High temperatures change the perspective: Integrating hormonal responses in citrus plants under co‐occurring abiotic stress conditions

Plants growing in the field are subjected to multiple stress factors acting simultaneously. Abnormally high temperatures are expected to affect wild plants and crops in the next years due to global warming. In this work, we have studied physiological, hormonal and molecular responses of the citrus rootstock, Carrizo citrange (Poncirus trifoliata L. Raf. × Citrus sinensis L. Osb.) subjected to wounding or high salinity occurring individually or in combination with heat stress. According to our results, combination of high salinity and heat stress aggravated the negative effects of salt intoxication in Carrizo. The high transpiration rate caused by high temperatures counteracted physiological responses of plants to salt stress and increased Cl^? intake in leaves. In addition, 12‐oxo‐phytodienoic acid accumulated specifically under combination of wounding and heat stress, whereas at low temperatures, wounded plants accumulated jasmonic acid (JA) and JA‐isoleucine (JA‐Ile). Moreover, an antagonism between salicylic acid (SA) and JA was observed, and wounded plants subjected to high temperatures did not accumulate JA nor JA‐Ile whereas SA levels increased (via isochorismate synthase biosynthetic pathway). Wounded plants did not accumulate abscisic acid (ABA) but its catabolite phaseic acid. This could act as a signal for the upregulation of (ABA)‐RESPONSIVE ELEMENT (ABRE)‐BINDING TRANSCRIPTION FACTOR 2 (CsAREB2) and RESPONSIVE TO DISSECATION 22 (CsRD22) in an ABA‐independent way. This work uncovers some mechanisms that explain Carrizo citrange tolerance to high temperatures together with different hormonal signals in response to specific stresses. It is suggested that co‐occurring abiotic stress conditions can modify (either enhance or reduce) the hormonal response to modulate specific responses.     

Transmissible salt tolerance traits identified through reciprocal grafts between sensitive Carrizo and tolerant Cleopatra citrus genotypes

In this work, reciprocal grafts between the chloride-tolerant Cleopatra mandarin (Citrus reshni Hort. ex Tan.) and the chloride-sensitive Carrizo citrange (Citrus sinensis [L.] Osb. × Poncirus trifoliata [L.] Raf.) rootstocks were grown under saline conditions to identify major transmissible salt tolerance traits in citrus. The data indicate that lower chloride levels in leaves, attenuated shoot growth and smaller vessel size in xylem were the most important transmissible salt tolerance traits. Other tolerance attributes such as larger leaf area and lower transpiration rates were non-transmissible charac teristics. Leaf cation levels and gas interchange parameters were unrelated to salt tolerance. In com parison with sensitive Carrizo, tolerant Cleopatra plants showed reduced capabilities for water uptake as well as lower leaf Cl-concentrations. Carrizo on Cleopatra grafts also possessed these two attributes although they were slightly less tolerant than Cleopatra plants, which had higher shoot to root ratios than the grafted plants. Cleopatra on Carrizo plants showed high sensitivity to salt because they had higher ability for water uptake and accumulated higher Cl-concentrations in leaves.     

Growth and root hydraulic conductivity of several citrus rootstocks under salt and polyethylene glycol stresses

