Tree performance and fruit yield and quality of ‘Okitsu’ Satsuma mandarin grafted on 12 rootstocks

The citriculture in Brazil, as well as in other important regions in the world, is based on very few mandarin cultivars. This fact leads to a short harvest period and higher prices for off-season fruit. The ‘Okitsu’ Satsuma (Citrus unshiu Marc.) is among the earliest ripening mandarin cultivars, and it is considered to be tolerant to citrus canker (Xanthomonas citri subsp. citri Schaad et al.) and to citrus variegated chlorosis (Xylella fastidiosa Wells et al.). Despite having regular fruit quality under hot climate conditions, the early fruit maturation and absence of seeds of ‘Okitsu’ fruits are well suited for the local market in the summer (December through March), when the availability of citrus fruits for fresh consumption is limited. Yet, only a few studies have been conducted in Brazil on rootstocks for ‘Okitsu’. Consequently, a field trial was carried out in Bebedouro, São Paulo State, to evaluate the horticultural performance of ‘Okitsu’ Satsuma mandarin budded onto 12 rootstocks: the citrandarin ‘Changsha’ mandarin (Citrus reticulata Blanco) × Poncirus trifoliata ‘English Small’; the hybrid Rangpur lime (Citrus limonia Osbeck) × ‘Swingle’ citrumelo (P. trifoliata (L.) Raf. × Citrus paradisi Macfad.); the trifoliates (P. trifoliata (L.) Raf.) ‘Rubidoux’, ‘FCAV’ and ‘Flying Dragon’ (P. trifoliata var. monstrosa); the mandarins ‘Sun Chu Sha Kat’ (C. reticulata Blanco) and ‘Sunki’ (Citrus sunki (Hayata) Hort. ex. Tanaka); the Rangpur limes (C. limonia Osbeck) ‘Cravo Limeira’ and ‘Cravo FCAV’; ‘Carrizo’ citrange (Citrus sinensis × P. trifoliata), ‘Swingle’ citrumelo (P. trifoliata × C. paradisi), and ‘Orlando’ tangelo (C. paradisi × Citrus tangerina cv. ‘Dancy’). The experimental grove was planted in 2001, using a 6 m × 3 m spacing, in a randomized block design. No supplementary irrigation was applied. Fruit yield, canopy volume, and fruit quality were assessed for each rootstock. A cluster multivariate analysis identified three different rootstock pairs with similar effects on plant growth, yield and fruit quality of ‘Okitsu’ mandarin. The ‘Flying Dragon’ trifoliate had a unique effect over the ‘Okitsu’ trees performance, inducing lower canopy volume and higher yield efficiency and fruit quality, and might be suitable for high-density plantings. The ‘Cravo Limeira’ and ‘Cravo FCAV’ Rangpur limes induced early-ripening of fruits, with low fruit quality. ‘Sun Chu Sha Kat’ and ‘Sunki’ mandarins and the ‘Orlando’ tangelo conferred lower yield efficiency and less content of soluble solids for the latter rootstock.     

