Selectable marker-free transgenic orange plants recovered under non-selective conditions and through PCR analysis of all regenerants

Selectable marker (SM) genes have been considered necessary to achieve acceptable rates in the generation of transgenic plants. Genes encoding antibiotic or herbicide resistance are widely used for this purpose. In most cases, once transgenic plants have been regenerated, permanence of SM genes in the plant genome is no longer necessary, and it becomes a matter of public concern. Moreover, the removal of SM genes from transgenic plants could facilitate gene stacking through successive transformations, particularly when the availability of these markers is rather limited for most crop plants. In the genus Citrus, with highly heterozygotic species of long generation cycles, methods implying the segregation and removal of marker transgenes in the progeny are not feasible. Here, we have evaluated the direct production of SM-free citrus plants under non-selective conditions, using a “clean” binary vector carrying only the transgene of interest, and through the recovery of transformants by polymerase chain reaction (PCR) analysis of all regenerated shoots. The response of two different citrus genotypes, Carrizo citrange (intergeneric hybrid of C. sinensis L. Osb. X Poncirus trifoliata L. Raf.) and Pineapple sweet orange (C. sinensis L. Osb.), was evaluated. Our results indicate that, in this system, the competence between transgenic and non-transgenic cells is the main factor determining final transgenic regeneration frequencies. For Carrizo citrange, no transgenic plant could be recovered. For Pineapple sweet orange, marker-free transformation efficiency was 1.7%, paving the way for the viable production of orange transformants carrying only the transgene(s) of interest.     

Evaluation of selection strategies alternative to <Emphasis Type="Italic">nptII</Emphasis> in genetic transformation of citrus

The neomycin phosphotransferase (nptII) selection system has proved successful in citrus transformation; however, it may be recommendable to replace it given the pressure exerted against antibiotic-resistance selectable marker genes in transgenic plants. The present work investigates three different selection alternatives, comparing them to nptII selection in two citrus genotypes, Carrizo citrange and Pineapple sweet orange. The first method used the beta-glucuronidase (uidA) reporter marker gene for selection; the second attempted to generate marker-free plants by transforming explants with a multi-auto-transformation (MAT) vector, combining an inducible R/RS-specific recombination system with transgenic-shoot selection through expression of isopentenyl transferase (ipt) and indoleacetamide hydrolase/tryptophan monooxygenase (iaaM/H) marker genes; while the third exploited the phosphomannose isomerase (PMI)/mannose conditional positive selection system. Firstly, GUS screening of all regenerated shoots in kanamycin-free medium gave 4.3% transformation efficiency for both genotypes. Secondly, workable transformation efficiencies were also achieved with the MAT system, 7.2% for citrange and 6.7% for sweet orange. This system affords an additional advantage as it enables selectable marker genes to be used during the in vitro culture phase and later removed from the transgenic plants by inducible recombination and site-specific excision. Thirdly, the highest transformation rates were obtained with the PMI/mannose system, 30% for citrange and 13% for sweet orange, which indicates that this marker is also an excellent candidate for citrus transformation.     

Efficient production of transgenic citrus plants using isopentenyl transferase positive selection and removal of the marker gene by site-specific recombination

The presence of marker genes conferring antibiotic resistance in transgenic plants represents a serious obstacle for their public acceptance and future commercialization. In citrus, selection using the selectable marker gene nptII, that confers resistance to the antibiotic kanamycin, is in general very effective. An attractive alternative is offered by the MAT system (Multi-Auto-Transformation), which combines the ipt gene for positive selection with the recombinase system R/RS for removal of marker genes from transgenic cells after transformation. Transformation with a MAT vector has been attempted in two citrus genotypes, Pineapple sweet orange (Citrus sinensis L. Osb.) and Carrizo citrange (C. sinensis L. Osb. × Poncirus trifoliata L. Raf.). Results indicated that the IPT phenotype was clearly distinguishable in sweet orange but not in citrange, and that excision was not always efficient and precise. Nevertheless, the easy visual detection of the IPT phenotype combined with the higher transformation efficiency achieved in sweet orange using this system open interesting perspectives for the generation of marker-free transgenic citrus plants.     