The water relations responses to salt of several important citrus rootstocks such as Swingle citrumelo, sour orange, and Milam lemon have not been studied in detail before. Studies were set up to compare growth and root hydraulic properties of these rootstocks to other citrus rootstocks by exposing them to NaCl and polyethylene glycol (PEG) stresses. Seedlings of 7 citrus rootstocks were irrigated for 5 months with nutrient solutions containing NaCl or PEG that had been adjusted to osmotic potentials of -0.10, -0.20 or -0.35 MPa. The 7 rootstocks studied were sour orange (Citrus aurantium), Cleopatra mandarin (Citrus reticulata Blanco), Swingle citrumelo (C. paradisi x P. trifoliata), Carrizo citrange (C. sinensis x P. trifoliata), rough lemon (Citrus jambhiri Lush), Milam lemon (C. jambhiri hybrid), and trifoliate orange (Poncirus trifoliata [L.] Raf.). In both shoot and root growth, Cleopatra mandarin and sour orange were the least sensitive to salt, Milam and trifoliate orange were the most sensitive, and rough lemon, Swingle, and Carrizo were intermediate in sensitivity. Even though the roots were exposed to solutions of equal osmotic potentials, plant growth and root conductivity were reduced more by the PEG treatments than the corresponding NaCl treatments. At -0.10 and -0.20 MPa, shoot and root dry weights were reduced 16 to 55% by NaCl and 24 to 68% by PEG. Shoot root ratio was lowered at the higher concentrations, particularly by PEG. There was a major decrease in root conductivity caused by NaCl at -0.10 MPa (19 to 30% in sour orange and Cleopatra mandarin and 78 to 85% in trifoliate orange and Milam). Conductivity decreased more at -0.20 and -0.35 MPa, but not proportionally as much as at -0.10 MPa. Root weight per unit length increased at the higher salt levels, particularly in trifoliate orange. Water flow rate through root systems followed the same trend as root conductivity; salt affected sour orange and Cleopatra mandarin the least and trifoliate orange and Milam the most. However, reductions in fibrous root length by salt treatment differed. Root lengths of Swingle and Carrizo were least affected by salt while sour orange. Milam, and rough lemon were the most affected. Hence, even though sour orange and Cleopatra mandarin were more tolerant than the other rootstocks in terms of water flow rate or root conductivity, these 2 rootstocks showed a proportionally greater decrease in root length than Carrizo, Swingle, or trifoliate orange.     

Morphological factors determining salt tolerance in citrus seedlings: the shoot to root ratio modulates passive root uptake of chloride ions and their accumulation in leaves

The results presented in this work were obtained with two citrus genotypes, the chloride-tolerant Cleopatra mandarin (Citrus reshni Hort. ex Tan.) and the chloride-sensitive Carrizo citrange [Citrus sinensis (L.) Osb. × Poncirus trifoliata (L.) Raf.]. The data show that chloride uptake under salinization is driven by passive forces. In both species, net rates of chloride root uptake increased linearly, without saturation, with the increase of external NaCl concentrations (30–240 mol m^–3). Uptake rates, on a μ g g root dry weight^–1 h^–1 basis, in Cleopatra and Carrizo decreased (from 38 to 21) and increased (from 21 to 35), respectively, with the increase (about three-fold) of the shoot to root ratio. With the appropriate shoot to root ratio in each genotype, it was demonstrated that at identical external doses of NaCl, Cl^– uptake rates and Cl^– xylem concentrations in the two species were very similar. Root pruning and defoliation showed that the amount of chloride taken by the plant was a function of the size of the root system, whereas leaf chloride concentration, the parameter responsible for salt damage, was dependent upon leaf biomass. Measurements of water transpiration suggested that chloride root uptake and leaf accumulation might be linked to water absorption and transpiration rates, respectively. The data indicate that plant morphology is a crucial factor determining salt-tolerance in citrus.     

Restoration of vigor and rooting competence in stem tissues of mature citrus by repeated grafting of their shoot apices onto freshly germinated seedlings in vitro

Summary Repeated grafting of 0.2-cm shoot tips from fruiting-age trees ofCitrus reticulata Blanco ‘Ponkan’ mandarin andC. sinensis Osbeck ‘Liu Tseng’ sweet orange onto freshly germinated ‘Troyer’ citrange [Poncirus trifoliata (L.) Raf. X.C. sinensis Osbeck] seedlings in vitro resulted in progressive restoration of rooting competence and vigor of regenerated roots and shoots. The restored traits were retained through the course of the investigation and suggested a phase reversal phenomenon.     

