired resistance (SAR) in plants. The hrpN gene was inserted in a construct made up of gst1, a pathogen-inducible promoter (so the gene would not be expressed constitutively and hence the SAR response would only be induced in the presence of the pathogen), a signal peptide for protein secretion to the apoplast (the canker bacterium does not penetrate the cell and remains apoplastic). Several of the hrpN transgenic lines showed reduction in their susceptibility to citrus canker as compared to wild type plants, and one line in particular displayed very high resistance to the pathogen (up to 79% reduction in disease severity) (Barbosa-Mendes et al., 2009).
Fungal pathogens also affect citrus production. In particular, Phytophthora spp can cause root rot and gummosis in mature trees and damping-off in seedlings. For the control of Phytophthora nicotianae\n\t\t\t\t\tAzevedo et al (2006) used a bacterio-opsin (bO) gene to transform ‘Rangpur’ lime. The bO gene is derived from Halobacteria halobium and can spontaneously activate programmed cell death and enhance broad-spectrum disease resistance accompanied by pathogenesis-related (PR) protein accumulation. In two of the transgenic lines, higher levels of tolerance to this pathogen with significantly smaller lesions were observed; however, these lines also exhibited HR-like lesions in the absence of pathogen (Azevedo et al., 2006). It remains to be seen if this strategy will work under field conditions given the fact that the transgenic plants develop spontaneous lesions.
Upon recognition of a potential pathogen plants naturally respond by triggering defense mechanisms that can, in some instances, halt pathogen colonization. One such defense mechanism is SAR, a form of inducible defense in which infection by a pathogen leads to an enhanced defense state that is durable and provides resistance or tolerance to a wide range of pathogens in subsequent challenges (Durrant & Dong, 2004).
A gene that has been identified as critical in the establishment of SAR is the NON-EXPRESSOR OF PATHOGENESIS RELATED 1 (NPR1). NPR1 is a transcription co-activator and plays a key role in regulating defense gene transcription and signal transduction pathways that lead to SAR (Despres et al., 2000; Zhang et al., 1999). Under normal conditions the NPR1 protein does not induce SAR, however in the presence of a pathogen and increased levels of salicylic acid (SA) NPR1 is translocated to the nucleus where it interacts with transcription factors that ultimately induce the expression of SAR-associated genes (Kinkema et al., 2000).
A number of studies have demonstrated that the over-expression of the Arabidopsis NPR1 provides a broad-spectrum enhanced resistance to various pathogens (Cao et al., 1998; Lin et al., 2004). Our laboratory and others have invested a considerable amount of time and effort investigating the nature of SAR in citrus and the full length sequences of five citrus NPR1-like genes has been cloned and sequenced. Their expression levels are differentially affected by pathogen and other treatments (Febres and Khalaf, unpublished results).
Zhang et al. (2010) reported transforming the Arabidopsis NPR1 gene into ‘Duncan’ grapefruit and ‘Hamlin’ sweet orange. The over-expression of this gene increased resistance to citrus canker and the observed resistance correlated with the expression levels of the transgene. Our results of transgenic ‘Carrizo’ citrange plants, also transformed with the Arabidopsis NPR1 gene indicated that the transgenic lines were as well more tolerant to citrus canker (slower lesion development) and had higher levels of pathogenesis-related (PR) genes than wild type plants (Febres, unpublished).
As mentioned above, attempts to use pathogen-derived sequences for the control of CTV have not rendered consistent results. A different approach has recently been tested (Cervera et al., 2010) by using single-chain variable fragments (scFv) from two monoclonal antibodies that in combination seem to detect the major CP from most CTV isolates. ‘Mexican’ lime plants were transformed with each scFv either individually or in combination. Essentially all constructs conferred some level of protection when the plants were challenged with a severe strain of CTV. Between 40 to 60% of the plants tested did not get infected, compared to 95% infection in control plants. In addition a delay and attenuation in symptom development was also observed. Although complete resistance was not observed in this case either it is still a promising approach that needs further investigation.
The production of new varieties via transformation in citrus and many other woody perennials poses a challenge not found in the breeding of annuals and other fast-growing plants. Due to combinations of long juvenile periods, biological barriers to crossing, and the difficulty of reconstituting favored types, such as the complex hybrids sweet orange and grapefruit in citrus, new cultivars will probably have to be selected from T0 transformants. There are several implications to this, discussed below.
One of the greatest challenges of producing and testing transgenic Citrus plants is the long juvenile periods observed in this genus. As discussed above, most citrus transformation techniques utilize explants derived from juvenile tissue, and the transgenic plants must be grown for many years, in most cases, for their horticultural attributes to be evaluated. Two approaches are being investigated to overcome this problem. The first is efforts to decrease the juvenile periods of transgenic plants. There are both historical work and ongoing efforts to use horticultural methods to bring citrus plants into bearing earlier. Another alternative for shortening the juvenile period is to produce transgenic plants that over-express a flower meristem identity gene that causes them to flower earlier. The Arabidopsis\n\t\t\t\tLEAFY and APETALA1 genes have been over-expressed in ‘Carrizo’ citrange (Peña et al., 2001) and transgenic Poncirus plants over-expressing a citrus orthologue of Arabidopsis\n\t\t\t\tFLOWERING LOCUS T (FT) have been produced (Endo et al., 2005; Nishikawa et al., 2010). However, in most cases, the expression of these genes, while dramatically reducing time to flowering, also conferred deleterious morphological phenotypes to the transgenic plants. Thus this approach may benefit citrus breeding efforts and early testing of traits designed to be evinced in fruit, it may not produce T0 transgenic plants that could directly be used in production. However, all possibilities of this approach have not been explored. For instance, citrus genomes contain at least three orthologues of FT that produce quite different phenotypes when overexpressed in transgenic tobacco (Kamps and Moore, unpublished). Also, Carrizo plants transformed with APETALA1 displayed normal morphology (Peña & Séguin, 2001).
The second approach for overcoming juvenility is to use explants from mature plants for transformation. However, taking explants directly from mature trees is not likely to be successful due to the low regeneration potential of such explants and perhaps also of lower competence for transformation. Success has been achieved by reinvigorating mature citrus types by grafting mature buds on vigorous juvenile rootstocks and using the first flushes for Agrobacterium-mediated transformation (Cervera et al., 1998a, 2005). However, this is a technically demanding approach. The plant material must be in excellent condition, which is particularly difficult to achieve in humid climates, where the pathogen load on tissue, even when grown under greenhouse conditions, may make disinfection of explants difficult. Even then, only a relatively small number of explants can be obtained from the first flush or two of the grafted plant. In some genotypes, a lack of bud uniformity in sprouting and morphology is problematic (Cervera et al., 2008). In other cases, culture requirements for regeneration may be quite different for even closely related citrus types (Almeida et al., 2003; Rodríguez et al., 2008). Kobayashi (2003) circumvented some of these problems by using already grafted ‘Pera’ sweet orange nursery plants for harvest of explants and by thin segments (1 to 2 mm) of stems as explants. In all cases, transgenic plants in the greenhouse began to flower after 14 months or less after micrografting the transgenic scions on rootstock. Experiments are underway in several laboratories to improve still further on the production of transgenic plants from mature tissue. The importance of the cambium in producing transgenic tissue in many of the above reports and the recent description of the cambium cells of several plants as analogous to vascular stem cells (Lee et al., 2010) suggest that one research direction could be exploration of other types of explants where the cambium cells are maximally exposed to Agrobacterium and subsequent growth hormones in the culture medium.