A Dark Incubation Period Is Important for Agrobacterium-Mediated Transformation of Mature Internode Explants of Sweet Orange,Grapefruit, Citron,and a Citrange Rootstock

Background

Citrus has an extended juvenile phase and trees can take 2–20 years to transition to the adult reproductive phase and produce fruit. For citrus variety development this substantially prolongs the time before adult traits, such as fruit yield and quality, can be evaluated. Methods to transform tissue from mature citrus trees would shorten the evaluation period via the direct production of adult phase transgenic citrus trees.

Methodology/Principal Findings

Factors important for promoting shoot regeneration from internode explants from adult phase citrus trees were identified and included a dark incubation period and the use of the cytokinin zeatin riboside. Transgenic trees were produced from four citrus types including sweet orange, citron, grapefruit, and a trifoliate hybrid using the identified factors and factor settings.

Significance

The critical importance of a dark incubation period for shoot regeneration was established. These results confirm previous reports on the feasibility of transforming mature tissue from sweet orange and are the first to document the transformation of mature tissue from grapefruit, citron, and a trifoliate hybrid.     

Identification of closely related citrus cultivars with inter-simple sequence repeat markers

 Inter-simple sequence repeat (ISSR) markers generated by 22 primers were tested for their ability to distinguish among samples from 94 trees of 68 citrus cultivars. Within each of the six cultivar groups studied, most of these cultivars are so closely related that they are difficult to distinguish by other molecular-marker techniques. ISSR markers involve PCR amplification of DNA using a single primer composed of a microsatellite sequence anchored at the 3′ or 5′ end by 2–4 arbitrary, often degenerate, nucleotides. The amplification products were separated on non-denaturing polyacrylamide gels and detected by silver staining. ISSR banding profiles were very repeatable on duplicate samples. Different citrus species had very different fingerprint patterns. Within Citrus sinensis (L.) Osbeck and C. paradisi Macf., in which all cultivars have originated by the selection of mutants, ISSR markers distinguished 14 of 33 sweet orange and 1 of 7 grapefruit cultivars. Five of six lemon cultivars were discriminated by ISSR markers. Many differences were found among mandarin cultivars; however, all five satsuma cultivars analyzed had identical ISSR fingerprints. Four of five citrange cultivars were distinguishable, but ‘Troyer’ and ‘Carrizo’ had identical ISSR fingerprints. ‘Kuharske Carrizo’ citrange, which has better citrus nematode resistance than other ‘Carrizo’ citrange accessions, had unique ISSR fingerprints. Three ISSR markers that differentiated certain sweet orange cultivars were hybridized to Southern blots of sweet orange DNA digested with different restriction endonucleases. The sweet orange cultivars tested could be distinguished by these ISSR-derived RFLP markers. Moreover, one ISSR marker unique to ‘Ruby’ blood orange was observed in its progeny trees. Received: 9 September 1996 / Accepted: 4 April 1997     