Variation in Radopholus citrophilus Population Densities in the Citrus Rootstock Carrizo Citrange

Seedlings of the hybrid citrus rootstock, Carrizo citrange (Citrus sinensis × Poncirus trifoliata) do not uniformly limit development of the citrus burrowing nematode, Radopholus citrophilus. Variation in nematode population densities in roots of seedlings germinating from the same seed suggests that factors responsible for nematode incompatibility are not functional or are not inherited uniformly among progeny. Seeds which produced a single seedling were more likely to produce plants which suppressed citrus burrowing nematode population increase than were seeds which produced two or three seedlings.     

NaCl effects on 4-desmethylsterol composition of plasma-membrane-enriched preparations from citrus roots

Abstract The free 4-desmethylsterol composition of plasma-membrane-enriched preparations from white fibrous roots of Rangpur lime (Citrus reticulata var. austera hybrid?), Kharna khatta (C. kharna Raf.) and Etrog citron (C. medica L.) seedlings grown in the presence of 0, 50, or 100 mol m^?3 NaCl for 28 d was quantitated by gas chromatography (GC) on analytical capillary (SE-54 fused silica) columns and the sterols were identified by combined gas chromatography-mass spectrometry (GC-MS). Only three 4-desmethylsterols were positively identified by GC-MS, viz. campesterol, stigmasterol and sitosterol. Cholesterol could not be positively identified in any of the membrane preparations. Campesterol levels were generally similar for all treatments and for all three genotypes, approximating 30% of the total free 4-desmethylsterol content of the plasma membranes. At all levels of salinity (0, 50 or 100 mol m^?3 NaCl) sitosterol levels decreased in the order Rangpur lime > Kharna khatta > Etrog citron and stigmasterol levels decreased in the reverse order. The ratio of sitosterol to stigmasterol was highest in Rangpur lime and lowest in Etrog citron at each level of salinity and was reduced by salt treatment in all three genotypes. Salt-induced reductions in the ratio of ‘more planar’ to ‘less planar’ sterols correlated inversely with the accumulation of Cl^? in the leaves of the three genotypes suggesting a role for plasma membrane sterols in the Cl^? exclusion mechanism. A model relating sterol structure, membrane sterol composition and membrane permeability to Cl^? exclusion ability in citrus is presented.     

Influence of Chloride and Transpiration on Net15NO3-Uptake Rate byCitrus Roots

Three-month-old Carrizo citrange (hybrid of Citrus sinensisL. OsbeckxPoncirus trifoliata Blanco) seedlings were grown incontrolled environment chambers in pots of fine sand. Plantswere irrigated with either non-saline or saline solutions overa 3-week period. After these treatments, plants were transferredto vessels containing a 5 m M¹⁵NO₃K (96% atom excess¹⁵N) solution,and transpiration as well as concentration of¹⁵N and Cl^-in roots,stem and leaves were measured after 24 h. Transpiration and¹⁵NO₃^-uptakerates were inhibited after exposure to NaCl and the concentrationof salt pre-treatment determined the intensity of this inhibitoryeffect. To determine the effect of transpiration on NO₃^-absorption,net¹⁵NO₃^-uptake rate was measured in salt stressed and non-stressedplants exposed to different light intensities or relative humiditiesand also in detached roots. Reduction in NO₃^-uptake was moreclosely related to Cl^-antagonism from salt stress than to reducedtranspiration rate. Copyright 1999 Annals of Botany Company Nitrate, absorption, inhibition transport system, salt, light and humidity.     

