Characterisation of citrus rootstock germplasm introduced as seeds to Australia from the People's Republic of China

Seeds were collected from a range of citrus rootstock types in China and sent to Australia for evaluation. Before evaluation proceeded, however, the seeds and their seedlings were characterised for uniformity and trueness-to-type. The seeds were cultured in vitro and/or germinated in sand beds to assess whether or not they were mono- or polyembryonic. Seedlings were then rogued on the basis of vigour and morphology before isozyme profiles were developed from leaf extracts to assess seedling uniformity within introductions. Isozyme profiles were also compared to those of the source trees, as well as hybrids generated between some of them in China, to assess and verify trueness-to-type. The degree of variability between the seedlings propagated from the imported seeds and their source trees in China differed between accessions, which highlighted some of the issues that need to be considered when importing citrus germplasm as seeds. Regardless of whether the seedlings could be shown to be identical to their source tree in China, three uniform seedlings of polyembryonic types were established in arboreta as Australian source trees to provide propagules for evaluation and possibly commercial use.     

Cloning and characterization of 5 MADS-box cDNAs isolated from citrus fruit tissue

We isolated and characterized 5 MADS-box cDNA clones of CitMADS1, CitMADS3, CitMADS5, CitMADS6 and CitMADS8 from fruit tissues of Satsuma mandarin (Citrus unshiu Marc.). Sequence analysis revealed that they have high sequence identities with known MADS-box genes and that their predicted proteins possess the general structural features of the M domain and the K domain. In a phylogenetic tree with 24 known Arabidopsis MADS-box genes, 5 Citrus MADS-box genes were classified into 4 clades of the MADS-box gene with independent gene functions. Their broad expression profiles in the Citrus life cycle suggested that they play roles in flower and fruit development. Most of them, except CitMADS1 and CitMADS3, are expressed in seedlings before the phase transition from vegetative to reproductive growth. CitMADS1 and CitMADS3 are not expressed in vegetative organs and could serve as a molecular marker for reproductive development.     

DNA content,morphometric and molecular marker analyses of Citrus limonimedica, C. limon and C. medica for the determination of their variability and genetic relationships within the genus Citrus

This work investigated the fingerprinting and phenotyping of Citrus germplasm; species selected were of historical importance belonging to Citrus limonimedica Lush. and its supposed ancestors, along with some other species of the Citrus genus. An integrated approach based on the exploitation of nuclear DNA content, morphological traits and molecular markers, such as RAPD fingerprints and ITS-based SNPs, was employed. We studied a core collection of 54 distinct accessions, including 43 genotypes of the Citrus species (18 species or supposed species) and 11 genotypes of the Poncirus genus, which was used as the reference outgroup. Morphological trait analysis and statistical analysis of DNA content and markers were useful for reconstructing a Citrus phylogeny. In particular, our experiments aimed at estimating the genetic variation within and the genetic relatedness among C limon (L.) Burm., C. limonimedica and C. medica L. to shed light on the hybrid origin hypothesis of C. limonimedica. The results of the multidisciplinary analyses allowed us to confirm a remarkable differentiation between Poncirus and Citrus genera and to highlight a close relationship among the three investigated Citrus species but a distinct difference between these three species and other species in the Citrus genus. RAPD fingerprints and ITS polymorphisms enabled us to point out a variation gradient between lemon and citron, with C. limonimedica as a possible intermediate species. Some accessions of C. medica and C. limonimedica that deviate from such a trend suggest recurrent introgression and/or hybridisation with other species of Citrus.     

Facultative apomixis and chromosome doubling are sources of heterogeneity in citrus rootstock trials: Impact on clementine production and breeding selection

All commercial citrus rootstocks are polyembryonic and propagated by seeds. Although these seeds produce uniform plant material most of the time, zygotic or polyploid citrus seedlings may arise. The aim of this study was to understand how zygotic or tetraploid rootstock could affect a citrus rootstock selection field trial. A trifoliate orange selection field trial, which was planted in 1974 and grafted with clementine, was re-investigated with respect to the presence of rootstocks that were not true-to-type. Among the 288 trees investigated, flow cytometry identified 2.4% of rootstocks as tetraploid and SSR markers indicated that 6.6% were zygotic. Yield data showed that the presence of tetraploid rootstock dramatically decreased (by about 45%) clementine fruit production. However, zygosity did not always affect fruit production and a range of effects were observed, from a slight increase in production to a 24% decrease. Exclusion of non-true-to-type genotypes from the production analysis indicated that the best candidate for rootstock was a clone previously ranked in the middle of the 32 rootstocks under evaluation. However, the presence of zygotic rootstock did not appear to cause any significant differences in fruit quality during the first 5 years of the investigation, which suggests that non-true-to-type plants cannot be identified by fruit quality parameters. This study indicates that tetraploid and zygotic rootstocks have a strong impact on citrus fruit production in orchards, and removal of off-type seedlings is required prior to planting in any agronomic trial.     