Citrus Leprosis and its Status in Florida and Texas: Past and Present

According to published reports from 1906 to 1968, leprosis nearly destroyed the Florida citrus industry prior to 1925. This was supported with photographs showing typical leprosis symptoms on citrus leaves, fruit, and twigs. Support for the past occurrence of citrus leprosis in Florida includes: (1) presence of twig lesions in affected orange blocks in addition to lesions on fruits and leaves and corresponding absence of similar lesions on grapefruit; (2) yield reduction and die-back on infected trees; and (3) spread of the disease between 1906 and 1925. Transmission electron microscopy (TEM) examination of tissue samples from leprosis-like injuries to orange and grapefruit leaves from Florida in 1997, and fruits from grapefruit and sweet orange varieties from Texas in 1999 and 2000 did not contain leprosis-like viral particles or viroplasm inclusions. In contrast, leprosis viroplasm inclusions were readily identified by TEM within green non-senescent tissues surrounding leprosis lesions in two of every three orange leaf samples and half of the fruit samples obtained from Piracicaba, Brazil. Symptoms of leprosis were not seen in any of the 24,555 orange trees examined across Florida during 2001 and 2002. The authors conclude that citrus leprosis no longer exists in Florida nor occurs in Texas citrus based on: (1) lack of leprosis symptoms on leaves, fruit, and twigs of sweet orange citrus varieties surveyed in Florida: (2) failure to find virus particles or viroplasm inclusion bodies in suspect samples from both Florida and Texas examined by TEM; (3) absence of documented reports by others on the presence of characteristic leprosis symptoms in Florida; (4) lack of its documented occurrence in dooryard trees or abandoned or minimal pesticide citrus orchard sites in Florida. In view of the serious threat to citrus in the U.S., every effort must be taken to quarantine the importation of both citrus and woody ornamental plants that serve as hosts for Brevipalpus phoenicis (Geijskes), B. californicus (Banks), and B. obovatus Donnadieu (Acari: Tenuipalpidae) from countries where citrus leprosis occurs.     

Susceptibility and Resistance of Citrus Genotypes to Alternaria alternata pv. citri

A survey of citrus cultivars in Israel in orchards where Alternaria brown spot was common on Minneola tangelos (mandarin × grapefruit), revealed the occurrence of the disease as typical foliar and fruit lesions on Dancy and Ellendale (mandarins), on Murcott tangor (mandarin × sweet orange), on Nova and Idith (mandarin hybrids), on Calamondin, and on Sunrise and Redblush (grapefruit). Isolates of Alternaria alternata from each of these hosts were proven to be pathogenic to Minneola tangelo.
The host range of A. alternata pv. citri from Israel was assayed by inoculating leaves of diverse citrus genotypes. Several mandarins and their hybrids (Dancy, Kara, King, Wilking, Satsuma, Minneola, Orlando, Mikhal, Idith, Nova, Page, Murcott), grapefruit (Marsh seedless), grapefruit × pummelo (Oroblanco), sweet orange (Shamouti, Valencia, Washington navel) Calamondin, and Volkamer citrus were susceptible. Several mandarins and their hybrids (Clementine, Avana, Yafit, Ortanique), Cleopatra, one sweet orange cultivar (Newhall), pummelo (Chandler), lemon (Eureka), Rough lemon, Rangpur lime, sweet lime, citron, limequat, sour orange, Troyer citrange and Alemow were resistant.     

Ecological impacts of trees with modified lignin

Few experiments have yet been performed to explore the potential ecological impacts of genetic modification in long-lifespan species such as trees. In this paper, we review the available data on GM trees with modified lignin focussing on the results of the first long-term field trials of such trees. These trials evaluated poplars expressing antisense transgenes to reduce the expression of the lignin biosynthesis genes cinnamyl alcohol dehydrogenase (CAD) or caffeic acid/5-hydroxyferulic acid O-methyltransferase (COMT) with the aim of producing trees with improved pulping characteristics. The trees were grown for 4 years at two sites in France and England, and their ecological impacts and agronomic performance were assessed. Modifications to lignin in the poplars were maintained over the 4 years of the trial. The trees remained healthy throughout and growth was normal. The lignin modifications had no unexpected biological or ecological impacts. Interactions with leaf-feeding insects, microbial pathogens and soil organisms were unaltered although the short-term decomposition of transgenic roots was slightly enhanced. Investigation of the ecological impacts of the GM trees was curtailed by the early termination of the field trial when it was attacked and largely destroyed by anti-GM protestors. To supplement our work on the decomposition of GM plant materials with modified lignin, we have therefore turned to the study of transgenic tobacco lines where we can perform more comprehensive and controlled analyses of the biological and ecological effects of lignin-gene suppression.     