Horticultural performance of ‘Folha Murcha’ sweet orange onto twelve rootstocks

Despite its outstanding position, the Brazilian citriculture is established on a very limited pool of varieties that limits its expansion and restricts the fruit availability throughout the year. This situation determines the urgent necessity of developing alternative scion and rootstock cultivars, with good performance under local conditions. ‘Folha Murcha’ sweet orange (Citrus sinensis (L.) Osbeck) is a late-harvest cultivar, suitable both for the juice processing industry and the fresh fruit market, being described as tolerant to citrus canker (Xanthomonas citri subsp. citri Schaad et al.), and less affected by citrus variegated chlorosis (Xylella fastidiosa Wells et al.). A study was conducted in Bebedouro, São Paulo State, Brazil, to evaluate the horticultural performance of ‘Folha Murcha’ sweet orange budded onto 12 rootstocks: the citrandarin ‘Changsha’ mandarin (Citrus reticulata Blanco) × Poncirus trifoliata ‘English Small’; the hybrid ‘Rangpur’ lime (Citrus limonia Osbeck) × ‘Swingle’ citrumelo (P. trifoliata (L.) Raf. × Citrus paradisi Macfad.); the trifoliates (P. trifoliata (L.) Raf.) ‘Rubidoux’, ‘FCAV’, and ‘Flying Dragon’ (P. trifoliata var. monstrosa); the ‘Sun Chu Sha Kat’ mandarin (C. reticulata Blanco); the ‘Sunki’ mandarin (Citrus sunki (Hayata) Hort. ex. Tanaka); the ‘Rangpur’ limes (C. limonia Osbeck) ‘Cravo Limeira’ and ‘Cravo FCAV’; ‘Carrizo’ citrange (C. sinensis × P. trifoliata), ‘Swingle’ citrumelo (P. trifoliata × C. paradisi), and ‘Orlando’ tangelo (C. paradisi × Citrus tangerina cv. ‘Dancy’). The experimental grove was planted in 2001, using a 7 m × 4 m spacing, in a randomized block design, with five replications and two plants per plot. No supplementary irrigation was applied. Fruit yield, canopy volume, tree tolerance to drought and to citrus variegated chlorosis, and fruit quality were assessed for each rootstock. Trees grafted onto the ‘Flying Dragon’ trifoliate were smaller in size, but had largest yield efficiency when compared to those grafted onto other rootstocks. Lower alternate bearing index was observed on trees budded onto ‘Cravo FCAV’ ‘Rangpur’ lime. Both ‘Rangpur’ lime rootstocks and the ‘Sunki’ mandarin induced higher tree tolerance to drought. The ‘Flying Dragon’ trifoliate induced better fruit quality and higher tolerance to citrus variegated chlorosis (CVC) to ‘Folha Murcha’ trees. A cluster multivariate analysis identified three groups of rootstocks with similar effects on ‘Folha Murcha’ tree performance. Among the 12 evaluated rootstocks, the ‘Flying Dragon’ trifoliate has a unique effect on plant growth, tolerance to drought and CVC, fruit yield and fruit quality of ‘Folha Murcha’ trees, and may be better suited for high-density plantings.     

Agrobacterium tumefaciens-mediated genetic transformation and plant regeneration from a complex tetraploid hybrid citrus rootstock

Agrobacterium-mediated genetic transformation of a tetraploid “tetrazyg” citrus rootstock selection ‘Orange #16’ [Nova mandarin (Citrus reticulata Blanco) + Hirado Buntan pummelo (Citrus grandis L. Osbeck)] × [Cleopatra mandarin (C. reticulata Blanco) + Argentine trifoliate orange (Poncirus trifoliata (L.) Raf.)] was performed. Juvenile epicotyl segments were transformed with a construct containing a bifunctional egfp–nptII fusion gene under the control of an enhanced double CaMV 35S promoter. Our protocol resulted in a reasonable transformation efficiency of 18%. Stable integration of the transgene was confirmed by visual observation of EGFP expression, PCR and Southern blot hybridization. The purpose of this work was to investigate the amenability of novel citrus rootstock germplasm being developed for improved tree size control, soil adaptation, and disease resistance, to existing transformation technologies. Seed trees of such transgenic tetraploids also have potential as trap plants containing potent insecticidal transgenes, due to their inedible fruit and inherent crossing barriers with conventional commercial diploid scion cultivars, and could be planted around producing citrus groves.     

Cybrid/hybrid plants regenerated from somatic fusions between male sterile Satsuma mandarin and seedy tangelos

Somatic hybridization provides an alternative for transferring mitochondria-encoded cytoplasmic male sterility (CMS). Herein, symmetric protoplast electrofusion was conducted between embryogenic callus protoplasts of Citrus unshiu Marc. cv. Guoqing No. 1 (G1), a CMS cultivar, and mesophyll-derived protoplasts of seedy ‘Page’ tangelo [C. reticulata Blanco × (C. reticulata Blanco × C. paradisi Macf.)] or ‘Nova’ tangelo [C. reticulata Blanco × (C. reticulata Blanco × C. paradisi Macf.)], to transfer CMS trait. Flow cytometry analysis showed that 14 plants recovered from G1 + ‘Page’ tangelo that displayed typical morphological character of ‘Page’ were diploid, and 6 plants regenerated from G1 + ‘Nova’ tangelo were tetraploid. Genetic compositions of regenerated plants from the two fusions were determined by SSR, CAPS and chloroplast-SSR analysis. Cybrid nature of diploids from G1 + ‘Page’ tangelo with nuclear DNA from ‘Page’, mitochondrial DNA (mtDNA) from the G1 and chloroplast DNA (cpDNA) derived from either parent was confirmed. Tetraploid plants from G1 + ‘Nova’ tangelo were identified as somatic hybrids with random cpDNA inheritance. The regenerated cybrid and hybrid plants hold great potential for Citrus seedless breeding at diploid or triploid levels.     

