Citrus somatic hybridization with potential for improved blight and CTV resistance

Summary The production of five new somatic hybrids with potential for improved disease resistance is reported herein. Protoplast isolation, fusion, and plant regeneration was achieved from Caipira sweet orange (Citrus sinensis L. Osbeck) as an embryogenic parental source and Volkamer lemon (C. volkameriana Pasquale), Cleopatra mandarin (C. reticulata Blanco), and Rough lemon (C. jambhiri Lushington) as non-embryogenic parental sources. Fusion involving Cleopatra mandarin and Rangpur lime (C. limonia L. Osbeck) as embryogenic parental sources with Sour orange (C. aurantium L.) also resulted in somatic hybrid plants. Somatic hybridization was confirmed by leaf morphology evaluation, chromosome counting, and randomly amplified polymorphic DNA (RAPD) analyses. Somatic hybrids may combine complementary characteristics from both parental sources and have potential for tolerance to blight and citrus tristeza virus (CTV).     

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.     

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.     

Analysis of volatile compounds released from embryogenic cultures and somatic embryos of sweet oranges by head space SPME

Volatile compounds released from callus and nucellar embryo tissues of ‘Valencia late’ and ‘Washington Navel’ sweet oranges (Citrus sinensis, L. Osbeck) were collected/concentrated by head space solid phase micro extraction and analysed by gas chromatography-mass spectrometry. Friable, white embryogenic cultures released a number of volatile compounds, including some essential oils. Different samples of the same embryogenic culture showed variability, possibly related to the presence of tissues undergoing differentiation. Analyses of the somatic embryos permitted the identification of several components, including limonene and methyl anthranilate. Considering the simplicity and the very small sample required (0.3 g of fresh tissue) head space solid phase micro extraction is suitable for studies and comparisons of volatile metabolites released from in vitro Citrus tissue cultures suggesting its potential in Citrus biochemical, genetic and breeding research. This revised version was published online in June 2006 with corrections to the Cover Date.     

Contrasts between Citrus species in response to salinisation: An analysis of photosynthesis and water relations for different rootstock-scion combinations

Leaf gas exchange, water relations and ion content were measured on two-year-old Valencia orange (Citrus sinensis [L.] Osbeck), Washington Navel orange (C. sinensis) and Marsh grapefruit (C. parodisi Macfad) scions budded to either Trifoliata (Poncirus infoliata [L] Raf) or Cleopatra mandarin (C. reticuLua Blanco) rootstoeks. Trees were watered with dülute nutrient solution containing either 0 or 50 _mM NaCl for 77 days. Leaf chloride concentrations (cell sap basis) were higher in all scions budded on “Trifoliata but sodium levels were lower than in equivalent foliage budded on Cleopatra mandarin rootstock. Foliar salt levels also varied according to scion. Leaves of Marsh grapefruit had higher levels of both sodium and chloride than leaves of either Valencia orange or Washington Navel orange on both rootstocks. Accumulation of sodium and chloride in salinised leaves caused a reduction in leaf osmotic potential of 0.2–1.4 MPa. and leaf water potential declined by as much as 0.5 MPa. Turgor pressure in salinised leaves was thus maintained at or above the control level. Osmotic potentials determined by psychrometry compared with pressure-volume curves were taken to imply that some accumulation of sodium or chloride in the apoplast of salinised leaves may have occurred. Despite turgor maintenance both _co2 assimilation and stomatal conductance were reduced by salinity. Following onset of leaf response to salinisation, gas exchange was impaired to a greater extent in scions budded to Cleopatra mandarin compared to those on Trifoliata. Amongst those scions. leaves of salt-treated Marsh grapefruit showed greater reductions in gas exchange than Valencia orange or Washington Navel orange budded on either rootstock. Increased sensitivity of 1Marsh grapefruit was correlated with a higher foliar sodium and chloride content in this scion. Scion differences in sensitivity of leaf gas exchange to solute concentration were independent of rootstock and appeared unrelated to leaf prolinebetaine concentrations. This implies an inherent difference between scion species with respect to salt tolerance, rather than variation in their capacity to acquire that type of compatible solute. In terms of rootstock effects, all scions proved more sensitive to salinity when budded to Cleopatra mandarin compared with Trifoliata. That response was attributed to a disproportionately higher concentration of leaf sodium in scions on Cleopatra mandarin.     