Transgene stability and dispersal in forest trees

Transgenics from several forest tree species, carrying a number of commercially important recombinant genes, have been produced, and are undergoing confined field trials in a number of countries. However, there are questions and issues regarding stability of transgene expression and transgene dispersal that need to be addressed in long-lived forest trees. Variation in transgene expression is not uncommon in the primary transformants in plants, and is undesirable as it requires screening a large number of transformants in order to select transgenic lines with acceptable levels of transgene expression. Therefore, the current focus of plant transformation is toward fine tuning of transgene expression and stability in the transgenic forest trees. Although a number of studies have reported a relatively stable transgene expression for several target traits, including herbicide resistance, insect resistance, and lignin modification, there was also some unintended transgene instability in the genetically modified (GM) forest trees. Transgene dispersal from GM trees to feral forest populations and their containment remain important biological and regulatory issues facing commercial release of GM trees. Containment of transgenes must be in place to effectively prevent escape of transgenic pollen, seed, and vegetative propagules in economically important GM forest trees before their commercialization. Therefore, it is important to devise innovative technologies in genetic engineering that lead to genetically stable transgenic trees not only for qualitative traits (herbicide resistance, insect resistance), but also for quantitative traits (accelerated growth, increased height, increased wood density), and also prevent escape of transgenes in the forest trees.     

重庆三峡库区柑橘硼营养状况及其影响因子

为了解重庆三峡库区柑橘叶片硼营养状况及其影响因子,在该区域12个主产县(区)的代表性果园采集叶片样品954份和土壤样品302份,测定叶片硼含量,并分析了土壤有效硼、土壤pH值、品种、砧木和树龄对叶片硼营养的影响.结果表明: 该区域柑橘叶片硼含量不足(<35 mg·kg^-1)的果园比例达41.6%,土壤有效硼含量不足(<0.5 mg·kg^-1)的果园比例高达89.4%,柑橘叶片硼含量与土壤有效硼含量的相关性未达显著水平.土壤pH值、品种、砧木和树龄均影响叶片硼含量.pH值4.5～6.4土壤上的柑橘叶片硼含量显著高于pH值6.5～8.5土壤上的柑橘;品种间叶片硼含量为:温州蜜柑>柚类>夏橙>普通甜橙>杂柑>脐橙;枳砧和酸柚砧柑橘叶片硼含量显著高于枳橙砧和红橘砧柑橘;3～8年生柑橘树叶片硼含量适宜(35～100 mg·kg^-1)样品比例比8年生以上柑橘树高6.6%.     

Pollen competition as a reproductive isolation barrier represses transgene flow between compatible and co-flowering citrus genotypes

Background/Objective

Despite potential benefits granted by genetically modified (GM) fruit trees, their release and commercialization raises concerns about their potential environmental impact, and the transfer via pollen of transgenes to cross-compatible cultivars is deemed to be the greatest source for environmental exposure. Information compiled from field trials on GM trees is essential to propose measures to minimize the transgene dispersal. We have conducted a field trial of seven consecutive years to investigate the maximum frequency of pollen-mediated crop-to-crop transgene flow in a citrus orchard, and its relation to the genetic, phenological and environmental factors involved.

Methodology/Principal Findings

Three different citrus genotypes carrying the uidA (GUS) tracer marker gene (pollen donors) and a non-GM self-incompatible contiguous citrus genotype (recipient) were used in conditions allowing natural entomophilous pollination to occur. The examination of 603 to 2990 seeds per year showed unexpectedly low frequencies (0.17–2.86%) of transgene flow. Paternity analyses of the progeny of subsets of recipient plants using 10 microsatellite (SSR) loci demonstrated a higher mating competence of trees from another non-GM pollen source population that greatly limited the mating chance of the contiguous cross-compatible and flowering-synchronized transgenic pollen source. This mating superiority could be explained by a much higher pollen competition capacity of the non-GM genotypes, as was confirmed through mixed-hand pollinations.

Conclusions/Significance

Pollen competition strongly contributed to transgene confinement. Based on this finding, suitable isolation measures are proposed for the first time to prevent transgene outflow between contiguous plantings of citrus types that may be extendible to other entomophilous transgenic fruit tree species.     