QTL analysis and regression model for estimating fruit setting in young Citrus trees based on molecular markers

This study was conducted to assess the genetic basis and develop a regression model for a QTL trait, fruit setting of a full sib population of 164 hybrids obtained between ‘Clementine’ mandarin (Citrus reticulata Blanco ‘Clementine) and ‘Orlando’ tangelo’ (Citrus paradisi Macf. ‘Duncan’ × C. reticulata Blanco ‘Dancy’). Fruit setting of a 164 full sib population field-planted in 2007 was evaluated by visual counting of fruits in 2008, 2009, and 2010. We estimated linkage groups and effects of QTLs by using MapQTL5. Based on interval mapping, seven linkage groups of the maternal and eight linkage groups of the paternal maps harboured QTLs of the fruits setting, covering a total 300 of 1744 cM Citrus map. Only three segments were associated with all three-year data (one in ‘Clementine’ and two in ‘Orlando’ map) spanning 60 cM of 1744 cM of Citrus linkage map. Twenty-seven (17%) and 13 (8%) hybrids consistently produced less than 5 fruits and the highest number of fruits (≥50) in their 2nd, 3rd, and 4th ages. Based on BACKWARD elimination procedure of PROC REG option nested in SAS program, regression models constructed for 2008, 2009, and 2010s fruit setting explained 35, 81, and 38% of the total phenotypic variation (R²) observed in the 164 full-sib hybrids, respectively (P ≤ 0.05). This study indicated that early fruit setting was a complex trait affected by many QTLs and the regression model developed in this study might be used to predict performances of hybrids with similar genetic background.     

Cellular wall metabolism in citrus fruit pericarp and its relation to creasing fruit rate

Citrus creasing results in serious economic loss in many citrus orchards. Based on the different incidence of creasing, two sweet orange cultivars ‘Hong Jiang’ (grafting chimaera of which flesh is mostly from Citrus reticulata Blanco and peel is from Citrus sinensis Osbeck in) and ‘An Liu’ (Citrus sinensis Osbeck) were used to investigate the creasing rate. The ultrastructure in cellular wall, cellular wall component, cellular wall degradation-related enzymes and expansin (Ct-Exp1) of sweet orange during fruit ripening in field were comparatively analyzed. ‘Hong Jiang’ sweet orange had a higher creasing rate than ‘An Liu’ during ripening. The activities of polygalacturonase, cellulase and pectinesterase in cellular wall of ‘Hong Jiang’ increased more markedly compared with ‘An Liu’. The increases in the content of soluble pectin, ionically associated pectin, covalently bound pectin, hemicellulose and cellulose of ‘Hong Jiang’ were higher than those of ‘An Liu’. Furthermore, the enhanced degradation of the ultrastructure in the albedo cellular walls was observed in ‘Hong Jiang’. Moreover, the northern blot analysis indicated that the Ct-Exp1 gene expressed more strongly in peel of ‘Hong Jiang’ than ‘An Liu’ during fruit ripening. These data suggest that enhanced loss of pectin and cellulose in the cellular walls of peel tissue of sweet orange could result in fruit creasing.     

Evaluation of ‘Hamlin’ sweet orange + ‘Montenegrina’ mandarin somatic hybrid for tolerance to Xanthomonas axonopodis pv. citri and Xylella fastidiosa

