Citrus tristeza virus (CTV) resistance in transgenic citrus based on virus challenge of protoplasts

Summary A strategy for the sereening of candidate virus-derived sequences to provide RNA-mediated citrus tristeza virus (CTV) resistance and early selection of virus-resistant citrus is presented. The system is based on the polyethylene glycol-(PEG) mediated cotransformation of protoplasts using virus-derived sequences and green fluorescent protein as a single selectable marker, followed by an in vitro assay of virus inoculation into transgenic protoplasts to determine the level of citrus tristeza virus replication. A cotransformation rate higher than 20% allowed selection of several clones carrying the desired transgenes. Efficient in vitro inoculation of virus in transgenic protoplasts was performed. Tobacco mosaic virus virions were used as a control in order to check eitrus protoplast viability. Different CTV replication levels were detected in transgenic clones. Only one clone showed no replication of CTV. Considerations regarding selection of candidate virusderived sequences and virus challenge of transgenic cells are presented.     

Corrections to Morris and Mound (2004) 'Molecular relationships between populations of South African citrus thrips (Scirtothrips aurantii Faure) in South Africa and Queensland, Australia'

Abstract  Contrary to the statements by Morris and Mound (2004), Scirtothrips aurantii were first found outside the quarantine facility on mother-of-million plants ( Bryophyllum spp.) sourced from the western suburbs of Brisbane and in January 2003 the distribution of the thrips in south-eastern Queensland was from at least 20 km south-east and 70 km west of the detection site. Some records in South Africa of thrips from mother-of-million plants may not refer to this species.     

Early events in Agrobacterium-mediated genetic transformation of citrus explants

BACKGROUND AND AIMS: Genetic transformation of plants relies on two independent but concurrent processes: integration of foreign DNA into plant cells and regeneration of whole plants from these transformed cells. Cell competence for regeneration and for transformation does not always fall into the same cell type/developmental stage, and this is one of the main causes of the so-called recalcitrance for transformation of certain plant species. In this study, a detailed examination of the first steps of morphogenesis from citrus explants after co-cultivation with Agrobacterium tumefaciens was performed, and an investigation into which cells and tissues are competent for regeneration and transformation was carried out. Moreover, the role of phytohormones in the co-cultivation medium as possible enhancers of gene transfer was also studied. METHODS: A highly responsive citrus genotype and well-established culture conditions were used to perform a histological analysis of morphogenesis and cell competence for transformation after co-cultivation of citrus epicotyl segments with A. tumefaciens. In addition, the role of phytohormones as transformation enhancers was investigated by flow cytometry. KEY RESULTS: It is demonstrated that cells competent for transformation are located in the newly formed callus growing from the cambial ring. Conditions conducive to further development of this callus, such as treatment of explants in a medium rich in auxins, resulted in a more pronounced formation of cambial callus and a slower shoot regeneration process, both in Agrobacterium-inoculated and non-inoculated explants. Furthermore, co- cultivation in a medium rich in auxins caused a significant increase in the rate of actively dividing cells in S-phase, the stage in which cells are more prone to integrate foreign DNA. CONCLUSIONS: Use of proper co-cultivation medium and conditions led to a higher number of stably transformed cells and to an increase in the final number of regenerated transgenic plants.     

扁桃优良砧木离体快繁

对扁桃优良砧木“Hansen”侧芽进行离体快繁研究,结果表明：外植体取材时间不同,成活率差异明显,以春季芽即将萌动的3月下旬为最佳时间,在MS 6-BA1．0mg／L GA30．2mg／L培养基上诱导芽萌发,萌发率在75％以上;以MS 6-BAl．5mg／L IAA0．1mg／L进行芽的增殖培养效果最好;1／2MS IBA0．5mg／L诱导生根,并给以黑暗预处理可使生根率达85％以上,每株生根数量多,根系质量好;采用分步炼苗移栽,提高了小苗成活率,有利于小苗后期生长。     

Immature embryo rescue,culture and seedling development of acid citrus fruit derived from interploid hybridization

Interploid sexual hybridizations were completed in 2001 and 2002 between seven lemon (Citrus limon(L.) Burm. f.) varieties, Key lime (C. aurantifolia (Cristm.) Swing), Palestine sweet lime (C. imettioides Tan.), Lakeland limequat (C. aurantifolia x Fortunella japonica (Thumb.) Swing.), and Etrog citron (C. medica L.) as diploid progenitors and four allotetraploid somatic hybrids (Key lime + Valencia orange, Hamlin orange + Femminello lemon, Valencia orange + Rough lemon, and Valencia orange+ Femminello lemon) in efforts to generate improved seedless triploid acid fruit hybrids. Efficient recovery of triploid progeny from such crosses requires embryo rescue to avoid embryo abortion due to endosperm failure. Germination of rescued genetically diverse immature embryos was induced on two culture media (EME and Gamborgs B5), with two sucrose concentrations (50 or 70 g l^–1). All media contained 0.5 g l^–1 malt extract and 4.50 M GA₃. Germination of globular, heart and torpedo shaped embryos (defined as small embryos) was significantly (p < 0.05) affected by medium and genotype. Gamborgs medium induced 82.89% germination. Of germinated embryos, 11–65% developed into normal plants with differences among crosses. Cotyledonary embryos (defined as immature embryos with fully developed cotyledons) germinated and developed into normal plants at higher rates than less-developed embryos. In efforts to improve the efficiency of plant recovery, small embryos from Todo el año × HF and Lisbon × HF crosses conducted during 2002 were rescued and cultured on three media (MS, Gamborgs, and RMA) for comparison. Media did not significantly affect the proportion of normal plant recovery.     