Essential oils of citrus fruit stimulate oviposition in the Mediterranean fruit fly Ceratitis capitata (Diptera: Tephritidae)

Female Mediterranean fruit flies (medfly) Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) perceive both qualitative and quantitative aspects of citrus fruit chemistry. However, the behavioural and biological adjustments of this response remain largely unknown. In the present study, the ovipositional responses of gravid female medflies to essential oils (i.e. the most critical resistance factor to medfly infestation) of various citrus fruits are investigated. In dual‐choice (treatment versus distilled water control) experiments, females lay significantly more eggs into pre‐punctured hollow oviposition hemispheres (domes) provided with 1 µL of citrus peel oil from sweet orange, satsuma mandarin, bitter orange, grapefruit and lemon compared with odourless domes. No‐choice tests show a weak effect of lemon essential oils in stimulating oviposition. The female ovipositional response to sweet orange oil (the most active in eliciting oviposition) is dose‐dependent. Additionally, limonene, the most abundant chemical in all citrus oils, stimulates oviposition, whereas linalool, a representative compound of immature citrus fruit associated with high toxicity against immature stages of fruit flies, has a significant deterrent effect. In further no‐choice tests, females lay approximately 23% fewer eggs in limonene (93%) (amount found in orange oil) and 60% fewer eggs in limonene 93% plus linalool 3% (approximately 10‐fold the amount found in orange oil) mixtures, relative to sweet orange oil. The results suggest that the limonene content accounts largely (but not completely) for the ovipositional responses observed in sweet orange oil, whereas high linalool proportions are capable of significantly masking and/or disrupting its stimulatory effects in citrus oils. The importance and practical implications of these findings with respect to understanding how citrus fruit chemistry influences the ovipositional responses of medfly is discussed.     

Engineering of gibberellin levels in citrus by sense and antisense overexpression of a GA 20-oxidase gene modifies plant architecture

Carrizo citrange (Citrus sinensisxPoncirus trifoliata) is a citrus hybrid widely used as a rootstock, whose genetic manipulation to improve different growth characteristics is of high agronomic interest. In this work, transgenic Carrizo citrange plants have been produced overexpressing sense and antisense CcGA20ox1 (a key enzyme of GA biosynthesis) under control of the 35S promoter to modify plant architecture. As expected, taller (sense) and shorter (antisense) phenotypes correlated with higher and lower levels, respectively, of active GA1 in growing shoots. In contrast, other phenotypic characteristics seemed to be specific to citrus, or different from those described for similar transgenics in other species. For instance, thorns, typical organs of citrus at juvenile stages, were much longer in sense and shorter in antisense plants, and xylem tissue was reduced in leaf and internode of sense plants. Antisense plants presented a bushy phenotype, suggesting a possible effect of GAs on auxin biosynthesis and/or transport. The main foliole of sense plants was longer, although total leaf area was reduced. Leaf thickness was smaller in sense and larger in antisense plants due to changes in the spongy parenchyma. Internode cell length was not altered in transgenic plants, indicating that, in citrus, GAs regulate cell division rather than cell elongation. Interestingly, the phenotypes described were not apparent when transgenic plants were grafted on non-transgenic rootstock. This suggests that roots contribute to the GA economy of aerial parts in citrus and opens the possibility of using the antisense plants as dwarfing rootstocks.     