Asiatic citrus canker (ACC), caused by Xanthomonas axonopodis Starr & Garces pv. citri (Hasse) Vauterin et al., and citrus variegated chlorosis (CVC), caused by Xylella fastidiosa Wells et al., are considered the main diseases affecting sweet orange scion varieties in Brazil. Among commercial varieties, mandarins and tangerines are recognized as tolerant to these pathogens. We report herein the production of ‘Hamlin’ sweet orange (Citrus sinensis L. Osbeck) + ‘Montenegrina’ mandarin (Citrus deliciosa Ten.) allotetraploid somatic hybrid plants by protoplast fusion with improved disease tolerance that could be used as a donor of resistance genes in interploid hybridisation. Somatic hybridisation was confirmed by leaf morphology, flow cytometry and RAPD analyses. The somatic hybrid was propagated by grafting and cultivated in a screenhouse for tolerance assays. For X. axonopodis pv. citri assays, buds were collected from both ‘Hamlin’ sweet orange and the somatic hybrid and grafted onto ‘Cleopatra’ mandarin (Citrus reshni hort. ex Tanaka). As a negative control, buds from ‘Mexerica Tardia’ mandarin (C. deliciosa) were collected and grafted onto ‘Cleopatra’ mandarin. Two-month old plants with at least one young vegetative flush were individually spray-inoculated with a 10⁶ CFU mL⁻¹X. axonopodis pv. citri suspension and incubated in a growth chamber, at 27 °C, under 16-h photoperiod. The somatic hybrid showed a statistically significant reduction in susceptibility to ACC 30 days after inoculation. Compared to ‘Hamlin’ sweet orange, disease severity was reduced by 70%, with similar tolerance to that of the mandarin negative control. For X. fastidiosa assays, buds were collected from the somatic hybrid and its parental plants and grafted onto ‘Rangpur’ lime (Citrus limonia Osbeck). The developed plants were needle-inoculated with a X. fastidiosa suspension (8.7 × 10¹⁰ CFU mL⁻¹) into the new growth flush stem. Bacterial population was quantified both at 4 (at the inoculation point) and 8 months (50 cm above the inoculation point) after inoculation. The first evaluation detected X. fastidiosa in 63% of ‘Hamlin’ sweet orange and ‘Hamlin’ + ‘Montenegrina’ mandarin samples. In the second evaluation, X. fastidiosa was detected in 47.4% of ‘Hamlin’ sweet orange and 10.5% of ‘Hamlin’ + ‘Montenegrina’ somatic hybrid samples, suggesting that bacterial movement was restricted in the somatic hybrid. X. fastidiosa was not detected in both evaluations in samples collected from leaves of ‘Montenegrina’ mandarin. These results indicate that the ‘Hamlin’ sweet orange + ‘Montenegrina’ mandarin somatic hybrid has potential for improved disease tolerance that should enhance its value regarding future use in citrus breeding programs.     

Production of mandarin + pummelo somatic hybrid citrus rootstocks with potential for improved tolerance/resistance to sting nematode

Sting nematode (Belonolaimus longicaudatus Rau) has become a primary factor limiting citrus production in localized regions of the central Florida sandridge citrus production area, making the development of resistant rootstocks a new breeding objective. In efforts to develop a replacement rootstock for the widely adapted sour orange, our focus has been on somatic hybridization of selected mandarin + pummelo combinations [Grosser, J.W., Gmitter, Jr., F.G., 1990. Protoplast fusion and citrus improvement. Plant Breed. Rev. 8, 339–374; Ananthakrishnan, G., Calovic, M., Serrano, P., Grosser, J.W., 2006. Production of additional allotetraploid somatic hybrids combining mandarins and sweet oranges with pre-selected pummelos as potential candidates to replace sour orange rootstock. In Vitro Cell. Dev.: Plant 42, 367–371], since sour orange is probably an introgression hybrid of mandarin and pummelo as suggested by molecular marker analyses [Nicolosi, E., Deng, Z.N., Gentile, A., La Malfa, S., Tribulato, E., 2000. Citrus phylogeny and genetic origin of important species as investigated by molecular markers. Theor. Appl. Genet. 100, 1155–1166; Gulsen, O., Roose, M.L., 2001. Lemons: diversity and relationships with selected Citrus genotypes as measured with nuclear genome markers. J. Am. Soc. Hort. Sci. 126, 309–317]. Somatic hybrid plants were produced from four new mandarin (C. reticulata Blanco) + pummelo (C. grandis L. Osbeck) parental combinations by fusing embryogenic suspension culture-derived protoplasts isolated from selected mandarins with leaf protoplasts of pummelo seedlings previously selected for tolerance/resistance to the sting nematode (B. longicaudatus Rau) as follows: Amblycarpa mandarin + ‘Liang Ping Yau’ (seedling) pummelo seedling SN7; Amblycarpa mandarin + ‘Hirado Buntan Pink’ (HBP) pummelo seedling SN3; Murcott tangor + pummelo seedling SN3; and Shekwasha mandarin + pummelo seedling SN3. Somatic hybridization was verified by ploidy analysis (via flow cytometry) and RAPD analyses. Mandarin parents were selected for wide soil-adaptation and ability to produce friable embryogenic callus lines. Pummelo seedlings used as leaf parents were identified from a previous screen of large seed populations (200 each) from four pummelos for resistance to sting nematode as follows: ‘Hirado Buntan Pink‘; ‘Red Shaddock‘; ‘Large Pink Pummelo’ and a seedling pummelo of ‘Liang Ping Yau‘. Ten resistant/tolerant pummelo seedlings were selected from the 800 pummelo seeds planted in the screen for further study. The four new somatic hybrids have been propagated to evaluate their horticultural performance and resistance to the sting nematode. These potential somatic hybrid rootstocks should also have potential to control tree size due to polyploidy.     