Fertile fruit trees obtained by somatic hybridization: navel orange (Citrus sinensis) and Troyer citrange (C. sinensis x Poncirus trifoliata)

Summary Nucellar cell suspension protoplasts of navel orange (Citrus sinsensis Osb.) were chemically fused with mesophyll protoplasts of Troyer citrange (C. sinensis x Poncirus trifoliata) and cultured in hormone-free Murashige and Tucker medium containing 0.6 M sucrose. Two types of plant were regenerated through embryogenesis. One type showed intermediate mono-and difoliate leaves and the other types was identical to Troyer citrange. The regenerated plants with intermediate morphology were demonstrated by chromosome counts and rDNA analysis to be amphidiploid somatic hybrids. Five clones of these somatic hybrids were grafted in the field. After 4 years, they set flowers having a morphology intermediate between those of the two parents. The pollen grains showed high stainability and sufficient germinability, and were larger than those of Troyer citrange. The fruits of the somatic hybrids were large and spherical with thick rinds. Most of them contained seeds with normal germinability. These results indicate that somatic hybridization is a useful tool for Citrus breeding.     

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.     

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.     

Genetic transformation of lime (Citrus aurantifolia Swing.): factors affecting transformation and regeneration

We have previously developed procedures for the efficient production of sweet orange (Citrus sinensis L. Osbeck) and Carrizo citrange (C. sinensis L. Osbeck×Poncirus trifoliata L. Raf.) transgenic plants using an Agrobacterium tumefaciens-mediated transformation and shoot tip grafting in vitro regeneration system. We now report on the optimization of the cocultivation, regeneration and selection conditions for efficient and reliable production of transgenic lime (C. aurantifolia Swing.) plants. Improved transformation frequencies were obtained by cocultivating the explants with Agrobacterium on feeder plates. Optimum regeneration of transgenic shoots was obtained by exposing the explants to darkness for 2 weeks and by using kanamycin at 100 mg/l as selective agent. Attempts to use geneticin as selection antibiotic were not successful. Shoot tip grafting of regenerated shoots on Troyer citrange seedlings resulted in 100% successful production of transgenic plants. The presence and expression of the transferred genes in the regenerated plants was verified by β-glucuronidase histochemical and fluorimetric assays, neomycin phosphotransferase ELISA assays, PCR and Southern analyses. Received: 10 December 1996 / Revision received: 10 February 1997 / Accepted: 25 February 1997     

De novo arginine biosynthesis in leaves of phosphorus-deficient citrus and poncirus species

Young, fully expanded leaves from 7-month-old P-deficient citrus rootstock seedlings had levels of nonprotein arginine that were 10- to 50-fold greater than those from P-sufficient control plants. Arginine content of the protein fraction increased 2- to 4-fold in P-deficient leaves. Total arginine content, which averaged 72 ± 6 micromoles per gram dry weight of P-sufficient leaf tissue (mean ± se, n = the four rootstocks) was 207, 308, 241, and 178 micromoles in P-deficient leaves from Citrus limon cv rough lemon, Poncirus trifoliata × C. sinensis cv Carrizo citrange and cv Troyer citrange, and P. trifoliata cv Australian trifoliate orange, respectively. For each rootstock, the accumulation of arginine paralleled an increase in the activity of the pathway for the de novo biosynthesis of arginine. The ratio of the nanomoles NaH¹⁴CO₃ incorporated into the combined pool of arginine plus urea per gram fresh weight intact leaf tissue during a 3-hour labeling period for P-deficient to P-sufficient plants was 91:34, 49:11, 35:11, and 52:41, respectively. When P-deficient plants were supplied with P, incorporation of NaH¹⁴CO₃ into arginine plus urea was reduced to the level observed for the P-sufficient control plants of the same age and arginine ceased to accumulate. Arginase and arginine decarboxylase activity were either unaffected or slightly increased during phosphorus deficiency. Taken together, these results provide strong evidence that arginine accumulation during phosphorus deficiency is due to increased activity of the de novo arginine biosynthetic pathway.     

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.     