Somatic hybrid plants from sexually incompatible woody species: Citrus reticulata and Citropsis gilletiana

Summary Allotetraploid intergeneric somatic hybrid plants between Citrus reticulata Blanco cv. Cleopatra mandarin and Citropsis gilletiana Swing. & M. Kell. (common name Gillet's cherry orange) were regenerated following protoplast fusion. Cleopatra protoplasts were isolated from an ovule-derived embryogenic suspension culture and fused chemically with leaf-derived protoplasts of Citropsis gilletiana. Cleopatra mandarin and somatic hybrid plants were regenerated via somatic embryogenesis. Hybrid plant identification was based on differential leaf morphology, root-tip cell chromosome number, and electrophoretic analyses of phosphoglucose mutase (PGM) and phosphohexose isomerase (PHI) isozyme banding patterns. This is the first somatic hybrid within the Rutaceae reported that does not have Citrus sinensis (sweet orange) as a parent, and the first produced with a commercially important citrus rootstock and a complementary but sexually incompatible, related species.Abbreviations PGM phosphoglucose mutase - PHI phosphohexose isomerase - MES 2[N-morpholino] ethane sulfonic acid - BH3 protoplast culture medium (Grosser and Chandler, 1987) - PEG polyethylene glycol - MT Murashige and Tucker (1969) basal medium - NAA 1-naphthaleneacetic acid - GA3 gibberellic acid - H+H and EME citrus embryogenic cell culture media (Grosser and Gmitter, 1990b) - B embryo germination medium - RMAN rooting medium Florida Agricultural Experiment Station Journal Series No. R-00298.     

Etiology of three recent diseases of citrus in São Paulo State: sudden death, variegated chlorosis and huanglongbing