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.     

Comparative analysis of genetic diversity in Citrus germplasm collection using AFLP,SSAP, SAMPL and SSR markers

In this study we evaluate the informativeness and efficiency of Amplified Fragment Length Polymorphism (AFLP), Sequence-Specific Amplified Polymorphism (S-SAP), Selectively Amplified Microsatellite Polymorphic Loci (SAMPL) and Simple Sequence Repeat (SSR) markers for genetic diversity, phylogenetic relationship among the Citrus species and mapping ability of the marker system. The SSR exhibited relatively higher level of polymorphism information content in terms of the expected heterozygosity, than that of the AFLPs, SSAPs and SAMPLs. For each marker system, average level of the discriminating potential was very close to the actual discriminating potential. Similarity matrices showed weak, yet significant correlations when Mantel's test was applied. The highest positive (0.72) correlation was found between the AFLP and SSAP markers. The SSR and SAMPL markers were poorly correlated. The dendrogram topology among the four marker systems had high similarity. Taken together, the SSAP and SAMPL were highly efficient in detecting genetic similarity in Citrus, while the SSR may be more useful for segregation studies and genome mapping in Citrus. The SSAP and SAMPL markers could be useful for Citrus genome mapping in combination with AFLP and SSR markers. To our knowledge, this was the first detail report of a comparison of performances among AFLP, SSR and retrotrasposon based molecular marker technique on a set of samples of Citrus. Our result provides guidance for future efficient use of these molecular methods in genetic analysis of Citrus sp. and its relatives.     

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.     

Light quality affects in vitro adventitious rooting and ex vitro performance of cherry rootstock Colt

The effect of light quality on in vitro rooting of cherry rootstock “Colt” was studied to evaluate adventitious root development, ex vitro acclimatization and plant growth performance. Adventitious rooting was dependent on the light quality to which the microcutting were exposed. Highest number of roots per microcuttings was recorded under dichromatic light (blue + red). This response could be ascribed to the control of a strong synergistic interaction between phytochromes and blue light photoreceptors. Red light was more effective on root elongation than blue light. In this response a strong synergistic interaction between phytochromes and blue light photoreceptors was suggested. The effect of light quality on the number of root/explant affected the plant during acclimation, scoring the highest level of plant survival in the blue-, red- and blue + red-exposed plantlets. The light quality effect was also observed under greenhouse culture conditions, as shown by growth parameters at the end of six months in plants growth. No specific role was observed for the photoequilibrium of phytochrome. The results reported in this work show that plantlets exposed to different light quality, during the in vitro rooting phase, retain the light quality effects in the subsequent plant acclimatization and greenhouse plant growth phase.     

Genome composition and genetic diversity of Musa germplasm from China revealed by PCR-RFLP and SSR markers

Banana (Musa spp.) is one of the most important crops in the world. In this study, 216 banana accessions, 184 from the National Banana Germplasm Collection of China (NBGCC) and 32 from the International Network for the Improvement of Banana and Plantain (INIBAP), were used to determine the genome composition of banana plants in these collections and to estimate their genetic diversity. The genome composition was examined using PCR-RFLP markers. The molecular data for all but one accession (ITC 1231) from INIBAP were in agreement with the initial records based on phenotypic characteristics. Microsatellite (SSR) markers were used to investigate the genetic variability and relationships among these banana accessions. Ten of the 47 primer pairs tested consistently produced reproducible and discrete fragments. We identified a total of 92 alleles, ranging from 5 to 15 per locus. The genetic similarity between the accessions ranged between 0.1 and 1, when estimated using Jaccard's coefficient. The UPGMA method based on genetic similarities, grouped the NBGCC accessions according to those containing the ‘A’ and ‘B’ genomes. However, this analysis could not separate all the accessions, especially the somatic mutations, using the primers in this study. These data indicated that limited genetic variation exists within these accessions and the collections of NBGCC should include a much wider range of banana plant material.     