Another problem with using T0 plants is that the gene insertion site(s) is unknown. This can affect the expression of the transgene and could lead to altered morphology that was not intended. However, genomic changes that are not selected for also may happen during conventional breeding due to, for instance, transposon activity or irradiation and mutation breeding.
Of course there are also advantages to utilizing T0 transformants in perennials. With the explosion in genomic information, the functions of more and more genes are being elucidated (Talon & Gmitter Jr, 2008), so choosing a transgene that will impart a particular trait should be more targeted in the future. It has also been found in both conventional and molecular breeding that valuable genes or alleles are found in plant relatives or wild species. In such cases using T0 transgenics circumvents the problem of linkage drag that may result from the transfer of unknown and undesirable genes that are linked to the desirable gene or allele from the donor parent. It might also be possible to “stack” valuable genes or alleles in a desirable citrus type via multiple transformations or multiple genes inserted in a single transformation.
Another important area of research has been to increasing the cold hardiness of citrus. This could potentially extend production areas to new regions where pathogens or other limiting factors are not present. As in the case with disease resistance there are some citrus relatives that can endure freezing temperatures. While most commercially important citrus varieties are susceptible to freezing, P. trifoliata for instance can tolerate temperatures well bellow freezing if cold acclimated prior to the exposure (Talon & Gmitter Jr, 2008).
Genes associated with cold acclimation have been identified in citrus as an initial milestone in a multistep approach to ultimately incorporate some of these genes in the genome of selected citrus varieties that are naturally susceptible to freezing. Our laboratory and others have studied the effect of cold stress or freezing on gene expression. For instance, in an attempt to minimize the chilling injury during citrus fruits storage, a genome-wide transcriptional profiling analysis was performed (Maul et al., 2008). Grapefruit flavedo RNA was used to study the responses of citrus fruit to low temperatures. The study applied a pre-storage conditioning treatment of 16°C for 7 days and utilized an Affymetrix Citrus GeneChip microarray. While the applied treatment seemed to have halted the expression of general cellular metabolic activity, it induced changes in the expression of transcripts related to membranes, lipid, sterol and carbohydrate metabolism, stress stimuli, hormone biosynthesis, and modifications in DNA binding and transcription factors.
Our laboratory provided the first evidence of an association in citrus between C-repeat binding factors (CBF) expression levels and the extent of cold tolerance (Champ et al., 2007). CBFs have been identified in many species and they function as transcriptional activators regulating the expression levels of a number of genes that impart cold and stress tolerance. P. trifoliata, a Citrus relative, can survive freezes of -20°C when fully cold acclimated. On the other hand, grapefruit cannot withstand temperatures lower than 0°C. In P. trifoliata transcripts of CBF1 and CORc115 (a cold-induced group II LEA gene, and a likely target of CBF1) accumulate both earlier and to higher levels than in grapefruit when exposed to cold temperatures. Additionally, using subtractive hybridization we identified a number of new, differentially cold-regulated genes from P. trifoliata (Sahin-Cevik & Moore, 2006). Although several of the genes identified were unique sequences, many were homologous to cold and environmental stress-induced genes from other species. Taken together, our results indicate that similar pathways are present and activated during cold acclimation in diverse plant species.
In a more recent study (Crifo et al., 2011) performed a transcriptome analysis based on subtractive hybridization to study cold stress response of pigmented sweet oranges (blood oranges) in order to study the overall induction in gene expression after the exposure to low temperatures. On the whole, the expression of transcripts related to defense, oxidative damage, osmo-regulation, lipid desaturation and primary and secondary metabolism were induced. In addition, cold stress induced flavonoid biosynthesis, including those reactions involved in anthocyanin biosynthesis and metabolic pathways supplying it. Several transcription factors were identified for the first time as cold responsive genes in plants.
In summary, cold stress has been linked to signaling pathways where gene expression can further interrelate with additional stress related pathways. The entire signaling network throughout the plant affects its response(s) to biotic or abiotic stress. Along with the mentioned gene annotations, additional functional analyses are crucial to study the nature of the expected phenotype before we can introduce new genes into the Citrus genome using transformation techniques.
Antimicrobial peptides (AMPs) are currently the subject of intense research for the control of diseases in citrus, particularly canker and huanglongbing (HLB) or citrus greening. There is no known resistance in Citrus to HLB (caused by Candidatus Liberibacter spp); however, it can have devastating effects by reducing overall production. Infected trees have smaller fruits with less juice, the flavor of the juice is changed and it eventually leads to micronutrient deficiencies, defoliation and tree death. It has been known for years that AMPs play a vital role in plant defense. Plant AMPs are monomer or oligomer building units that have mostly three-dimensional or tertiary structures of either amphipathic or amphiphylic nature (Sitaram & Nagaraj, 1999, 2002). The latter characteristic and folding are essential for the peptides antibacterial activity (Epand & Vogel, 1999). Different scenarios for their function have been suggested but they all agree on the fact that these AMPs operate by the formation of membrane pores that ultimately cause the disruption of the membrane and subsequently cell death through ion and metabolite leakage (Yeamn & Yount, 2003). A number of studies have confirmed the inhibitory effect of these peptides to fungal and bacterial pathogens when expressed in different plant species such as rice, wheat, and tomato fruits (Jha & Chattoo, 2010; Jha et al., 2009; Ramamoorthy et al., 2007). In a recent study, two AMP genes, Shiva A and Cecropin B, were transformed into ‘Jincheng’ and ‘Newhall’ sweet orange. Subsequently, the transgenic plants were challenged with Xanthomonas axonopodis pv. citri, the causal agent of citrus canker. In both greenhouse and field experiments with artificial or natural inoculation, respectively, some transgenic lines were highly resistant to canker and either did not develop canker lesions or the number of lesions was significantly reduced compared to wild types. The plants were also phenotypically normal, flowered after two years (grafted on Poncirus), borne fruit and the juice was no different in solid and sugar content and acidity from non-transgenic plants (He et al., 2011).
Transient expression systems are beneficial for some purposes, such as rapidly and easily assaying promoter function or gene expression under some conditions. Although it has been surprisingly difficult to implement transient expression in citrus leaves it has been possible to transiently express genes in the fruit, particularly young fruit (Ahmad & Mirza, 2005; Spolaore et al., 2001).
Finally, a vector based on CTV has been developed (Folimonov et al., 2007). Such vectors have been used in herbaceous plants to study gene function, expression, and silencing, but have not been available for woody plants. This can be seen as a hybrid strategy between transient expression and stable transformation. Although the virus vector nucleic acid is not incorporated into the genome of the citrus host, Folimonov et al. (2007) reported that expression of GFP continued for up to four years after introduction of the scorable marker into CTV vectors.
Ultimately the use of genetic engineering is just another tool in the improvement of citrus. Genetic transformation has the advantage of potentially reducing breeding time, particularly important in the case of a perennial crop such as citrus with a long juvenile period, and also facilitating the introduction of traits not readily available in the particular species. Breeding programs take into consideration the needs of both farmers and consumers. Production of genetically modified citrus should also take into consideration the needs of both; however, genetically modified organisms (GMOs) tend to be more controversial and subjected to more public scrutiny than traditionally produced varieties. For instance, a recent European survey indicated that among respondents GMOs were considered unnatural (70%), made them feel uneasy (61%), harmed the environment (59%) and were unsafe for people\'s health (59%) (European Comission, 2010). Regardless of whether these concerns are just perceived or real they will have to be addressed in order to fully implement the benefits of genetic engineering in solving real and important problems for citrus farmers and at the same time delivering desirable products to consumers.