Effect of the insecticides abamectin and lufenuron on eggs and larvae of Chrysoperla externa under laboratory conditions

The side effects of twoinsecticides/acaricides, abamectin andlufenuron, on the eggs and larvae of Chrysoperla externa (Hagen) were studied inthe laboratory (25 ± 2°C, 62 ±10% RH and 12-h photophase). The analyticalmethods used were those proposed by theInternational Organization for BiologicalControl (IOBC) – Working Group for Pesticides and Beneficial Organisms. Chrysoperla externa eggviability was not affected by abamectin.Neonate larvae from abamectin sprayed eggs aswell as first, second and third instar larvaethat were directly treated, developed normallyand yielded normal adults. Lufenuron presentedno adverse effects on egg survival. However,lufenuron induced high mortality in neonatelarvae from treated eggs. These neonates, aswell as lufenuron treated first and secondinstar larvae could not molt. In the thirdinstar, high pupal mortality occurred. Theresults showed that abamectin is innocuous andthat lufenurom is toxic to Chrysoperlaexterna eggs and larvae.     

Citrus replant problem in Iraq III. Interactive effect of soil fungi and allelopathy

The interactive effects of citrus root residues and soil fungi on citrus replant problems were investigated. The results indicated that incorporation of citrus root residues in combination with the pathogenic fungiPhytophthora citrophthora, Pythium aphanidermatum andFusarium solani in soil caused more reduction to sour orange growth than did the root residues alone. Subsequent experiments showed that extracts of different parts of sour orange and leachates of some soil fungi increased the disease index of citrus roots grownin vitro. The citrus extracts did not affect growth of the test fungi.Thus, it appears that allelopathic compounds of plant and microbial origins build up in old citrus soil and may act as predisposal agents for the infection of citrus roots by soil pathogens.     

Xenobiotic absorption and binding by proteins in hemolymph of the weevil Diaprepes abbreviatus

A synthetic coumarin, 7-amino-3-phenyl coumarin (coumarin-10), was used to study the uptake of ingested xenobiotics into hemolymph. Larvae were forcefed coumarin-10 in peanut oil, and hemolymph was extracted and analyzed by fluorescence spectroscopy. Coumarin-10 entered hemolymph within 5 min, reaching a steady state of concentration within 1 h. Assayed 2 h after feeding, hemolymph titers of 1–5 ng/μl were proportional to log dose between 10 and 100 ng/mg body weight; hemolymph did not reach saturation. Fluorescence spectra of hemolymph in saline revealed that energy was readily transferred from hydrophobic residues of hemolymph proteins to coumarin-10. Ultracentrifugal density gradients revealed that 94% of absorbed coumarin-10 was bound to sedimenting proteins while 6% bound to lipophorin. Native polyacrylamide gel electrophoresis (N-PAGE) on minigels identified two major proteins responsible for binding. Though readily separated by native electrophoresis, these proteins were not fully separable by HPLC using a wide variety of columns. Gel permeation-HPLC of the sedimenting proteins from hemolymph revealed a single major peak of 480,000 M_r. When upper and lower electrophoretic bands were isolated by preparative N-PAGE, the upper band (band I) yielded subunits of 75,000 and 71,000 M_r, while the lower band (band II) yielded only one size subunit of 75,000 M_r on denaturing (SDS) PAGE. The fluorescent products bound by sedimenting proteins were identified by thin-layer chromatography and scanning fluorescence densitometry as coumarin-10 (80% of total) and a polar metabolite (20%). In addition, lipophorin-containing fractions contained an apolar metabolite (3% of total fluorescence). In vitro binding studies utilizing fluorescent energy transfer demonstrated saturation binding with a K_D of 1.5 μM.     

Influence of root zone oxygen stress on potassium and ammonium absorption by Myrobalan plum rootstock

Summary Growth chamber experiments were conducted with French prune (Prunus domestica L.) scions grafted on Myrobalan 29C (P. cerasifera Ehrh.) rootstocks grown in nutrient solution to characterize K and NH₄ uptake before, during, and after anaerobiosis. Conditions of oxygen stress were imposed by removing the source of aeration and bubbling solutions with nitrogen gas.At solution oxygen concentrations less than 1%, K leaked from plant roots. After 18 h of anaerobic conditions, aeration was restored and K depletion from solution occurred within 2 h. Uptake of K the following day was similar to that before oxygen stress was imposed.Under similar conditions with solution oxygen concentrations less than 1%, both K and NH₄ uptake were inhibited. Potassium leakage from roots was significantly greater than that of NH₄. The presence of NH₄ had no significant effect on K leakage from roots. Signs of wilting during oxygen stress appeared first on those trees that received NH₄. Potassium uptake by rootstocks in the presence of NH₄ was inhibited prior to and following anaerobiosis. However, the extent of NH₄-induced inhibition of K uptake before anaerobiosis was similar to the K uptake inhibition after anaerobiosis.