Identification and distribution of S haplotypes in Brassica vegetables from China

The amplification efficiency and identification diversity with gene-specific primers derived from S locus glycoprotein gene (SLG) and S locus receptor kinase gene (SRK) were compared, and the geographical distribution for S haplotypes was investigated by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) in 72 genotypes of 5 Brassica vegetables from China. The amplification efficiency and identification diversity with class I SRK primers were generally higher than that with class I SLG in most crops tested. Class I alleles were found in total 66 genotypes and they were classified into 16, 10, 7 and 10 groups for Chinese cabbage, purple flowering stalk, cauliflower and cabbage respectively. The number of amplification accessions and identification diversity using the primers of class II SLG and SRK were quite similar. Class II alleles were detected in 55 genotypes and further grouped into one type in mustard and three in other crops. The nucleotide sequences showed high similarity between identical S haplotypes determined by reciprocal pollination and PCR-RFLP tests. It demonstrated that the PCR-RFLP analysis was feasible for identification of S alleles, and SRK should be considered as a better marker for the identification of S haplotypes than SLG. The types of S haplotypes are highly diverse in Brassica vegetables from China. Nevertheless, they were geographically limited in some Brassica vegetables, so the exchange of germplasm resources should be enhanced for breeding.     

Relation of carbohydrate reserves with the forthcoming crop,flower formation and photosynthetic rate,in the alternate bearing ‘Salustiana’ sweet orange (Citrus sinensis L.)

The aim of this work was to assess the relation between carbohydrate levels and flower and fruit production, as well as the role of carbohydrates on CO₂ fixation activity, by analysis of leaves, twigs and roots from the alternate bearing ‘Salustiana’ cultivar of sweet orange (Citrus sinensis [L.] Osbeck). A heavy crop load (on year) did not affect photosynthesis activity when compared to non-fruiting trees (off year). Fruiting trees accumulated most of the fixed carbon in mature fruits, whilst no accumulation was observed in roots before harvest. Non-fruiting trees transported part of the fixed carbon to the roots and mobilize it for growth processes and, at the end of the season (December), store it as reserves. Reserve carbohydrates accumulation in leaves started by early December for both tree types, showing the same levels in on and off trees until spring bud sprouting. A heavy flowering after an off year caused the rapid mobilization of the stored reserves, which were exhausted at full bloom. We found no evidence on carbon fixation regulation by either fruit demand or carbohydrate levels in leaves. Carbohydrate reserves played little or no role over fruit set, which actually relied on current photosynthesis.     

Nutrient deficiency disorders in Pistacia vera seedling rootstock in relation to eco-physiological,biochemical characteristics and uptake pattern of nutrients

This study examines the effects of nitrogen (N), iron (Fe), magnesium (Mg), manganese (Mn) and molybdenum (Mo) deficiencies on the eco-physiological, biochemical and growth characteristics of pistachio seedlings (Pistacia vera) in sand culture situation. This experiment, which was carried out in Iran's Pistachio Research Institute (IPRI), seedlings of ‘Badami-e-Zarand’ were used as common rootstock. The following treatments were employed: (1) complete Hoagland's nutrient solution; nutrient solution lacking either: (2) N, (3) Fe, (4) Mg, (5) Mn, (6) Mo, (7) distilled water without Hoagland's solution (Control), (8) Fe–N, (9) Fe–Mn and (10) Fe–Mo. The main deficiency symptoms observed after 7 months of planting were; leaf discoloration, necrosis, scorching, defoliation and stunted growth. Seedlings that received complete nutrient solution were healthy with dark green foliage. Visual symptoms of N-deficient seedlings also coincided with the reduction in foliar levels of the concerned element. Shoot and root growth of the seedlings deficient in nutrients were also affected. Changes in shoot and root biomasses production due to treatments were statistically significant at 1% level. A reduction in height, leaf area and leaf number was noticed for various levels of nutrient element deficiencies. Analysis of some biochemical indices (chlorophyll content) and eco-physiological indices such as primary florescence chlorophyll (Fo), ratio of variable chlorophyll to maximum (Fv/Fm), leaf temperature, transpiration, stomata conduction and resistance of stomata, showed that fractions of chlorophyll, transpiration, stomata conduction and resistant of stomata in treated seedlings, particularly N-deficient seedlings, declined significantly. Chlorophyll (Fo) and ratio of variable chlorophyll to maximum (Fv/Fm) were significant at 5% level. The effects of Fe, Mn, Mg and Mo on dry matter production were statistically significant at 1% level. Synergic effects of certain elements were also evident in the present study. The typical symptoms of deficiencies of various nutrient elements can be used as a guideline for diagnosing nutrient deficiencies of pistachio in commercial nurseries and plantations. The present study also showed multi-deficiency of nutrients, produces new symptom in leaves, which differs from single deficiencies.     