Two major concerns regarding GMOs are: 1) impact to the environment, in the form of the transgene \'escaping\' and transferring to wild species and thus eroding the biodiversity of wild relatives of the crop or, on the other hand, creating \'super weeds\' of species that acquire the transgene and become better fitted and difficult to control (Azevedo & Araujo, 2003; Parrott, 2010; Sweet, 2009); and 2) impact to human health by a potentially toxic or allergenic transgenic protein (Domingo & Gine Bordonaba, 2011).
In the particular case of citrus there are ways to mitigate these concerns. Essentially all presently grown GMOs are transgenic in nature, with “trans” referring to genetic sequences that come from organisms that are not crossable with the plant in question, such as sequences from viruses or bacteria or even from a plant species that is not crossable, for instance the insertion of an Arabidopsis gene into a citrus plant. This has led to many countries and groups being resistant to the growth and consumption of GMOs. Thus, there are proponents of producing GMOs that are cisgenic, where all of the inserted genetic material comes from the original plant or a crossable type (Jacobsen & Schouten, 2008). Such genes could be perceived by the public as more “natural” and could potentially be less likely to be toxic or allergenic (although this would have to be tested experimentally on a case by case basis). Plants transformed this way do not appear to raise the fear and ethical concerns that the production of transgenic plants inspires (Conner et al., 2006; Rommens et al., 2007). However, this approach would rule out the use of most commonly used selectable and scorable marker genes, as well as the most commonly used promoters and termination sequences and the necessary T-DNA borders for Agrobacterium-mediated transformation. A cisgene consists of a native gene with its native promoter and termination. In these discussions, there is also mention of intragenes in which gene parts can originate from different genes as long as the donor is a crossable type (Jacobsen & Schouten, 2008). Many laboratories are now looking for plant DNA sequences that are homologous to the bacterial sequences present in T-DNA borders and for methods to produce genetically modified plants where selectable and scorable genes can be either removed after transformation or are of plant origin (Rommens et al., 2007).
There has been a small amount of research of this kind in citrus. Fleming et al. (2000) transformed sweet orange protoplasts with a construct containing the GFP scorable gene using a PEG method. Transformed regenerating somatic embryos were identified by their GFP expression and physically separated from nontransformed tissues, resulting in transgenic plants. No Agrobacterium was involved and there was no selective agent applied. Ballester et al. (2008) compared the most common citrus transformation and selection system, using kanamycin selection and scorable GUS staining to three methods that did not utilize antibiotic selection, in ‘Carrizo’ citrange and ‘Pineapple’ sweet orange. The alternative methods included scoring for GUS staining without applying selection, transforming explants with a multi-autotransformation (MAT) vector, combining an inducible recombinase-specific recombination system (R/RS) with transgenic-shoot selection through expression of isopentenyl transferase (ipt) and indoleacetamide hydrolase/tryptophan monooxygenase (iaaM/H) marker genes, and selection with the PMI/mannose conditional positive selection system (Boscariol et al., 2003). Transgenic plants were obtained from all treatments, but selection for nptII expression was by far the most efficient. The authors preferred the MAT vector, because with it they could obtain transformed plants where the selectable marker would recombine out (Ballester et al., 2007). However, all of the transgenic plants still contained some sequences of bacterial origin.
Another approach is the use of promoters that do not express the transgene in the edible parts (fruits). Again this would potentially reduce the possibility of becoming harmful to human health. Several groups are actively searching for such promoters in citrus, including inducible promoters that would be turned on at will by chemical application, etc. As explained before the genomic information currently available should facilitate this endeavor. A third strategy we are exploring is the use of transgenic rootstocks that could confer the desired trait to the wild type (non transgenic) scion, without the need of incorporating and expressing transgenes in the scion and edible parts of the plant. This would prevent or at least reduce the chances of spreading transgenic pollen into the wild. There is evidence for the transfer of genetic material between rootstock and scion but this seems to be limited to the graft union region (Stegemann & Bock, 2009). However, it is unlikely that this grafting approach would work with all transgenes since not all expressed proteins are translocated and/or have a systemic effect. One case in which it could work in citrus is the reduction of juvenility using the FT protein. Transgenic FT is capable of inducing flowering through graft unions (Notaguchi et al., 2008; Notaguchi et al., 2009). Induction of pathogen defense could potentially be tackled this way as well since some of the proteins activate systemic signaling (Xia et al., 2004).
These approaches take into consideration consumer’s perception about GMOs, educated concerns about the release of GMOs and the needs of citrus farmers for better, disease resistant crops. Citrus production faces important challenges due to climate change and disease and genetic engineering has the potential, as has been the case in other crops, of becoming an important weapon in the arsenal against these major challenges.
Research in our laboratory is funded in part by the Citrus Research & Development Foundation of Florida and a USDA Special Grant.
Choledochal cysts (CCs) are congenital dilatations of extrahepatic and/or intrahepatic bile ducts defined by Vater and Ezler in 1723. It is a rare biliary entity with an estimated incidence of 1:100–150,000 live births in Western countries. In the Asian population, the incidence can be as high as 1:1000 live births. CC is primarily a childhood disease—up to 80% of patients are diagnosed before 10 years of age. The original classification, first described by Alanso-Lej and colleagues in 1959, was changed in 1977 by Todani and colleagues that classified the CCs as five types. In addition, isolated cystic dilatation of the cystic canal was identified subsequently and proposed as type VI, apart from the revised Todani classification. Although the etiology is controversial, the main elements in the natural historical emergence of the type I and type IV, which make up the majority of all types, have become clearer. A common symptom is nonspecific abdominal pain in older children. When the cyst is complicated, the diagnosis is delayed, the treatment becomes complex, and the results are affected. External drainage (ED), internal drainage (ID), total cyst excision (CE) + hepaticoduodenostomy (HD), and total CE + hepaticojejunostomy (HJ) were defined according to historical development of treatment. Resection is considered that is necessary to prevent further complications and long-term sequel.
The anatomist Abraham Vater first described the normal anatomy of the bile ducts and the fusiform dilatation of the common bile duct (CBD) in 1723 [1]. Then, Doctor Halliday Douglas first described clinically CBD dilatation in 1852. Douglas had detected a large tenderness cystic mass on the right upper quadrant by physical examination of a 17-year-old girl who had an intermittent right-sided pain, obstructive jaundice, and fever complaints in her history. Despite performing external drainage promptly, she died within 1 month. Subsequently, Douglas detected a CC with her autopsy [2]. In 1894, British surgeon William Swain performed the first successful operation in a 17-year-old girl presented with CC, by anastomosing the jejunum to a giant CC. This patient had been reported 2 months later with no jaundice. In 1922, Golder McWhorter underwent hepaticoduodenostomy after excising the CC in a 49-year-old patient who had complaints since infancy. In 1959, Alanso-Lej and colleagues first published the series of CCs. In this publication, they reviewed 94 cases, published previously, together with their own 2 cases and classified the congenital cystic dilatations of the bile ducts anatomically for the first time [3]. In 1977, Todani and colleagues modified the classification of CCs according to cholangiographic images [4, 5, 6, 7, 8].
CCs are more common in Asian populations with an incidence of 1 in 13,000, even 1:1000 in Japan [9], versus 1 in 100,000–150,000 in Western populations [10]. The reason for this Asian preponderance is still unknown [11]. Although predominately diagnosed in children, CCs are found with increasing frequency in adults such that adults comprise the majority of patients in recent series [12, 13], which in part may be due to the increased use of diagnostic imaging [14]. In both adult and children [15], females are higher risk for the disease with a nearly 4:1 female preponderance [10, 16]. Nearly, 80% of CCs are diagnosed in early infancy [10, 17].