The state of S?o Paulo (SSP) is the first sweet orange growing region in the world. Yet, the SSP citrus industry has been, and still is, under constant attack from various diseases. In the 1940s, tristeza-quick decline (T-QD) was responsible for the death of 9 million trees in SSP. The causal agent was a new virus, citrus tristeza virus (CTV). The virus was efficiently spread by aphid vectors, and killed most of the trees grafted on sour orange rootstock. Control of the disease resided in replacing sour orange by alternative rootstocks giving tolerant combinations with scions such as sweet orange. Because of its drought resistance, Rangpur lime became the favourite alternative rootstock, and, by 1995, 85% of the SSP sweet orange trees were grafted on this rootstock. Therefore, when in 1999, many trees grafted on Rangpur lime started to decline and suddenly died, the spectre of T-QD seemed to hang over SSP again. By 2003, the total number of dead or affected trees was estimated to be over one million. The new disease, citrus sudden death (CSD), resembles T-QD in several aspects. The two diseases have almost the same symptoms, they spread in time and space in a manner strikingly similar, and the pathological anatomy of the bark at the bud union is alike. Transmission of the CSD agent by graft-inoculation has been obtained with budwood inoculum taken not only on CSD-affected trees (grafted on Rangpur lime), but also on symptomless trees (grafted on Cleopatra mandarin) from the same citrus block. This result shows that symptomless trees on Cleopatra mandarin are tolerant to the CSD agent. Trees on rootstocks such as Sunki mandarin or Swingle citrumelo are also tolerant. Thus, in the CSD-affected region, control consists in replacing Rangpur lime with compatible rootstocks, or in approach-grafting compatible rootstock seedlings to the scions of trees on Rangpur lime (inarching). More than 5 million trees have been inarched in this way. A new disease of sweet orange, citrus variegated chlorosis (CVC), was observed in 1987 in the Triangulo Mineiro of Minas Gerais State and the northern and north-eastern parts of SSP. By 2000, the disease affected already 34% of the 200 million sweet orange trees in SSP. By 2005, the percentage had increased to 43%, and CVC was present in all citrus growing regions of Brazil. Electron microscopy showed that xylem-limited bacteria were present in all symptomatic sweet orange leaves and fruit tissues tested, but not in similar materials from healthy, symptomless trees. Bacteria were consistently cultured from twigs of CVC-affected sweet orange trees but not from twigs of healthy trees. Serological analyses showed the CVC bacterium to be a strain of Xylella fastidiosa. The disease could be reproduced and Koch's postulates fulfilled, by mechanically inoculating a pure culture of X. fastidiosa isolate 8.1.b into sweet orange seedlings. The genome of a CVC strain of X. fastidiosa was sequenced in SSP in the frame of a project supported by FAPESP and Fundecitrus. X. fastidiosa is the first plant pathogenic bacterium, the genome of which has been sequenced. Until recently, America was free of huanglongbing (HLB), but in March 2004 and August 2005, symptoms of the disease were recognized, respectively in the State of S?o Paulo (SSP) and in Florida, USA. HLB was known in China since 1870 and in South Africa since 1928. Because of its destructiveness and its rapid spread by efficient psyllid insect-vectors, HLB is probably the most serious citrus disease. HLB is caused by a phloem sieve tube-restricted Gram negative bacterium, not yet available in culture. In the 1990s, the bacterium was characterized by molecular techniques as a member of the alpha proteobacteria designated Candidatus Liberibacter africanus for the disease in Africa, and Candidatus Liberibacter asiaticus for HLB in Asia. In SSP, Ca. L. asiaticus is also present, but most of the trees are infected with a new species, Candidatus Liberibacter americanus.     

Doubled haploid callus lines of Valencia sweet orange recovered from anther culture

Homozygous genotypes are valuable for genetic and genomic studies in higher plants. However, obtaining homozygous perennial plants using conventional breeding techniques is currently a challenge because of a long juvenile period, high heterozygosity and the substantial inbreeding depression. In vitro androgenesis has been used to develop haploid and doubled haploid plants. In this study, we report the regeneration of doubled haploid lines of Valencia sweet orange cv. Rohde Red (Citrus sinensis [L.] Osbeck) via anther culture. Anthers at the uninucleate stage were induced and two embryogenic calli were obtained that further regenerated to embryoids (2/400). Plantlets were obtained after transferring the embryoids to a shoot regeneration medium, but were short-lived. Ploidy analysis via both flow cytometry and chromosome counting verified that these two lines were diploids. Additionally, 43 simple sequence repeat (SSR) markers which showed to be heterozygous in the Valencia sweet orange donor line confirmed homozygosity and doubled haploids in the anther-derived lines. Furthermore, analysis of the doubled haploids via cleaved amplified polymorphic sequence (CAPS) markers and target region sequencing confirmed the allelic state of two genes (LCYE and LCYB) involved in the carotenoid biosynthesis of sweet oranges.     

Somatic embryogenesis from ovaries of sweet orange cv. Tobias

Callogenesis, somatic embryogenesis, and regeneration were obtained from tissues of unfertilized ovaries of sweet orange (Citrus sinensis Osbeck.) cv. Tobias. The influence of two modified basal media, woody plant medium (WPM) and N6 medium, to induce callus formation from pistils was determined. Overall, high frequencies of callogenesis were observed when either medium was used. However, initial culture of explants in WPM medium followed by transfer of callus to N6 medium resulted in higher frequency of callus induction (of 2.30 callus per explant that were larger than 0.5 cm in size), and of subsequent development of embryogenic callus (10%). A total of 125 somatic embryos were obtained. After 6 months of culture, 72% of somatic embryos germinated into plantlets. These plantlets were subsequently micrografted in vitro, and then acclimatized. Ploidy of these plants were determined using flow cytometry and TRAPS molecular markers were used to confirm their maternal origin.     