Response of Vitis vinifera L. plants inoculated with Phaeoacremonium angustius and Phaeomoniella chlamydospora to thiabendazole,resveratrol and sodium arsenite

The effect of some pesticides (sodium arsenite, thiabendazole and ziram) and the natural phytoalexin (resveratrol) on mycelial growth of Phaeomoniella chlamydospora and Phaeoacremonium angustius was studied. Several strains of these species were grown on malt extract agar (MEA) plates containing different concentrations of inhibitory compounds and colony diameters were measured. While sodium arsenite and ziram had little effect on the growth of both species, thiabendazole inhibited colony growth, at minimum concentrations of 6 μM for Ph. chlamydospora and 15 μM for P. angustius. Resveratrol at concentrations equal to or grater than 867 μM also inhibited colony growth of both species. To assay the effect of these substances on plant response to infection, in vitro grapevines were inoculated with Ph. chlamydospora or P. angustius spores and were grown in the presence of sodium arsenite (0–30 μM), thiabendazole (0–30 μM) or resveratrol (0–876 μM). Infected untreated plants and sodium arsenite-treated plants developed symptoms of senescence (reduced growth, increased membrane lipid peroxidation, and decreased chlorophyll content and fluorescence). In contrast, infected plants treated with thiabendazole (30 μM) or with resveratrol (876 μM) showed similar characteristics of fresh weight, malondialdehyde accumulation and chlorophyll fluorescence to those of uninfected plants. These results are promising with respect to the use of thiabendazole-containing pesticides as alternatives to currently used pesticides for control of esca in vines. Results also suggest that the presence of resveratrol in grapevines may be useful to induce resistance to these fungi.     

Isoenzyme and microsatellite analysis of Vitis vinifera L. varieties from the Hungarian grape germplasm

The aim of our work was to investigate the genetic diversity of grapevine with biochemical and molecular markers (isoenzyme and SSR). The isoenzyme patterns of 4 enzyme systems (catechol-oxidase, glutamate-oxalacetate-transaminase, acid phosphatase and peroxidase) and the microsatellite profile in 6 loci (VVS2, VVS16, VVMD7, VMC4A1, VMC4G6, VrZag79) of 48 grapevine varieties were analysed. The results with CO, GOT, AcP and PER enzymes were reproducible and the zymograms obtained from the woody stems were independent from the time of sampling during the dormant period of the grape. Based on the isoenzyme patterns of these 4 enzymes most of the investigated varieties (40/48) were identified. A correlation was found between the isoenzyme patterns and the classification to convarietas of the varieties. It was established, that while the varieties of the convarietas pontica differed from those of the convarietas orientalis and occidentalis, the two latter groups could have not been differentiated from each other. Based on the SSR (simple sequence repeat) analyses 46 of the 48 investigated varieties were identified. Even ‘Pinot blanc’ and ‘Pinot gris’ cultivars belonging to the same conculta (Pinot) could be differentiated in their VMC4A1 locus.     

Genetic transformation of Citrus sinensis cv. Hamlin with hrpN gene from Erwinia amylovora and evaluation of the transgenic lines for resistance to citrus canker

Transgenic Citrus sinensis (L.) Osb. cv. Hamlin plants expressing the hrpN gene were obtained by Agrobacterium tumefaciens (Smith and Towns) Conn-mediated transformation. hrpN encodes a harpin protein, which elicits the hypersensitive response and systemic acquired resistance in plants. The gene construct consisted of gst1, a pathogen-inducible promoter, a signal peptide for protein secretion to the apoplast, the selection genes nptII or aacC1 and the Nos terminator. The function of gst1 in citrus was evaluated in transgenic C. sinensis cv. Valencia harboring the reporter gene uidA (gus) driven by this promoter. Histochemical analysis for gus revealed that gst1 is activated in citrus leaves by both wounding and inoculation with Xanthomonas axonopodis Starr and Garces pv. citri (Hasse) Vauterin et al. Genetic transformation was confirmed by Southern blot hybridization in eight cv. Hamlin acclimatized plants. RT-PCR confirmed hrpN gene expression in seven cv. Hamlin transgenic lines before pathogen inoculation. Some hrpN transgenic lines showed severe leaf curling and abnormal growth. Six hrpN transgenic lines were propagated and evaluated for susceptibility to X. axonopodis pv. citri. RT-PCR confirmed gene expression in all six hrpN transgenic lines after pathogen inoculation. Several of the hrpN transgenic lines showed reduction in susceptibility to citrus canker as compared with non-transgenic plants. One hrpN transgenic line exhibited normal vegetative development and displayed very high resistance to the pathogen, estimated as up to 79% reduction in disease severity. This is the first report of genetic transformation of citrus using a pathogen-inducible promoter and the hrpN gene. Further evaluations of the transgenic plants under field conditions are planned. Nevertheless, the evidence to date suggests that the hrpN gene reduces the susceptibility of citrus plants to the canker disease.     