Grossly, CCs appear as a diffuse dilatation of the bile ducts [18]. In the congenital dilatations of the bile ducts, the cyst wall thickness is between 2 and 7 mm in diameter and usually involves an inflammatory reaction (80%) [18] that becomes severe after 10 years of age. Cysts, especially infected, are usually being adherent to the surrounding tissues. Bile ducts and columnar epithelium can be seen in the microscopic examination of the cyst wall. Choledochocele that is covered with duodenal mucosa appears different from other types of CCs according to epithelial histology. Liver biopsy findings usually vary with the age in patients with CC. Although, newborn liver is mostly normal, mild periportal fibrosis may be seen in older children. Varying degrees of histological hepatic changes severity may be seen in most patients with CC when it is diagnosed [19]. Higher degree of liver damage associates with the presence of an anomalous pancreaticobiliary ductal union (APBDU), more severe symptoms, type IVa CC, and younger age [19, 20, 21]. It has been observed by investigating liver biopsies that most of these changes resolve after surgical excision; however, preoperative portal fibrosis and central venous distension may remain stable or increase in severity [19]. It is considered that carcinoma of the cyst wall may develop due to recurrent chronic inflammation attacks. However, it can also be seen in non-APBDU-associated CC patients. Additionally, the cancer can develop from anywhere such as the cyst wall, the gall bladder, or the common channel junction nearly pancreas, as a consequence of chronic inflammation due to cholangitis [22, 23].
Despite the existence of numerous theories and laboratory works to explain the etiology of the disease, the exact etiology remains incompletely understood. Initial theories, put forward in this regard, were congenital weakness of the choledochal wall, distal obstruction, oligoganglionosis, and disturbances in the process of recanalization. However, more accepted theories have been produced parallel to the progress in radiological imaging methods [24, 25]. Today, there are two main theories that are widely accepted; (1) reflux of trypsin and other pancreatic enzymes to the bile ducts due to an APBDU; (2) obstruction of distal CBD [26, 27, 28]. The idea that the choledochal and pancreatic ducts’ abnormality about joint and angle was first reviewed by Babbitt in 1969, and subsequently many number of studies, supporting this view, were made [22, 23, 29, 30, 31, 32]. A normal pancreaticobiliary junction usually has an acute angle between the CBD and the pancreatic duct [33] and is located within the duodenal wall [34]. The common channel (distance from between the junction of the CBD and the pancreatic duct to ampulla Vater) length is 4 mm or less, normally. In patients with CCs, this distance (common channel length) increases 5–20 mm [22, 23, 35, 36] that makes the common channel longer [37]. Okada defined it as “common channel syndrome” [36, 38]. It is considered that an abnormal long common channel (especially >15 mm proximal to ampullary sphincter) [39, 40] causes the pancreatic duct communicating with the choledochal duct without the support of ampulla Vater’s circular muscular layer (sphincter of Oddi) [41], which protects the biliary tree from reflux of pancreatic enzymes and bile [34]. Another observation is that the junction angle of the two ducts that should be acute normally, however, is close to 90° in these patients. It also causes the pancreatic fluids to flow into the CBD due to the higher pressure of pancreatic duct (Figure 1) [41]. Eventually, it is considered that APBDU has a tendency to cause reflux of the pancreatic enzyme into biliary tree with consequent biliary duct inflammation and increased duct pressure, leading to duct wall damage and cystic changes [42, 43]. In animal models of murine APBDU, this mechanism has also been demonstrated [44, 45]. APBDU is seen in up to 90% of patients with CC [25, 46], compared with 2% in the general population [10] and this seems to have important clinical implications. In a comparison of APBDU-associated CC versus non-APBDU-associated CC, APBDU-associated CC patients were significantly more likely to have evidence of pathologically confirmed inflammation including hepatitis, cholangitis, and pancreatitis [47, 48]. However, APBDU is not enough to explain the etiology of all CCs, such as diverticular cyst (type II), where the bile ducts were normal except cyst and the cyst is considered to be a sequel of an intrauterine CC rupture, such as meconium pseudocyst [28]. Additionally, type V CCs are possibly due to dysfunctional remodeling of the ductal plate during embryogenesis [40, 49].
Anomalous pancreaticobiliary union.
In addition, it is considered that primary strictures of the CBD may also play a role in the development of CCs. The types of the CCs are determined according to the location, severity, and length of the stricture. Detection of these strictures preoperatively is important because treating the CC without addressing the stricture may lead to recurrent episodes of cholangitis. This mechanism has been under estimated and is now believed to be more significant in the pathophysiology of CCs [50].
The association of CC with congenital anomalies remains ambiguous. Previous reports have demonstrated an association of pediatric CC and congenital anomalies. Murphy et al. reported in 2012 that screening for cardiac anomalies may be prudent in CC patients [51]. Other reports have postulated an association of CC with duodenal atresia, colonic atresia, gastroschisis, annular pancreas, and pancreatic cysts [10, 52, 53, 54, 55, 56, 57].
CCs are first classified in 1959 by Alanso-Lej and colleagues [58]. The original classification identified four types of biliary cysts (types I–IV). In 1977, Todani and colleagues [8] modified this classification and added a fifth category of CC, type V biliary cysts or Carol disease (Figure 2) [10]. Apart from types I–V included in the revised Todani classification, isolated cystic dilatation of the cystic duct has been described and suggested as type VI [59].
Types of choledochal cysts, classified by Todani et al.
Type I CC, most commonly seen, 80–90% of all CC, is a dilatation of the extrahepatic biliary tree. Importantly, the intrahepatic biliary tree is sometimes dilated secondarily due to biliary stasis. Type I cyst can be further subdivided into type Ia, Ib, and Ic cysts [10, 60]. Type Ia CCs are composed of the gallbladder arising directly from the CC, dilated extrahepatic biliary tree, and a nondilated intrahepatic tree. Type Ib CCs are focal segmental dilatation of the CBD and contain no evidence of APBDU [50, 60]. Finally, type Ic CCs are represented by a fusiform dilatation of the common hepatic duct and CBD in the presence of APBDU [10, 50, 60], and often also a low-grade stricture at the distal CBD [50]. In type I CC, usually, gallbladder is involved in cyst structure, and cyst extends from hepatic bifurcation to duodenum [61]. Most commonly, the ducts above (right, left, and intrahepatic ducts) and below of the CC are not dilated [14], except type Ic. In type Ic, cyst extends continuously to the common hepatic duct or intrahepatic ducts [62]. Type I CCs, along with type IV cysts, have the highest risk of malignancy [10]. This is not surprising because both of them have extrahepatic involvement associated with APBDU [63].
Type II CCs (2% of all CCs) consist of a diverticular dilatation of the extrahepatic bile duct system and is considered true diverticulum. During the diagnostic cholangiography study, the diverticulum is filled with radiopaque substance and can be confused with the gall bladder duplication seen rarely [10].
Type III CCs (choledochoceles), 4% of all CCs, are characterized by distal (located at the pancreaticobiliary junction) CBD dilatation, confined to the wall of the duodenum and often bulging into the duodenal lumen [25]. Type III choledochal cysts are dissimilar to other types of CCs, with features such as appearing in both sexes equally and low malignancy incidence [10, 64]. Cysts are mostly not associated with APBDU. Because of all these characteristics, it has been suggested that type III CCs should not be classified as a type of CCs revised by Todani [25, 64, 65]. In addition, Ziegler et al. reported that choledochoceles occur more frequently in older male patients presenting with acute pancreatitis [64].