Histological characterization of in vitro adventitious organogenesis in Citrus sinensis

The adventitious bud development was induced in epicotyl segments of Valencia sweet orange (Citrus sinensis L. Osbeck). Seeds were cultured in vitro for three weeks in the dark, followed by one week at a 16-h photoperiod. Epicotyl segments were cultured horizontally for the induction of organogenesis in Murashige and Tucker (1969, MT) culture medium supplemented with 1.0 mg dm⁻³ benzylaminopurine. Samples were observed by light and scanning electron microscopy from day zero to day 25, when buds were well grown. It was shown that the adventitious buds originated directly from the cambial region on the cut ends of the explants.     

Effects of medium composition and culture duration on in vitro morphogenesis of sweet potato

In vitro morphogenesis of sweet potato (Ipomoea batatas) shoot explants after cultures in callus initiation medium (CIM) with two sucrose contents and plant regeneration medium (PRM) with three growth regulator combinations for different durations was studied. After 4 weeks, explants on 5 % sucrose CIM had significantly more shoots but similar or lower root fresh mass and callus fresh mass than those on 3 % sucrose CIM subsequent to transfer for 6 weeks on all three PRM. Cultures transferred to growth regulator-free PRM after 4 and 12 weeks on 5 % sucrose CIM formed plants through organogenesis and embryogenesis, respectively. Embryogenic cultures from 4 weeks on CIM + 10 weeks on callus proliferation medium when transferred to PRM without growth regulator for 4 and 8 weeks produced multiple embryos in the prior and both embryos and shoot buds in the later.     

Regeneration of somatic embryos from sweet orange (C. sinensis) protoplasts using semi-permeable membranes

Sweet orange (C. sinensis L. Osbeck) protoplasts were isolated from nucellar-derived embryogenic callus, cultured in alginate beads for 5–30 days, and the resulting p-calli released by liquefaction and cultured on semi-permeable membranes overlaid on MT culture medium. Somatic embryos did not develop from 5- to 10-day-old p-calli but did develop from 15-, 20-, 25-, and 30-day-old p-calli. There were no significant differences in the numbers of embryos produced among the 15- to 30-day-old p-calli and no abnormal embryo morphologies were observed. The minimum size of p-calli to form embryos was 77.84 μm in diameter. Embryos were smaller from p-calli than those produced from embryogenic callus; p-calli-derived embryos ranged in size between 0.5 and 0.8 mm, while embryos derived from embryogenic callus ranged between 1 and 2 mm.     

Cryopreservation and storage of embryogenic callus cultures of several Citrus species and cultivars

Nucellus-derived embryogenic callus cultures of Salustiana sweet orange were subjected to cryoconservation assays. Cryoprotection with 10%(vol/vol) dimethylsulfoxide, freezing by slow cooling and thawing by fast warming was suitable to recover viable growing cultures and whole plants through embryogenesis. Evaluation of liquid phase R ₁ and solid phase R ₂ cooling rates using a programmable freezing unit indicated that 100% of embryogenic cultures survived when frozen using a range of cooling rates (R ₁ not above 0.5°C min^–1 and R ₂ not above 1°C min ¹) and thawed by fast warming. Storage up to 2 years in liquid nitrogen did not affect the growth of the cryopreserved cultures and the recovery of whole plants. Cultures of four cultivars of sweet orange (C. sinensis Osb.), three cultivars of grapefruit (C. paradisi Macf.), and one cultivar each of lemon [C. limon (L.) Burm. f.], Cleopatra mandarin (C. reshni Hort. ex Tan.), sour orange (C. aurantium L.) and Mexican lime [C. aurantifolia (Christm.) Swing.] have been successfully cryopreserved. Problems using a viability assessment using fluorescein diacetate staining are discussed. Received: 15 April 1996 / Revision received: 22 July 1996 / Accepted: 6 August 1996     