Chloride and sodium excluding capacities of citrus rootstock germplasm introduced to Australia from the People's Republic of China

The responses to root zone salinity (0, 25 and 50 mM NaCl) by 40 citrus rootstock genotypes introduced from the People's Republic of China, measured as shoot chloride (Cl⁻) and sodium (Na⁺) ion accumulation, growth and dry matter accumulation, were investigated under glasshouse conditions. Two experiments, one using aerated nutrient solutions and the other irrigated sand cultures, were conducted with plants grown from rooted cuttings taken from representative trees of different mandarin (Citrus reticulata Blanco and C. erythrosa Hort. ex Tan.), yuzu (C. junos Sieb. ex Tan.), Ichang papeda (C. ichangensis Swing.), sour orange (C. aurantium L.) and trifoliate orange (Poncirus trifoliata (L.) Raf.) genotypes. Two standard genotypes, viz. Rangpur lime (Citrus x limonia Osbeck.) and William's trifoliate orange were included in the experiments as reference controls.     

Effect of a cytochrome P450 inhibitor on abscisic acid biosynthesis and stomatal regulation in citrus trees in water-stressed conditions

The effect of diniconazole, an inhibitor of cytochrome P450, on drought tolerance was examined in ‘Shiranui’ [(Citrus unshiu Marc. × Citrus unshiu Osbeck) × Citrus reticulate Blanco] trees. Diniconazole treatment increased abscisic acid (ABA) concentration in leaves compared with untreated controls in water-stressed conditions after 20 days of water-stress treatment. Diniconazole significantly decreased the stomatal aperture at 9 h after application compared with untreated controls in water-stressed conditions. The photosynthetic rate decreased in water-stressed conditions; however, regardless of the earlier stomatal closure induced by diniconazole application, the decrease of photosynthetic rate was delayed by the application.     

‘Swingle’ citrumelo propagation by cuttings for citrus nursery tree production or inarching

‘Swingle’ citrumelo [Citrus paradisi MacFaden × Poncirus trifoliata (L.) Raf.] has been extensively used as a rootstock in several citrus growing regions of the World, including Southern Brazil where ‘Rangpur’ lime (Citrus limonia Osbeck) is still the predominant variety despite being affected by several important pathogens. In this case, ‘Swingle’ citrumelo is used to produce nursery trees to establish new orchards or to be inarched in adult and healthy groves in order to change the rootstock. We report herein a system to produce trees on ‘Swingle’ citrumelo more rapidly by budding onto non-rooted cuttings, as well as assessing potential to rapidly multiply ‘Swingle’ through rooting of non-budded cuttings. Therefore, two potential products are described: budded trees and rooted rootstock cuttings. ‘Valencia’ sweet orange [Citrus sinensis (L.) Osbeck] was budded at different heights on cuttings derived from eight-month old rootstocks. Grafted and additional non-budded cuttings were then treated with indole-3-butyric acid (500 mg L⁻¹) or left untreated before rooting. Three types of cuttings were evaluated: softwood, semi-hardwood and hardwood. The use of nursery trees derived from pre-budded hardwood cuttings of ‘Swingle’ citrumelo is an alternative grafting method on this cultivar. Softwood cuttings with one leaf pair were considered the most adequate material for rapid multiplication of ‘Swingle’ citrumelo by cutting. This could be particularly useful for inarching production or conventional budding after transplant of cutting-derived rootstocks.     

Growth,yield and fruit quality of ‘Marisol’ clementine grown on four rootstocks in Egypt

To evaluate the most appropriate rootstocks for mandarin production in Egypt, vegetative growth, yield and fruit quality of ‘Marisol’ clementine (as newly introduced cultivar in Egypt) grafted on Sour orange (the common rootstock), Cleopatra mandarin, Carrizo citrange and ‘Swingle’ citrumelo were evaluated under the Egyptian conditions during 2004/2005 and 2005/2006 seasons. Trees were grown in a private farm at ‘Wady El-Mullak’ region, Ismailia Governorate (Latitude, 30°36′ N; longitude, 32°14′ E; Altitude, 10 m above sea level).     