Type IV CCs, the second most common (15–20% of all), are multiple cysts which can involve both the intrahepatic and extrahepatic biliary trees. Type IV CC can be further subdivided into type IVa and IVb cysts depending on intrahepatic involvement. Type IVa CC refers to multiple segmental communicating biliary dilatations located in the intra- and extrahepatic biliary tracts, and relative stricture at the junction that is used to distinguish the true type IVa CC from type I [11]. Type IVa CCs are usually associated with APBDU [50]. Type IVb CC refers to multiple extrahepatic biliary cysts without intrahepatic involvement [25]. Some recent studies have shown that intrahepatic ductal dilatations seen on preoperative imaging are thought to have been caused by distal obstruction and not true intrahepatic biliary duct disease [11]. Additionally, the question of “Is the distinguishing type I from type IVa really necessary preoperatively?” has not answered yet. Because distinguishing between types I and IVa CCs is controversial for some authors due to complete excision of the extrahepatic bile ducts, and intensive long-term follow-up still remains standard of care for both types [11].
Type V CC or Carol disease, added by Todani, is characterized by multifocal segmental intrahepatic biliary ductal dilatation [14] without the evidence of extrahepatic dilatation [66]. Caroli disease is uncommon, accounting for less than 10% of cases. Patients often present in adolescence or early adulthood with recurrent cholangitis, abdominal pain, or jaundice. However, they may present later with the sequel of portal hypertension and cirrhosis [67]. Renal abnormalities, such as medullary sponge kidney, autosomal dominant polycystic kidney disease, and medullary cystic disease can be seen in Caroli disease [49]. Some authors call Caroli disease as Caroli syndrome when congenital hepatic fibrosis is also seen, as in half of the patients [68, 69].
Type VI CC, isolated cystic dilatation of the cystic duct, is rare with only several case reports describing it. Although it is not officially part of the revised Todani classification, it has been proposed to be called type VI CC [70]. If the cyst emerges from the cystic duct near a level close to the CBD, it can be confused with type II CCs. In such cases, the relation of the cyst with the cystic duct should be thoroughly evaluated to differentiate them [59].
Type IC cysts are the earliest cysts that can be detected by 15-gestational week fetal ultrasonography [71, 72]. There are two clinical forms of disease: adult and infant. In infant form, symptoms such as obstructive jaundice, clay colored stools, and hepatomegaly make it difficult to distinguish from biliary atresia. Adult form of CCs is also congenital, although they usually remain silent until the age of 2 years. There are three main symptoms in the classical clinical triad: recurrent jaundice 69–75%, right upper quadrant pain 47–60%, and right upper quadrant mass 47–80%. But the classic triad only presents in 10% of cases (6–25%) [46, 73, 74].
Abdominal pain is the most common symptom (93.8%) [75], especially in older patients and presents with colic pattern which has a variable interval time (between attacks) up to several years. When investigating the cause of CBD dilatation and differential diagnosis of unclear upper abdominal pain, jaundice, and pancreatitis in children, CCs must be considered [76]. Unfortunately, 29–62% of pediatric patients with CC have been reported that they have choledocholithiasis [77, 78], that is, distinguishing this two situation (CC-associated choledocholithiasis and -nonassociated choledocholithiasis) may be difficult. Choledocholithiasis can also lead to CBD dilatation which can be misdiagnosed as a CC [79].
In 1–2% of cases, especially in infants, CCs may present with rupture and biliary peritonitis prompting emergency biliary drainage [80, 81]. It is not a surprise in diagnosing pancreatitis in patients with CC, because of association of the presence of APBDU [10, 46, 82].
The risk for development of biliary carcinoma in the general population starts after the fourth decade and the incidence increase with age to 0.15% after the eighth decade. However, the risk for the development of carcinoma in patients with a CC starts in childhood and shows a significant increase with age. Interestingly, the age of biliary carcinoma development in patients who have undergone internal drainage without cyst excision has been reported to be 15 years earlier on average than patients who have never had surgery. This is thought to be associated with intestinal bacterial contamination and pancreatic enzymes added to biliary stasis [83]. The malignancy incidence in resected bile duct material has been reported as 7.5% for all age groups, and 0.4 and 11.4% for those under and over the age of 18, respectively. The incidence has been reported to gradually increase every decade to 38.2% over the age of 60, possibly related to chronic inflammation [5]. The incidence of a biliary malignancy development following CC excision is reported as 0.7–5.4%. The malignancy can arise from anywhere such as the porta hepatis, pancreas, or the intrahepatic bile ducts. The time to onset after primary surgical intervention is reported to be 1–34 years. The total excision of the cyst significantly decreases the probability of a malignancy although it does not eliminate it completely [84, 85].
Ultimately, multimodality imaging techniques are often utilized including computed tomography (CT), magnetic resonance imaging (MRI), and/or endoscopic retrograde cholangiopancreatography (ERCP) to confirm the extent of ductal involvement or the presence of extrahepatic disease [25]. Frequently, further imaging techniques are used to differentiate type I CC from type IVa, in the presence of intrahepatic biliary dilatation [11]. A cyst, presenting in the porta hepatitis, separated from the gallbladder and continuing with enlarged biliary ducts can be shown by ultrasound (US). Additionally, fusiform dilatation of choledoch, intrahepatic biliary dilatation (60–80%), biliary stones, and state of liver parenchyma can be shown by US [86].
Other intraabdominal cysts, such as pancreatic pseudocysts, echinococcal cysts, or biliary cystadenomas should also be differentiated from CCs, whether the cyst has continuity with the biliary tree or not [40]. CT is not only useful for demonstrating continuity of the cyst with the biliary tree, but also demonstrates relation of the cyst with the surrounding structures and the presence of associated malignancy [87]. In order to correctly plan surgery, CT cholangiography can be used to identify the full anatomy of the biliary tree but unfortunately it has been reported to be less sensitive for imaging the pancreatic duct which is responsible for the reflux of contrast into the biliary ducts [87]. As it is well known today, the nephro-hepatotoxicity of the contrast and the ionized radiation exposed are the restrictions of CT utilization in pediatric population (Figure 3) [62].
Type III CC. (a) MRCP; (b) CT image.
MRCP is noninvasive and highly sensitive (70–100%) and specific (90–100%) in the diagnosis of CCs [88, 89], so, is considered the current gold standard imaging even for initial evaluation [62]. Additionally, there is no irradiation, and modern scanners have alleviated the need for protracted breath-hold making it more amenable to the pediatric population [10, 90]. Although both ultrasound and CT are highly sensitive and specific in the diagnosis of CCs, MRCP can better identify the CCs subtypes and coexisting abnormalities [89]. For example, MRI can easily identify the pancreaticobiliary ductal anatomy, while ultrasound cannot accurately demonstrate the APBDU [89, 91]. Additionally, MRCP is preferred modality in the pediatric population due to invasive nature and inherent risks of endoscopic ultrasound and ERCP, despite their ability of detecting the abnormality of the common channel [89]. MRCP has also been shown to be as effective as intraoperative cholangiography in planning surgery [18]. In addition, lower cost and morbidity compared to other imaging/diagnostic modalities, and reliability for detecting abnormalities associated with CCs such as cholangiocarcinoma and choledocholithiasis, are some of the favorable features of MRCP [89, 92]. Unfortunately, as seen often in patients with CCs, intraductal air, debris, stones, or protein plugs can interfere with the signal and alter visualization (Figure 4) [93].
PTC reveals the detailed anatomy of the biliary tracts and associated CC.