Normalizing sweet orange (C. sinensis (L.) osbeck) somatic embryogenesis with semi-permeable membranes

Summary Development of citrus somatic embryos initiated from embryogenic callus generally results in abnormal morphogenesis of somatic embryos. To normalize development, glycerol-induced globular-stage somatic embryos of sweet orange [C. sinensis (L.) Osbeck cv. ‘Hamlin’] were cultured on 6000–8000 MW cutoff cellulose acetate, >400 000 MW cutoff cellulose acetate, nitrocellulose, polyvinylidene fluoride (PVDF), cellulose filter paper, or positive or neutral charged nylon membranes. Only the two cellulose acetate membranes resulted in the development of normal, two-cotyledon, bipolar, heart-shaped embryos, and no aberrant teratoma-like structures. Heart-shaped embryos developed and germinated normally on Murashige and Tucker basal medium with 0.5% sucrose and 1 μM gibberellic acid. Culture of embryogenic callus directly onto cellulose membranes also resulted in the development of normal heart-shaped embryos, indicating that glycerol induction of globular-stage embryos is not necessary. Heart-shaped embryos were not observed when the osmotic potential of the medium was increased by the addition of 2.5–15% polyethylene glycol; neither were they observed when the matric potential of the medium was increased by increasing the gelling agent concentrations of agar and Gelrite from 0.8% to 3% and 0.15% to 0.9%, respectively.     

Tissue Culture of Maize I. Callus Induction and Growth

Callus induction and subculture was successful with mature embryos and stem sections of seedlings of Zea mays L. on Linsmaier and Skoog's medium modified to contain 4 mg/I of 2,4-D and 1 g/I of casamino acids. — 2,4-D was superior to NAA and IAA for both callus induction and growth. Callus subcultured on NAA formed abundant roots on agar-solidified media and numerous root-like primordia in liquid cultures. — Kinetin had no effect on callus induction in the presence of 2,4-D and neither kinetin nor gibberellic acid stimulated callus growth during subculture. — Callus grew equally well on the medium of Linsmaier and Skoog, that of Schenk and Hildebrandt, and the B-5 medium of Gamborg and Eveleigh containing 2% sucrose, 4 mg/I of 2,4-D and 1 g/I of casamino acids. — The callus grew more rapidly at 25°C than at 30°C or 35°C. Little difference was noted at any temperature in callus growth in alternating light (16 h) and dark (8 h) or continuous dark. — Sucrose was superior to glucose and maltose in both liquid and agar-solidified cultures. Lactose and galactose failed to support callus growth.     

Delay expression of limonoid UDP-glucosyltransferase makes delayed bitterness in citrus

Genes encoding limonoid UDP-glucosyltransferase from albedo of six Citrus species with different levels of delayed bitterness are isolated and cloned in vector pTZ57R/T. Our results indicate that gene sequence of sweet lime (with intense juice delayed bitterness) have complete identity with Satsuma mandarin (without distinctive juice delayed bitterness). Also gene sequence of Marsh seedless grapefruit, local orange and Thompson navel orange (with mild juice delayed bitterness) have very similarity with Satsuma mandarin. On the other hand, this gene started to express 60, 120, and 210 days after full blooming in albedo of Satsuma mandarin, sweet oranges and sour orange, and both grapefruit and sweet lime, respectively. Expression pattern of limonoid glucosyltransferase gene in leaves was quite different with albedo. Thus, we supposed the delayed bitterness in this species was related to delay in expression of limonoid glucosyltransferase gene in albedo and lower limonoid glucoside accumulation in fruits.     