Bifunctional selection–reporter systems for genetic transformation of citrus: mannose- and kanamycin-based systems

Agrobacterium-mediated transformation of Carrizo citrange [Citrus sinensis (L.) Osbeck × Poncirus trifoliata (L.) Raf.] with a binary vector containing a novel bifunctional reporter–selection fusion gene comprising an in-frame fusion between the manA gene and egfp gene is detailed. This system combined the phosphomannose isomerase positive selection system with the ability to monitor gene expression in a non-destructive manner using EGFP. Transgenic plants stably expressing the EGFP protein were regenerated following Agrobacterium-mediated transformation using a vector containing this fusion gene. We also obtained comparable transformation efficiencies when Carrizo explants were transformed using another Agrobacterium strain containing a binary vector with a bifunctional egfp–nptII fusion gene. Regenerating shoots were selected on medium containing 15 g L⁻¹ mannose supplemented with 5 g L⁻¹ sucrose for the manA-based selection or on medium containing 100 mg L⁻¹ kanamycin for the nptII-based selection. Our results indicated that the mannose-based antibiotic-free selection combined with visual identification of transgenic shoots using EGFP allows for early elimination of escape non-transgenic shoots and can provide a viable alternative to antibiotic-based selection systems in the genetic transformation of citrus and other crops.     

Inheritance of citrus nematode resistance and its linkage with molecular markers

Eleven RAPD markers linked to a gene region conferring resistance to citrus nematodes in an intergen-eric backcross family were identified. Two sequence- characterized amplified region markers linked to a citrus tristeza virus resistance gene and one selected resistance gene candidate marker were evaluated for their association with citrus nematode resistance. A nematode-susceptible citrus hybrid, LB6-2 [Clementine mandarin (Citrus reticulata)×Hamlin orange (C. sinensis)], was crossed with the citrus nematode-resistant hybrid Swingle citrumelo (C. paradisi×Poncirus trifoliata) to produce 62 hybrids that were reproduced by rooted cuttings. The plants were grown in a greenhouse and inoculated with nematodes isolated from infected field trees. The hybrids segregated widely for this trait in a continuous distribution, suggesting possible polygenic control of the resistance. Bulked segregant analysis was used to identify markers associated with resistance by bulking DNA samples from individuals at the phenotypic distribution extremes. Linkage relationships were established by the inheritance of the markers in the entire population. A single major gene region that contributes to nematode resistance was identified. The resistance was inherited in this backcross family from the grandparent Poncirus trifoliata as a single dominant gene. QTL analysis revealed that 53.6% of the phenotypic variance was explained by this major gene region. The existence of other resistance-associated loci was suggested by the continuous phenotypic distribution and the fact that some moderately susceptible hybrids possessed the resistance-linked markers. The markers may be useful in citrus rootstock breeding programs if it can be demonstrated that they are valid in other genetic backgrounds. Received: 4 May 1999 / Accepted: 21 September 1999     

Tetraploid citrus rootstocks are more tolerant to salt stress than diploid

Citrus trees are subject to several abiotic constraints such as salinity. Providing new rootstocks more tolerant is thus a requirement. In this article, we investigated salt stress tolerance of three tetraploid rootstock genotypes when compared to their respective diploid rootstocks (Poncirus trifoliata, Carrizo citrange, Cleopatra mandarin). Plant growth, leaf fall and ion contents were investigated. At the end of the experiment, leaf fall was observed only for diploid Poncirus trifoliata plants as well as chlorosis symptoms for Poncirus trifoliata and Carrizo citrange diploid plants. The diploid Cleopatra mandarin plants growth rate was not affected by salt stress and has even been increased for tetraploid Cleopatra mandarin. Ion contents investigation has shown lower accumulations of chloride ions in leaves of the tetraploid plants when compared to diploid plants. Our results suggest that citrus tetraploid rootstocks are more tolerant to salt stress than their corresponding diploid. To cite this article: B. Saleh et al., C. R. Biologies 331 (2008).     