Although it is highly sensitive, invasiveness and associated risks including cholangitis, bleeding, pancreatitis, and perforation makes percutaneous transhepatic cholangiography (PTC) or ERCP utilization less frequently [94]. Moreover, PTC and ERCP can be technically challenging and require general anesthesia in the pediatric population. A lot of surgeons are finding the use of diagnostic ERCP and PTC in CCs unnecessary due to advantages of both MRCP and perioperative cholangiography (performed in nearly almost patients and give highly detailed information about biliary anatomy) [10, 95, 96]. Contemporary, ERCP should only be performed in cases where the appropriate diagnosis cannot be made by other less-invasive examinations, or when therapeutic performance (complications such as cholangitis or biliary stone obstruction [97, 98] and stabilization of the patients with preparing them to the next definitive surgery) is required (Figure 4) [39, 99, 100].
Another method that is not preferred now for diagnostic purposes is hepatobiliary scintigraphy. Although it is possible to do it with all IDA variants, DISIDA is the best. The radio isotopic substance is collected in the liver and is normally thrown into the biliary tract, but accumulates in the cyst space in patients with CC [101, 102].
There are many diseases including biliary atresia, infectious hepatitis, embryonal hepatic rhabdomyosarcoma, biliary lithiasis, pancreatitis, biliary hamartoma in the differential diagnosis of CCs, especially biliary atresia that is one of the two causes of neonatal obstructive jaundice in neonatal period [10]. Differentiating cystic biliary atresia (CBA), a subtype of biliary atresia and has an entirely different treatment approach, from CCs is particularly difficult. Therefore, prompt accurate diagnosis is critical [103]. While earlier presentation (<months of age), smaller cysts with less dilatation of the intrahepatic biliary system, and an atretic gallbladder with irregular and hypoplastic biliary radicles that is seen on ultrasound and cholangiography are characteristics of CBA patients [10, 20, 104, 105]; a dilated gallbladder communicated with the cyst in addition with a dilated intrahepatic biliary tree is mostly a determiner to infantile CC [104].
It is still important to differentiate biliary rhabdomyosarcoma, a rare soft tissue tumor that affects only 1% of children, from CC [106, 107]. In the presence of a mass or intraductal growth that causes obstructive jaundice, the possible diagnosis should return in favor of rhabdomyosarcoma in children and prompt evaluation is necessary [107].
“Children with CBD dilatation did not differ significantly in clinical characteristics compared with children who had obstructive CBD dilatation” said Oh and colleagues [78] by evaluating the cholangiographic characteristics of 85 children with CBD dilatation to differentiate obstructive and congenital CBD dilatation. Indeed, it can be difficult to distinguish them. Therefore, in the pediatric population with dilated biliary trees, ruling out a distal biliary obstruction that causes secondary biliary dilatation is prudent and essential [18].
As noted above, type I CC may present with intrahepatic biliary dilatation secondary to biliary stasis, thus resembling a type IVa CC. Some authors consider that this distinction is critical given the therapeutic implications and the need to include hepatic resection (in the case of type IVa CC) in addition to extrahepatic biliary tree excision [18], whereas the others consider that the need preoperatively distinguishing between type I and IVa CCs is controversial because for both, complete excision of the extrahepatic bile duct and intensive long-term follow-up remains standard of care [11].
The treatment time of antenatal diagnosed of CCs has been still a matter of debate. Some reports say that they can be operated within 2–6 weeks, even if they are asymptomatic, due to a potential complication risk of cysts, whereas, the others suggest that they can be followed-up for a time of period with US and regular monitoring of liver functions [108, 109].
Surgical treatment of CCs should be performed electively except complicated ones such as cyst perforation. Children, who have acute problems related to liver and pancreas, should be initially received appropriate medical treatment to remove inflammation and associated obstruction at the pancreaticobiliary system before surgery [110].
In the first half of this century, treatment methods such as cyst aspiration, marsupialization, and external drainage (cholecystostomy, tube drainage) had been used more extensively in the surgical treatment of CCs. And as expected, high mortality and morbidity rates had been detected in patient’s follow-up period. Surgical methods such as partial cyst excision and cystoduodenostomy were defined between 1920 and 1930 [111, 112]. Especially, cystoduodenostomy has been the preferred method by many surgeons until the early 1970s [113, 114] when the long-term morbidity was detected as higher (30–50%) [7]. Therefore, in those years, Roux-en-Y cystojejunostomy had identified with the idea of preventing the reflux of the duodenal contents into the bile ducts [7, 30, 115]. Indeed, the cholangitis had been significantly reduced with this method, but not completely eliminated [28]. After soon, it had been demonstrated that anastomosis with a large stoma, as possible as (at least 4 cm) is more important for protecting reflux-related cholangitis attacks than which intestinal segment it is performed (duodenum or jejunum) [116]. The recognition of the cancer development from the left cystic wall and Babbitt’s APBDU theory made the idea of cyst excision popular soon after. It had been reported that carcinoma develops after the internal drainage procedures at a frequency of 2.5–17.5% several years later as a consequence of chronic inflammation of the cyst wall. Therefore, cyst excision had gain popularity in a very short time [116].
Contemporarily, the definitive treatment for CCs are total excision that has become preferred management strategy over the internal drainage procedures (choledochocystoduodenostomy or choledochocystojejunostomy), which have an only historical value today despite they had been used as a treatment method in the past although caused high morbidity (probably because of not relieving biliary stasis sufficiently) [117, 118]. Furthermore, only complete resection can fully decrease the risk of malignant degeneration: a critical point in the pediatric population with a large number of expectant life years. The general aim is to remove the cyst completely and restore biliary enteric drainage either into the duodenum by hepaticoduodenostomy (HD) or jejunum by Roux-en-Y hepaticojejunostomy (RYHJ), although specific approaches for types vary minimally. Surgical intervention should be elective and patients should be medically optimized prior to operative intervention. İf patient has a cholangitis or pancreatitis attack preoperatively, the infection should be adequately treated with broad-spectrum intravenous antibiotics or biliary decompression if needed [118].
Surgery for CC disease can be performed open or laparoscopically based on patient characteristics and surgeon preference.
HD and RYHJ are the two most commonly utilized techniques of reconstruction [119], although other replacement conduits such as jejunal interposition HD, valved jejunal interposition HD, nonrefluxing biliary appendicoduodenostomy, hepaticoenterostomy, and wide hilar hepaticojejunostomy have been reported [120, 121, 122, 123, 124, 125]. HD has been favored by some groups [126, 127] but most series suggests significantly more bile reflux compared with RYHJ [121], which is currently the most commonly utilized reconstruction.