Morphogenesis and tissue cultures of three citrus species

Studies were performed to define tissue culture techniques and culture conditions for morphogenesis, callus culture and plantlet culture of sweet orange (Citrus sinensis (L.) Osb.), citron (C. medica L.) and lime (C. aurantifolia) (Christm. Swing). The optimal concentrations of NAA to induce root formation on stem segments were 10 mg l^-1 for sweet orange and lime, and 3 mg l^-1 for citron. The optimal BA concentration for shoot and bud proliferation was 3 mg l^-1 for sweet orange and citron, and 1 mg l^-1 for lime. Callus initiation was accomplished in a culture medium containing 10 mg l^-1 NAA and 0.25 mg l^-1 BA. Callus was maintained by periodical subculture into the same medium supplemented with 10% (v:v) organge juice. In vitro plantlets of the three species were obtained by rooting of shoots developed from bud cultures, and of citron and lime by development of shoots from root cultures. The plants were successfully established on soil.     

Tetraploid Rangpur lime rootstock increases drought tolerance via enhanced constitutive root abscisic acid production

Whole‐genome duplication, or polyploidy, is common in many plant species and often leads to better adaptation to adverse environmental condition. However, little is known about the physiological and molecular determinants underlying adaptation. We examined the drought tolerance in diploid (2x) and autotetraploid (4x) clones of Rangpur lime (Citrus limonia) rootstocks grafted with 2x Valencia Delta sweet orange (Citrus sinensis) scions, named V/2xRL and V/4xRL, respectively. Physiological experiments to study root–shoot communication associated with gene expression studies in roots and leaves were performed. V/4xRL was much more tolerant to water deficit than V/2xRL. Gene expression analysis in leaves and roots showed that more genes related to the response to water stress were differentially expressed in V/2xRL than in V/4xRL. Prior to the stress, when comparing V/4xRL to V/2xRL, V/4xRL leaves had lower stomatal conductance and greater abscisic acid (ABA) content. In roots, ABA content was higher in V/4xRL and was associated to a greater expression of drought responsive genes, including CsNCED1, a pivotal regulatory gene of ABA biosynthesis. We conclude that tetraploidy modifies the expression of genes in Rangpur lime citrus roots to regulate long‐distance ABA signalling and adaptation to stress.     

Efficient propagation and rooting of three citrus rootstocks using different plant growth regulators

The influence of various basal medium and plant growth regulators on the efficient micropropagation of nodal explants from mature trees of alemow, sour orange, and ??Cleopatra?? mandarin citrus rootstocks was studied. All three citrus rootstock shoot cultures showed a preference for high-salt media, like Murashige and Skoog or Driver and Kuniyuki Walnut medium. Several combinations of N ⁶-benzyladenine (BA) and adenine (AD), kinetin (KIN) or gibberellic acid (GA) were tested to optimize the shoot proliferation phase. BA/GA combinations improved the proliferation of all the rootstocks studied, especially alemow. The addition of BA and AD to the culture medium improved shoot proliferation in sour orange and ??Cleopatra?? mandarin in the same way as BA and GA. The addition of different combinations of BA/KIN did not result in further improvement of any of the studied variables. The transfer of in vitro shoots to rooting media, containing different concentrations of indolebutyric acid (IBA) and indoleacetic acid (IAA), resulted in regeneration of complete plantlets. Alemow and ??Cleopatra?? mandarin shoots rooted well using these plant growth regulators; however, all combinations of IBA and IAA tested resulted in very low rooting percentages in sour orange. To improve rooting in sour orange and ??Cleopatra?? mandarin, different combinations of naphthaleneacetic acid (NAA) and IBA were tested. All NAA/IBA combinations produced higher rooting percentages than did the IBA/IAA combinations, and in sour orange nearly 100 % of explants developed roots. An efficient and simple protocol for the micropropagation of three citrus rootstocks, alemow, ??Cleopatra?? mandarin, and sour orange, by culturing nodes from mature plants, has been established.