Evaluation of citrus somatic hybrids for tolerance to Phytophthora nicotianae and citrus tristeza virus

Somatic hybridization is a biotechnology tool that can be used in citrus breeding programs to produce somatic hybrids with the complete genetic combination of both parents. The goal of this work was to test the reaction of citrus somatic hybrids that may be useful as rootstocks to trunk and root infections caused by Phytophthora nicotianae van Breda de Haan (P. parasitica Dastur) and to citrus tristeza virus (CTV). The somatic hybrids evaluated were ‘Caipira’ sweet orange (Citrus sinensis L. Osbeck) + ‘Rangpur’ lime (C. limonia Osbeck), ‘Caipira’ sweet orange + ‘Cleopatra’ mandarin (C. reshni hort. ex Tanaka), ‘Caipira’ sweet orange + ‘Volkamer’ lemon (C. volkameriana V. Ten. & Pasq.), ‘Caipira’ sweet orange + rough lemon (C. jambhiri Lush.), ‘Cleopatra’ mandarin + ‘Volkamer’ lemon, ‘Cleopatra’ mandarin + sour orange (C. aurantium L.), ‘Rangpur’ lime + ‘Sunki’ mandarin (C. sunki (Hayata) hort. ex Tanaka), ‘Ruby Blood’ sweet orange (C. sinensis L. Osbeck) + ‘Volkamer’ lemon, ‘Rohde Red’ sweet orange (C. sinensis L. Osbeck) + ‘Volkamer’ lemon, and ‘Valencia’ sweet orange + Fortunella obovata hort. ex Tanaka. For P. nicotianae trunk and root infection assays, plants of the somatic hybrids, obtained from 9-month semi-hardwood cuttings, were evaluated and compared with diploid citrus rootstock cultivars after mycelia inoculation in the trunk or spore infestation in the substrate, respectively. ‘Cleopatra’ mandarin + sour orange, ‘Rangpur’ lime + ‘Sunki’ mandarin, ‘Cleopatra’ mandarin + ‘Volkamer’ lemon, ‘Ruby Blood’ sweet orange + ‘Volkamer’ lemon, ‘Rohde Red’ sweet orange + ‘Volkamer’ lemon, and ‘Caipira’ sweet orange + ‘Volkamer’ lemon had less trunk rot occurrence, whereas the somatic hybrids ‘Cleopatra’ mandarin + ‘Volkamer’ lemon, ‘Cleopatra’ mandarin + sour orange, ‘Caipira’ sweet orange + ‘Volkamer’ lemon, and ‘Caipira’ sweet orange + ‘Rangpur’ lime were tolerant to root rot. For CTV assays, plants of the somatic hybrids along with tolerant and intolerant rootstocks were budded with a mild strain CTV-infected or healthy ‘Valencia’ sweet orange budwood. Differences in average scion shoot length indicated that the hybrids ‘Cleopatra’ mandarin + sour orange and ‘Valencia’ sweet orange + Fortunella obovata were intolerant to CTV.     

Diploid female gametes induced by colchicine in Oriental lilies

The young flower buds of diploid Oriental cultivars: ‘Con. Amore’ and ‘Acapulco’ (Lilium) were treated with 0.02, 0.05, 0.1, and 0.2% colchicine to induce diploid egg (female gamete). The treated young buds between the two cultivars were crossed reciprocally as female parents with normal pollen respectively. The polyploid plants were identified by measuring stoma size and chromosome number. The results showed: that triploid progenies could be obtained through this way, and the fact that the treated young flower buds were successfully used as female parents indicated the formation of 2n or 2x egg cells. The above results implied that polyploidization by artificially induced diploid female gametes could be a powerful method to create novel variations in the breeding of Oriental lilies.     

Evaluation of rootstocks for the Cyprus local lemon variety ‘Lapithkiotiki’