Improved Transformation Efficiency in Citrus by Plasmolysis Treatment

Procedures for high efficiency production of transgenic citrus plants using an Agrobacterium tumefaciens system with plasmolysis treatment were developed. Longitudinally cut epicotyl segments of eight different citrus species [’Milam’ Rough lemon (Citrus jambhiri Lush), ‘Volkamer’ lemon (Citrus volkameriana L), Rangpur lime (Citrus limonia L), ‘Hamlin’ sweet orange (Citrus sinensis L Osbeck), ‘Duncan’ grapefruit (’Citrus paradisi’ Macf), Sour orange (Citrus aurantium L), ‘Cleopatra’ mandarin (Citrus reticulata Blanco) and Carrizo citrange (Citrus sinensis L Osbeck x Poncirus trifoliata L Raf) ] were plasmolyzed in different concentrations of sucrose and maltose [0, 3, 6, 8, 9, 10, 12 % (w/v) ] prior to Agrobacterium inoculation. Plasmolyzed epicotyl explants were cocultivated with either the hypervirulent Agrobacterium tumefaciens strain, the EHA-101 (harboring a binary vector pGA482GG) or Agl-1 (carrying pCAMBIA1303 vector). Both binary vectors contained neomycin phosphotransferase II (NPT II) and β-glucuronidase (GUS) genes. The binary vector, pCAMBIA1303 also contained a fused mGFP5 gene at the 3’ end of GUS gene as a reporter. Epicotyl explants of Rangpur lime, Rough and ‘Volkamer’ lemons plasmolyzed in 9–12 % maltose showed transient GUS gene expression comprising up to 95 % of the cut surface of explants, while Carrizo citrange showed 80 % expression when they were plasmolyzed in 6–10 % sucrose. On the other hand, epicotyl explants of ‘Hamlin’ sweet orange, Grapefruit, Sour orange and ‘Cleopatra’ mandarin showed transient GUS expession in 80–90 % of explants with 6–10 % sucrose. Basal portions of the regenerated putative transgenic shoots harvested from the cut surface of epicotyl explants within 2–3 months, were assayed for GUS, and apical portions were shoot-tip grafted in vivo for the production of whole plants. The transformation efficiencies in different species obtained are the highest so far reported for citrus.     

Influence of the Sting Nematode, Belonolaimus longicaudalus,on Young Citrus Trees

The sting nematode, Belonolaimus longicaudatus, was associated with poor growth of citrus in a central Florida nursery. Foliage of trees was sparse and chlorotic. Affected rootstocks included Changsha and Cleopatra mandarin orange; Flying Dragon, Rubidoux, and Jacobsen trifoliate orange; Macrophylla and Milam lemon; Palestine sweet lime; sour orange; and the hybrids - Carrizo, Morton, and Rusk citrange and Swingle citrumelo. Root symptoms included apical swelling, development of swollen terminals containing 3-5 apical meristems and hyperplastic tissue, coarse roots, and a reduction in the number of fibrous roots. Population densities as high as 392 sting nematodes per liter soil were detected, with 80% of the population occurring in the top 30 cm of soil; however, nematodes were detected to 107 cm deep. Although an ectoparasite, the nematode was closely associated with citrus root systems and was transported with bare root nursery stock. Disinfestation was accomplished by hot water treatment (49 C for 5 minutes).     

Evidence for host plant preference by Iphiseiodes quadripilis (Acari: Phytoseiidae) on Citrus

In this study, we present field and laboratory evidence on the preference of Iphiseiodes quadripilis (Banks) for grapefruit (Citrus paradisi Macfadyen) leaves compared with sweet orange (Citrus sinensis (L.) Osbeck) leaves. This preference was confirmed in four orchards whether leaf samples were taken from either border trees of contiguous grapefruit or sweet orange or interior row trees with both citrus species in adjacent rows. Iphiseiodes quadripilis was most abundant in grapefruit trees in spite of the greater abundance of the Texas citrus mite, Eutetranychus banksi (McGregor) (Acari: Tetranychidae) in sweet orange trees. Similar preference responses were observed in laboratory tests using a Y-tube olfactometer whether I. quadripilis were collected from sweet orange or grapefruit. Iphiseiodes quadripilis collected from grapefruit trees showed significant preference for grapefruit over sweet orange leaves in contact choice tests using an arena of alternating leaf strips (12 mm long × 2 mm wide) of sweet orange and grapefruit. However, I.␣quadripilis collected from sweet orange trees did not show preference for either grapefruit or sweet orange leaves. Based on these results, grapefruit leaves foster some unknown factor or factors that retain I. quadripilis in greater numbers compared with sweet orange leaves.