In all cases, cholangiography should be performed initially to obtain detailed anatomical information about the intra- and extrahepatic bile ducts, irrespective of preoperative examinations. Dissection of extrahepatic bile ducts starts from the gallbladder. The terminal end of the cyst opening to the duodenum should be isolated, clamped, cut, and transfixed, firstly. Some surgeons suggest that dissection should be continued until the appearance of pancreatic ducts, while the others not suggest. Additionally, some surgeons taking into account that dissection toward the lower end of the cyst may cause inevitable unplanned pancreatic duct injury that pancreaticoduodenectomy requirement should be in your mind, although very rarely [75]. After the distal portion of the cyst is ligated and cut, the posterior wall is dissected from the surface of the portal vein. In cases of marked inflammation, the cyst may be excised by leaving the posterior wall on the portal vein. The dissection should go on till the hepatic hilus. The best strategy to obtain a wide anastomosis stoma is to make a hepatic dissection more proximally until the left hepatic duct is seen. Although all parts of CCs need to be removed, sometimes residual proximal cyst walls can be left to facilitate biliary anastomosis [75]. Dilated bile ducts should be irrigated with heparinized saline to clear the gallstones before anastomosis. After the cyst is excised, one of the hepaticoenterostomy methods, such as hepaticojejunostomy, HD, jejunal interposition HD, valved jejunal interposition HD, nonrefluxing biliary appendicoduodenostomy, hepaticoenterostomy, and wide hilar hepaticojejunostomy [120, 121, 122, 123, 124, 125] is performed for biliary reconstruction. In RYHJ, 40-cm jejunal loop replaces to the hepatic hilus. In RYHJ surgery, to avoid the elongation of a blind pouch as the child grows, an end-to-end anastomosis of the jejunum to the CBD is recommended if technically possible [128]. If an end-to-side anastomosis is required (in some cases, the bile duct is too small), it should be as close as possible to the closed end of the jejunal limb. Additionally, although it is not possible to predetermine the length of the Roux limb, it should be appropriate to the child’s overall bowel length considering future growth. In HD, anastomosis is performed between the duodenum second part and the bile duct. The duodenum was mobilized to a limit. The duodenum is anchored to the liver at porta to avoid tension on the anastomosis (Figure 5).
Intraoperative pictures and drawn cartoon showing of the procedure.
The intraabdominal drain, kept in Morison’s pouch, may be removed on the seventh postoperative day [124].
Laparoscopic treatment of choledochal cysts was first described in 1995 [129] and demonstrated that it could be performed in children as young as 3 months [130] and as small as 6 kg [131]. As with most surgical diseases, longer operating time and shorter hospital stay [132] were comparable with open surgical approaches, and in the absence of cholangitis or pancreatitis, it becomes more suitable treatment [132]. While four or five ports are typically used in the traditional laparoscopic approach [130, 133], the use of single-port laparoscopy [134] and robotic surgical system [135] has also been reported. In a prospective randomized study of 121 children undergoing laparoscopic cyst excision with RYHJ, routine postoperative drainage has been shown to be unnecessary [136].
The most commonly performed operations for biliary reconstruction after complete surgical resection of CCs are RYHJ or HD [124]. There is a debate regarding the optimal technique for biliary reconstruction [124]. RYHJ is considered as an ideal technique for the repair of CC, but HD has gained wide acceptance and favored by many surgeons open as well as laparoscopically because of its advantages over hepaticojejunostomy. HD is more physiologic, but theoretically, the closeness of hepaticoenterostomy to stomach makes HD to have greater chance of cholangitis and bile gastritis, but in a meta-analyzed study [137], it has been shown that while the incidence of bile gastritis after HD is even higher when examined endoscopically, interestingly, there is no difference of cholangitis between HD and RYHJ. Additionally, HD is simpler to perform and associated with fewer complications such as adhesive bowel obstruction, anastomotic leakage, and peptic ulcer as compared to RYHJ [138]. HD requires less operative time, allows faster recovery of bowel function, and produces fewer complications requiring reoperation [139]. If there is an anastomotic stricture following HD, it can be easily managed by endoscopy as against hepaticojejunostomy [124]. But, when the diameter of the common hepatic duct more than 10 mm that lets duodenal contents more likely to reflux easily into the intrahepatic bile ducts through the HD anastomosis or when the intrahepatic biliary dilatation is present that lets refluxed duodenal contents remain longer in the intrahepatic bile ducts, HD is not recommended because of higher risk of cholangitis or anastomotic stricture formation [124]. Some studies have demonstrated high incidence of secondary bile reflux proven by endoscopy after HD [121]. Recently, a patient with hilary bile duct carcinoma, who was performed HD for the biliary reconstruction at the age of 13 months, has been reported in the 19 years follow-up after the primary cyst excision. Reflux of duodenal contents (including activated pancreatic enzymes) into the intrahepatic bile ducts through the HD anastomosis is thought to be hazardous to the bile duct mucosa in this patient [124]. Adhesive bowel obstruction is seen with a higher incidence in RYHJ that comprises a Roux-en-Y jejuna limb and two anastomoses, compared with HD. Cholangitis, peptic ulcer, fat malabsorption, diarrhea, and malnutrition are the other complications [126]. A significant incidence of long-term complications requiring reoperation such as anastomosis stenosis has been observed with the follow-up studies of patients who underwent hepaticojejunostomy after cyst excision [138, 140], and a wide hilar hepaticojejunostomy extending into the left hepatic duct is advocated for the way to prevent it [125].
Treatment of type I CC includes excision of the extrahepatic biliary tract, cholecystectomy, and reconstruction of the biliary system. If the duct is dilated at the distal margin, the mucosa may be left behind to prevent damage to the pancreaticobiliary system and can be striped. Infrequently, because of recurrent episodes of the cholangitis, the cyst wall may densely adherent to the portal vein, precluding safe resection [141]. In such cases, resection of the anterior wall with careful fulguration of the mucosa of the posterior wall can be performed [141]. Hepatic bifurcation is carefully evaluated for stricture and inflammation before performing anastomosis during proximal transection. If one of them is seen, more proximal transection should be considered [18].
For type II CCs, mostly, diverticulectomy or simple cyst excision is enough for the treatment. Primary or over a T-tube closure can be performed, and reconstruction is occasionally required if there is significant luminal narrowing [18].
One of the methods such as endoscopic sphincterotomy, sphincteroplasty, sphincteroplasty with cyst excision, or pancreaticoduodenectomy may be used to manage pediatric patients with type III CCs (choledochoceles) [10, 25]. Various reports denote adequate symptom control with this approach [142, 143]; however, long-term follow-up is lacking. Cysts not amenable to endoscopic intervention may benefit from lateral duodenotomy with sphincteroplasty and unroofing or marsupialization of the cavity [18].
Type IV CC is approached differently based on the presence or absence and location of intrahepatic disease [15]. Type IVb cysts are treated in the same fashion as type I. Management for IVa disease differs due to the presence of intra- as well as extrahepatic involvement, as well as the presence of functional liver disease. Of foremost importance is the characterization of actual type IVa as opposed to type I with upstream ductal dilatation due to stasis and functional obstruction [50]. If the dilatation is anatomic and isolated (limited; i.e., left hemiliver), partial hepatectomy with reconstruction to the remaining hepatic ducts may be warranted due to the ongoing risk of malignant transformation in the intrahepatic biliary system [85]. However, not all patients are appropriate candidates for partial hepatectomy [144]. Those patients with obvious dilatations and stenosis of intrahepatic ducts, intrahepatic duct stones, or parenchymal atrophy may benefit from hepatectomy [144]. If hepatectomy is planned concomitantly with extrahepatic duct excision, the distribution should allow removal of all disease (of the vast majority of the severe disease) with adequate future liver remnant [15]. If the pattern is more diffuse or imaging is inconclusive, treatment in a type I paradigm with close postoperative surveillance to follow intrahepatic ducts has been utilized [145]. This approach is justified by studies demonstrating that patients who progress to malignancy most commonly develop extrahepatic cholangiocarcinoma or gallbladder cancer (approximately 85% of malignancy), whereas intrahepatic cholangiocarcinoma rarely occurs [144]. It is reported that the intrahepatic component has actually resolved in 3–6 months with adequate drainage [146]. To differentiate type IVa from type I, while in adults, preoperative percutaneous biliary drainage to decompress the intrahepatic biliary ductal system has been advocated [147], in children, this practice has not been reported probability due to the difficulty in maintaining the external tube. Although long-term results are not known, intrahepatic cystojejunostomy, in addition to hepaticojejunostomy, has been described as a way of preventing liver resection in type IVa cysts [148]. Complete extrahepatic excision with hepaticoenterostomy and drainage of the remaining cyst externally or internally should adequately ameliorate biliary stasis in the presence of bilobar unresectable intrahepatic cyst [18].