The effect of various rootstocks on yield, yield efficiency, tree size and fruit quality of the local lemon variety ‘Lapithkiotiki’ (Citrus limon (L.) Burm. F.) was studied under Cyprus conditions. Total cumulative yield over 13 years of production was the highest on rough lemon (C. jambhiri Lush) followed by that on Volkameriana (C. volkameriana Ten. & Pasq.), Yuma Ponderosa lemon (C. limon (L.) Burm. F.), sour orange (C. aurantium L.), Citrus macrophylla Wester, Morton citrange (C. sinensis cv. Washington navel × Poncirus trifoliata (L.) Raf.), Yuma citrange, Rangpur lime (C. limonia Obs.), Palestine sweet lime (C. limettioides Tan.), C-32 citrange and Citremon 1449 (C. limon × P. trifoliata), although no statistically significant differences were found between sour orange, the commercial rootstock used in Cyprus, and the other above mentioned rootstocks. Next in order as regards to total cumulative yield was Cleopatra mandarin (C. reticulata Blanko.) followed by Carrizo citrange, with statistically significant differences compared with sour orange. Trees on Citrumelo CPB-4475 (C. paradisi Macf. × P. trifoliata) and C-35 citrange died 3–4 years after grafting. Canopy volume was the lowest for trees on Cleopatra mandarin and Carrizo citrange. Yield efficiency A, expressed as total cumulative yield per trunk cross-sectional area, was the highest on C. macrophylla and lowest on Carrizo citrange. Rootstock significantly affected fruit size and weight, rind thickness, juice content, brix, total acids and brix:acid ratio. The results of the present study reveal that the most promising rootstocks that may replace sour orange for the local lemon variety ‘Lapithkiotiki’ under Cyprus conditions are Volkameriana, Yuma Ponderosa lemon, C. macrophylla and Citremon 1449.     

Response of evergreen perennial tree crops to gibberellic acid is crop load-dependent. I: GA3 increases the yield of commercially valuable ‘Nules’ Clementine Mandarin fruit only in the off-crop year of an alternate bearing orchard

Worldwide, gibberellic acid (GA₃) is used routinely to increase fruit number and size of seedless mandarins. The efficacy of seven combinations of GA₃ concentrations and application times to maximize total yield and yield of commercially valuable fruit (diameter 57.2–76.2 mm) of ‘Nules’ Clementine mandarin (Citrus reticulata Blanco) was determined in a commercial orchard. GA₃ applied during the period of intense flower abscission failed to reduce the total number of abscised flowers in both the light off- and heavy on-bloom years. No GA₃ treatment reduced fruit abscission when trees were setting the low yield off-crop. However, all trees receiving GA₃ in the high yield on-crop year had fewer abscised fruit than untreated control trees (P = 0.0188) and GA₃ applied 10 days after 75% petal fall and in July increased the number of fruit retained on tagged branches >20% compared to control trees (P = 0.0005). Maximum air temperature was not related to flower or fruit abscission. In the off-crop year (548 fruit per untreated control tree), it was necessary to apply 15 or 25 mg L⁻¹ GA₃ at 60% bloom, 90% bloom, 75% petal fall and 10 days after 75% petal fall to significantly increase the number of fruit per tree and yield of commercially valuable fruit (kilograms and number per tree) (P < 0.0001) above that of control trees, with no reduction in total kilograms per tree. In the following on-crop year, it was better not to apply GA₃: no treatment increased total yield or fruit size and five of seven GA₃ treatments tested reduced total yield as kilograms and number of fruit per tree (P = 0.0003). The results provide strong evidence that GA₃ efficacy is crop load-dependent and dictate that crop load should be considered when using GA₃ to increase fruit set or fruit size of mandarins.     

Differential structure and physiological response to canker challenge between ‘Meiwa’ kumquat and ‘Newhall’ navel orange with contrasting resistance

Asiatic citrus canker caused by Xanthomonas citri subsp. citri (Xcc) is one of the most detrimental diseases affecting citrus production. ‘Meiwa’ kumquat (Fortunella crassifolia) and ‘Newhall’ navel orange (Citrus sinensis) are two genotypes with contrasting Xcc resistance. In the current study, efforts were made to perform pairwise comparison between them in terms of structural difference and biochemical response to Xcc attack. Pinprick inoculation demonstrated that ‘Meiwa’ had lower disease incidence rate and smaller lesion size, concurrent with significantly less bacterial number at the inoculation sites relative to ‘Newhall’. Microscopic observation demonstrated that stomatal density and size of ‘Meiwa’ were significantly smaller than ‘Newhall’. Stomatal apertures of ‘Meiwa’ were smaller than ‘Newhall’ measured at 2 h after their leaves were immersed in Xcc suspension, consistent with the bacteria number in the internal tissues counted at 4 d. Epicuticular wax content of ‘Meiwa’ was significantly higher than that of ‘Newhall’, while no difference in palisade and spongy tissue was observed between them. Enzymatic activity assay showed that after Xcc inoculation, ‘Meiwa’ exhibited significantly higher activities of catalase (CAT), peroxidase (POD) and phenylalanine ammonia-lyase (PAL) in comparison with ‘Newhall’ at most of the sampling time points. Our data showed that the two genotypes with contrasting Xcc resistance exhibited clear difference in the structure and physiological response, which may provide information to facilitate our understanding of Xcc resistance.     

