1. Introduction
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.
2. Citrus transformation: generalities
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
2.1. Protoplast transformation
Although,
2.2. Particle bombardment
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
2.3. Agrobacterium -mediated transformation
This system uses the ability of the
Generally, transformation efficiencies obtained by using
2.3.1. Species or cultivar specificity
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
2.3.2. Age and type of explant used
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).
2.3.3. Competence of the citrus cells or tissues
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).
2.3.4. Agrobacterium strains used and inoculation procedure
A study involving the use of three different strains of
The inoculation of the citrus explants with the
The optimal
2.3.5. Co-cultivation and pre-culturing conditions
Co-cultivation involves incubating both the explants and
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
2.3.6. Adequate selection conditions
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
2.3.7. Recovery of transgenic shoots
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
3. Genetic engineering and disease control in citrus
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.
3.1. Pathogen-derived genes
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
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
Resistance to another important viral disease,
Pathogen-derived genes have also been used to control bacterial diseases. Citrus canker, caused by
In another strategy, also to control citrus canker, a
Fungal pathogens also affect citrus production. In particular,
3.2. Plant defense genes
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
A number of studies have demonstrated that the over-expression of the
Zhang et al. (2010) reported transforming the
3.3. Additional strategies
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.
4. Emerging technologies
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
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
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,
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.
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
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
4.1. Transformation vs. transient expression
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.
5. Conclusions: The future of citrus transformation
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
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
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.
Acknowledgments
Research in our laboratory is funded in part by the Citrus Research & Development Foundation of Florida and a USDA Special Grant.
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