Effect of substrate,medium composition,irradiance and ventilation on jojoba plantlets at the rooting stage of micropropagation

The effect of substrate, medium composition, irradiance and ventilation on rooting percentage, and root morphology and functioning with respect to mineral uptake was investigated in jojoba [Simmondsia chinensis (Link) Schneider]. Plantlets grown on Sorbarod plugs embedded in perlite accumulated more biomass and showed better rooting than plantlets grown on Sorbarod plugs embedded in agar. In plants grown on the latter substrate, the addition of sucrose to water or salts media promoted rooting and accumulation of shoot and root biomass. On perlite, rooting was high in all treatments, while root growth was enhanced only by 2% sucrose. Sucrose and irradiance had a positive synergistic effect on plantlet growth. Doubling the irradiance from 58 to 116 μE m⁻² s⁻¹ and using high ventilation (22-mm membrane) failed to improve rooting but promoted the ability of the stomata to close, thereby reducing water loss under ambient conditions. High irradiance and ventilation lowered the chlorophyll content, but without impairing biomass accumulation. Plantlets grown under enhanced ventilation and 0.5% sucrose formed fine roots within the cellulose plug and thick roots on the vessel floor. With the 2% sucrose supplementation, the roots were generally thicker, with very few thin roots being formed. ³⁶Cl uptake into plantlets and transport to the leaves decreased in plants exposed to a high sucrose concentration. Acclimatization of jojoba plantlets was improved by enhancing ventilation.     

The effect of maturity,sunburn and the application of sunscreens on the internal and external qualities of pomegranate fruit grown in Australia

Consumer interest in pomegranate fruit (Punica granatum L.) is increasing in Australia as a result of its unique external and internal qualities. This work looked at the effect of applying sunscreen treatments to pomegranate fruit on the degree of sunburn damage and the effect of maturity and sunburn on the internal antioxidant concentration of the juice. The pomegranates, cultivar ‘Wonderful’ were grown in Condobolin, New South Wales, Australia.     

Effects of the commercial product based on Trichoderma harzianum on plant,bulb and yield characteristics of onion

Effects of the commercial product TrichoFlow WP™ (Agrimm Technologies Ltd., New Zealand), based on the fungus Trichoderma harzianum, on quality characteristics and yield of bulb onion was investigated. Bulb sets of the local cultivar Kantartopu was planted in soil with in and between row distances of 0.15 m and 0.40 m, respectively. The product, at considerably high dosages of 5 g m⁻², 10 g m⁻² and 15 g m⁻², was mixed with water and sprinkled once to the plots at planting. Analyses of data at harvest did not show statistical significance for Trichoderma effect on total bulb yield, bulb diameter, leaf length, number of shoot apex, %titratable acidity, number of internal (fleshy) leaves, number of external (papery) leaves, %soluble solids and %bulbs with diameters of 20–39 mm, 40–69 mm and ≥70 mm. The yields obtained from the plots treated with the dosages of 5 g m⁻², 10 g m⁻² and 15 g m⁻² and the control plots were 1063.7 kg da⁻¹, 1051.0 kg da⁻¹, 1066.5 kg da⁻¹ and 985.0 kg da⁻¹, respectively. Our results showed that high dosages of the Trichoderma product were not effective in enhancing onion bulb and yield characteristics under the given conditions.     

The culture of isolated microspores of ornamental kale (Brassica oleracea var. acephala) and the importance of genotype to embryo regeneration

The culture of isolated microspores of kale (Brassica oleracea var. acephala) was studied including the importance of genotype to embryo regeneration, medium composition chiefly the sucrose concentration and the use of colchicine, simultaneously medium renovation. It was initiated using 29 different genotypes as donor plants. Embryos were induced from six of the kale genotypes and these corresponded to the more out-bred genotypes. Embryogenesis was achieved using four different combinations of culture media: (a) microspores initially cultured in NLN medium supplemented with 13% (w/v) sucrose (NLN-13) for 48 h, followed by transfer to fresh NLN-13 medium; (b) microspores cultured for 48 h in NLN-13 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-13 medium; (c) microspores cultured for 48 h in NLN-16 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-16 medium; (d) microspores cultured for 48 h in NLN-16 medium supplemented with colchicines (50 mg/L) followed by transfer to unsupplemented NLN-13 medium. The embryos obtained from four of the genotypes developed into plantlets and these regenerated plants have been successfully transplanted to soil.