Cl‐ homeostasis in includer and excluder citrus rootstocks: transport mechanisms and identification of candidate genes

To reveal specific Cl^‐ transport activities in the symplastic pathway, uptake, long‐distance transport and distribution of Cl^‐ have been investigated in the citrus rootstocks Carrizo citrange (CC, Cl^‐ includer) and Cleopatra mandarin (CM, Cl^‐ excluder). Using an external concentration of 4.5 mm Cl^‐, both species actively transported Cl^‐ to levels that exceeded the critical requirement concentration by one and two orders of magnitude in the excluder and the includer rootstocks, respectively. Both CC and CM modulated Cl^‐ influx according to the availability of the nutrient as uptake capacity was induced by Cl^‐ starvation, but inhibited after Cl^‐ resupply. Net Cl^‐ uptake was higher in the includer CC, an observation that correlated with a lower root‐to‐shoot transport capacity in the excluder CM. The patterns of tissue Cl^‐ accumulation indicated that chloride exclusion in the salt‐tolerant rootstock CM was caused by a reduced net Cl^‐ loading into the root xylem. Genes CcCCC1, CcSLAH1 and CcICln1 putatively involved in the regulation of chloride transport were isolated and their expression analysed in response to both changes in the nutritional status of Cl^‐ and salt stress. The previously uncharacterized ICln gene exhibited a strong repression to Cl^‐ application in the excluder rootstock, suggesting a role in regulating Cl^‐ homeostasis in plants.     

Somatic hybrid plants obtained by protoplast fusion between Citrus sinensis and Poncirus trifoliata

Summary Somatic hybrid plants of Rutaceae were obtained by protoplast fusion between Citrus sinensis Osb. (Trovita orange) and Poncirus trifoliata. Protoplasts isolated from embryogenic cells of C. sinensis and from leaves of P. trifoliata, and the culture of fusion products in the presence of high concentrations of sucrose were essential requirements for the selection of hybrids. Green globular embryoids derived from protoplasts resulted in the regeneration of trifoliate plants. Other morphological characters of these plants were intermediate between both parents. The chromosome number in one of the hybrid plants was 36, which was the sum of C. sinensis (2n=18) and P. trifoliata (2n=18). EcoRI restriction analysis of rDNA confirmed the presence of parental nuclear DNAs in the hybrid.     

Effects of mixtures of citrus viroids as transmissible small nuclear RNA on tree dwarfing and commercial scion performance on Carrizo citrange rootstock

The term ‘transmissible small nuclear ribonucleic acids' (TsnRNAs) describes well characterised viroid RNA species that do not induce any disease syndromes in specific citrus hosts but rather act as regulatory genetic elements modifying tree performance. Twelve-year-old navel orange and 10-year-old Clementine mandarin trees on Carrizo citrange (Citrus sinensis×Poncirus trifoliata) rootstock treated with a mixture of three TsnRNAs (−Ia, syn. Citrus bent leaf viroid, +IIa, syn. Hop stunt viroid and +IIIb, syn. Citrus dwarfing viroid) were reduced in size by 33% and 43%, respectively. Clementine trees treated with a mixture of TsnRNA−Ia+IIa or −Ia+IIIb also had reduced canopy volume (CV) (∼38 and 31%, respectively), whereas trees treated with TsnRNA−IIa+IIIb showed little effect. The effects of the double TsnRNA treatments −Ia+IIa and −Ia+IIIb on Clementine canopy size and commercial performance were comparable and in some cases superior to that of the triple TsnRNA mixture. The TsnRNA−Ia+IIa treatment had the most attractive commercial traits with increased production of Clementine fruit per CV (23.6%), more fruit with high commercial value (31.7%), and more fruit optimally distributed in the canopy (68% of fruit between 0.5 and 2.5 m). None of the TsnRNA treatments affected the growth of Carrizo rootstock seedlings after 8 years in the field. Navel orange and Clementine scions treated with the same triple TsnRNA mixture expressed different trunk and fruit production patterns although effects on CV were similar.