Variability at the electron microscopic level in leaves of members of the genus Vitis

Scanning electron microscopy (SEM) was used to examine the characteristics of the upper and lower sides of leaves belonging to members of the genus Vitis – 11 vinifera varieties (‘Albariño’, ‘Treixadura’, ‘Caíño Blanco’, ‘Mencía’, ‘Chasselas’, ‘Cabernet Sauvignon’, ‘Alicante Bouschet’, ‘Godello’, ‘Torrontés’, ‘Blanco Legítimo’ and ‘Caíño Tinto’) and three non-vinifera varieties (‘110-Ritcher’, ‘SO4’ and ‘Jacquez’). All the genotypes studied grew in the same plot, were of the same age, and had been raised following the same cultivation practices. The leaves examined were taken from node 8 of a fruiting shoot growing from the previous year's wood. The same part of the leaf – the area between the main vein and the first right lateral vein – was examined in all plants. SEM observations were made at 200×, 655× and 2000×. Photomicrographs and drawings were prepared reflecting the observed morphology of the upper and lower leaf surfaces. Variables such as cell density, cell surface area (in the horizontal plane) and stomatal density and surface area (in the horizontal plane) were measured for both leaf surfaces, and intra- and inter-varietal differences recorded. The upper leaf surfaces of all the studied genotypes were similar, but clear differences were recorded for the lower sides.     

Endogenous plant growth substances content in young fruits of seeded and seedless Clementine mandarin as related to fruit set and development

The content of plant growth substances in the fruits of seedless (‘Fino’) and seeded (‘Monreal’) Clementine mandarin (Citrus reticulata Blanco) during the early stages of development was investigated. Auxin-like substances were determined with the oat mesocotyl bioassay, gibberellin-like substances with the α-amylase bioassay, and abscisic acid (ABA) by gas chromatography—electron capture detector. Methanolic extracts of fruits of seedless Clementine had higher content of auxin-like compounds in the acidic and butanolic fractions than the seeded Clementine extracts. In particular, a compound with R_f 0–0.25 in the chromatograms may be involved in the parthenocarpic fruit set development of ‘Fino’ Clementine ovaries. Diffusable gibberellin-like compounds (R_f 0.8–1.0), probably produced in the developing seeds, were obtained from seeded Clementine fruits after the time of anthesis. The content of ABA in the fruits was always low in ‘Monreal’ Clementine, whereas in fruits of ‘Fino’ Clementine and of ‘Monreal’ without seeds it increased dramatically after anthesis. It is suggested that the control of fruit set and development in Clementine mandarin is carried out through an equilibrium between auxin-like substances (‘Fino’) or gibberellin-like substances (‘Monreal’) and ABA.     

Identification of S-alleles in pear (Pyrus communis L.) cv. ‘Rocha’ and other European cultivars

In this work we report the cloning and identification of S-RNase alleles responsible for gametophytic self-incompatibility (GSI) of ‘Rocha’ pear and of 13 other European pear cultivars that might be used as its pollinators. Partial sequences of S-RNase alleles were amplified by PCR with specific primers hybridising in conserved regions of previously identified S-RNase alleles of Pyrus communis, cloned and sequenced and the S-genotype of eight pear cultivars was fully determined. Three cultivars (‘General Léclerc’ (SqSl), ‘Tosca’ (SbSl) and ‘Alexandrine Douillard’ (SbSk)) shared no S-alleles with ‘Rocha’ (SaSe) and shall be totally compatible with this cultivar. None of the cultivars analysed showed an identical amplification pattern to the one observed in ‘Rocha’, so the other cultivars shall be at least semi-compatible. One new allele was identified in P. communis cv. ‘Beurré d’Avril’ (designated as St). The determination of both S-RNase alleles of cvs ‘Rocha’, ‘Beurré Precoce Morettini’ (SeSk) and ‘Tosca’ and the identification of one S-RNase allele in cvs ‘Carapinheira’ (Sb), ‘Amêndoa’ (Se), ‘Pérola’ (Sk) and ‘Beurré d’Avril’ (St) are important contributions for the effort recently developed worldwide to establish groups of sexual compatibility among European pears.