Management of patients with Caroli’s disease can be particularly difficult given the location of the cysts and frequent necessity for surgery (considerable potential for cholangitis, liver complications, and biliary cirrhosis; moderate potential for neoplasia (7%)) [15]. In Caroli disease, intrahepatic cysts can be seen as limited disease restricted to a single segment/lobe or diffuse disease involving the entire intrahepatic biliary tree. If the patient has not developed cirrhosis and portal hypertension, the unilobar cystic disease should be treated with anatomic hepatectomy and biliary enteric bypass. However, bilobar disease should be treated with symptom-directed nonoperative treatment methods as including litholytic agents such as ursodiol, antibiotics, and percutaneous drainage if possible. Close follow-up is required for malignant transformation. Although there is no identification for prophylactically orthotopic liver transplantation in the treatment of the disease, it should be kept in mind for the choice of the treatment in patients who have diffuse symptomatic disease with cirrhosis or portal hypertension [149].
Patients should be monitored every 6 months during the postoperative 3 years and then annually. On initial follow-up, while all patients should be evaluated with complete blood count, liver function tests and abdominal US, on subsequent follow-up, investigations are done only in symptomatic ones. Long-term follow-up can be made by visits, telephonic conversations, and postal inquiry [124].
Resection of pediatric CC is generally well tolerated [18]. Despite recent advances in surgical techniques and perioperative management, short- and long-term complications are not rare in children, while they are more common in adults [6]. Complications such as recurrent cholangitis attacks, malignant transformation, intracystic or intrahepatic gallstone formation, cirrhosis development, and pancreatitis are common in patients who are not operated on. Complications such as anastomotic leakage, gastrointestinal or intraabdominal bleeding, acute pancreatitis, pancreatic leakage, wound infection, wound dehiscence, intraabdominal infection/abscess, intussusception can be seen in early postoperative period defined as short-term complications [150]. Most early complications can be treated conservatively [151].
However, most series are without early mortality and report rates of acute complications including wound infections from 0 to 17%, without significant difference between infants and children [46, 132].
Surgical inexperience and severe inflammation are often implicated in the development of anastomotic bile leakage [151]. The diagnosis of bile leakage is difficult and delayed in some cases due to nonspecific symptoms [151]. It may not always be possible to differentiate with the imaging findings of US and CT because not all of the intraabdominal fluid collection after surgery is associated with bile leakage, and so, this late diagnosis may result in mortality due to septicemia and septic shock [151]. However, it is reported that MRCP can be used to diagnose and accurately localize the site of bile leakage noninvasively [7, 151]. Bile leaks in the hepaticooenterostomy line can self-limited within a few weeks if they can be drained externally. If the bowel movement is sufficient during this period, the child can be fed by enterally. If the extracted bile is given back to the stomach with NG catheter, electrolyte losses can also be prevented. The bilirubin level of the child may remain high due to edema in the anastomosis line within the first 2 weeks, even if the operation has been successfully performed. If this takes longer, biliary tree, even anastomosis, can be evaluated with PTC [110]. Reoperation is considered only after the failure of conservative treatment [151]. The leakage can be repaired by a circumferential buried suture around the anastomotic site, peritoneal lavage, and effective drainage [151].
Gastrointestinal bleeding may be due to hepaticojejunostomy or stress ulcer.
Acute pancreatitis occurs in patients with CC, both preoperatively and due to injury of the pancreatic tissue during distal dissection of the cyst or to edema in the distal part of the pancreatic duct related postoperatively (4.2%) [151]. Therefore, it has been recovered with a conservative treatment for a short time period. Some reports say that CC excision without ligation of the distal stenotic stump decreases the incidence of pancreatic duct injury [75]. A probe inserted into the pancreatic duct through a duodenotomy may help to prevent pancreatic duct injury in difficult cases [75]. Additionally, Urushihara et al. [152] consider that using bipolar electro cautery to scrape pancreatic tissue away from the bile duct wall during the dissection of the intrapancreatic part of the bile duct causes minimal bleeding and enables clear identification of the narrow part of the CBD [151]. Eventually, complete resection of the distal portion of the cyst, removal of debris and protein plugs in the long common channel and pancreatic duct, and correction of anomalous arrangement of the pancreaticobiliary duct junction are essential to minimize pancreatic complications after the operations [151]. The pancreatic fistula occurs because of not closing the distal choledoch well after the cyst excision or injured pancreatic duct during the dissection. An external drainage of 3–4 weeks allows the fistulas closing.
Late/long-term complications (5–15%) [151] include anastomotic stricture, cholangitis, hepatolithiasis, ileus, cirrhosis, and malignancy. Benign anastomotic stricture with recurrent cholangitis is less common than in adults but is still seen in many as 10–25% of patients and can be associated with both intrahepatic and bile duct stone formation [117].
After intraabdominal surgery, small bowel obstructions, mostly due to adhesions, are common [154]. Patients should be closely monitored for any possible clinical deterioration [154]. If there is no improvement after 48 h of follow-up, there is a high risk of bowel resection due to bowel necrosis despite it has not been clearly defined [156]. Furthermore, it has been defined that during biliary reconstruction, the length and placement of the Roux loop is very important in adhesive bowel obstruction developing postoperatively [156].
In terms of anastomotic stricture, improvement of surgical skills, preservation of blood supply, no or mild inflammation cyst wall, and construction of wide (larger than 1 cm) and tension-free stoma are key factors to reduce anastomosis-related complication [138]. There should be no delay in surgical or endoscopic intervention once biliary obstruction develops postoperatively, but a great deal of planning and a thoughtful workup are required [151]. Kim et al. [155] reported that PTC with stone removal and balloon dilatation was useful in patients with anastomotic stricture. However, some investigators consider that recurrent anastomotic strictures may occur due to fibrosis even after balloon dilatation with PTCS, and repeated cholangitis may cause multiple intrahepatic biliary strictures, recurrent hepatic stones, and development of biliary carcinoma [152, 153]. Hence, especially in young patients, it is recommended that revision of the hepaticojejunostomy followed by ductoplasty, to create a wide stoma for sufficient bile drainage [152, 153].
Rigorous long-term follow-up after pediatric CC resection is limited, but the risk of biliary carcinoma (cholangiocarcinoma, squamous cell carcinoma, sarcoma, gallbladder cancer [12, 157], most often cholangiocarcinoma) clearly remains elevated even after CC excision compared to the general population [18]. The malignancy risk is considered to increase with age at surgery, and the cumulative biliary malignancy risk 25 years after primary surgery has been reported to be as high as 11% [75]. Malignant disease has been noted in up to 14% 0f patients after CC resection as a child [46]. In fact, cancer is the most frequent cause of late mortality in pediatric CC series [18]. Even after complete excision, patients are at higher risk for malignancy than general population [158]. Continued surveillance is, therefore, strongly recommended, though it is not known whether there are risk factors such as retained portion of cyst or not [18]. In those with known malignancy, oncologic principles should apply; patients who can undergo safe resection with negative margins are appropriate for operation [15]. Resection may include hepatectomy with regional lymphadenectomy, extirpation of extrahepatic bile ducts with regional lymphadenectomy (and cholecystectomy), or pancreaticoduodenectomy [15]. However, the 5-year survival rate for patients with CCs complicated by malignancy is high, up to 55% in patients with cholangiocarcinoma [159].
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