Production of transgenic tomato plants with enhanced resistance against the fungal pathogen Fusarium oxysporum

Tomato [Lycobersicum esculentum Mill] cultivar CastleRock was used to produce fungal resistant plants by introducing an antimicrobial chitinase gene. Hypocotyl with a part of cotyledon [hypocotyledonary] of young tomato seedlings were used as explant material. The transformation performed by delivering the vector pGL2 harbouring a class I rice chitinase gene [chi11] under the control of the CaMV 35S promoter and hygromycin resistant gene as a plant selectable marker, using biolistic bombardment mediated transformation system. Shoots were regenerated onto selective regeneration medium supplemented with 1 mg/l BAP, 1 mg/l Zeatin ripozide, 5mg/l AgNO[3] and 25mg/l hygromycin, then subjected to rooting medium for developing roots. Putative transgenic plantlets of R0 were confirmed by PCR analysis using specific primers for the transgene, and most regenerated plants showed positive results. However, PCR and dot blot analyses were conducted using R1 seedlings to confirm the stable integration of transgenes in R1 progeny. Bioassay for transgenic plants was performed on the transgenic R1 young seedlings and non-transgenic controls by challenging with a vigorous isolate of the fungal pathogen Fusarium oxysporum f. sp. Lycopersici to assay for the resistance against fusarium wilt disease among individuals. Data from transgenic plants showed lower percentages of infection ranging from 11 to 60% in lines 8 and 5 respectively compared to the control plants [80% infected]. The percentage of resistance ranged from 11 to 77.7% in lines 7 and 8, respectively compared to 10% in non-transgenic control plants. In addition, the average percent of the tolerant seedlings were19.69% that had been infected but showed delayed symptoms and suppression in symptom extension. The obtained results indicated that expression of the chitinase protein [Chi 11] in transgenic tomato plants acquired them antifungal activity against Fusarium

Evaluation of some cucumber inbred lines and their hybrids for cucumber mosaic virus [CMV] resistance

The major objective of our cucumber breeding program involves breeding for CMV disease resistance to reduce losses in quality and yield. Seven half diallel cross hybrids resistant to CMV were developed at the Vegetables Breeding Dept., Hort. Res. Inst., Egypt during March, 2006. Sixteen genetically different pure inbred lines of Cucumis sativus were used to develop the hybrids obtained from different sources and selected for their CMV disease-resistance trait. The inbred lines and one commercial [Beit alpha] cultivar as a susceptible control were examined to CMV resistance using biological, serological and molecular methods. The CMV isolate identified by Plant Path. Res. Inst. Virus and Phytoplasma Res. Dept. [ARC] was used in mechanical inoculation of all cucumber genotypes used during this study. The seeds of the genotypes were incubated and the seedlings were cultivated in foam trays with peat soil and kept under greenhouse conditions. At the cotyledon stage, i.e. before the development of the first true leaf, the seedlings were mechanically inoculated by rubbing with virus inoculum. Disease severity was assessed visually 7- 10 days [on cotyledons] and 14-25 days [on true leaves] after inoculation with CMV. The results revealed that six out of sixteen cucumber inbred lines [Cus 260/1980, 6-5-23-2 Kaha, 1-180-309- 18-105 Dokky, 5-57-22-17 Kaha, Cus 38/1991, and 25-2-1-90 Kaha] were found to be without systemic symptoms of CMV infection and proved to be resistant to CMV when tested by DAS-ELISA and RT-PCR. The promising accessions as sources of resistance have been intercrossed with leading commercial type [Beit-alpha] in half diallel system. In order to determine the genetic polymorphism and discriminate between cucumber inbred lines, RAPD-PCR analyses were conducted on the DNA isolated from each line. Dendrograms representing genetic distances were performed on the studied genotypes using the UPGMA [Unweighted Pair Group Method with Arithmetic Average]. Twenty one cucumber hybrids obtained from the half diallel crossing between the six resistant genotypes and the local commercial cultivar [Beit-alpha] were subjected to CMV artificial inoculation in a separate greenhouse and symptoms were visually monitored for two months. Only seven cucumber hybrids showed high a level of resistance to CMV were screened in the greenhouse and evaluated for CMV resistance. The resistant hybrids obtained did not develop visual symptoms of CMV infection on cotyledons and true leaves. These resistant lines could serve as potential sources of resistance in breeding programs

Identification and characterization of resistance gene analog [RGA] and the leaf rust resistance gene Lr21 from the wheat cultivar Giza 168

Leaf [brown] rust caused by Puccinia triticina is a fungal disease of wheat [Triticum aestivum L.] that causes significant yield losses annually in many wheat-growing regions of the world. Host-plant resistance is the most economically viable and environmentally responsible method for controlling Puccinia triticina, the causal agent of leaf rust in wheat. The identification and utilization of new resistance sources is critical to continue the development of improved cultivars. The objective of this work was to identify defense-related genes against rust in the Egyptian rust resistant cultivar Giza168. Specific primers were designed on the basis of converse motifs of cloned resistance genes of the resistance gene analog [RGA] and leaf rust resistance gene [Lr21] in wheat [Triticum aestivum L.]. The designed PCR primers were subsequently used for RT-PCR using RNA isolated from a resistant variety to amplify fragments of 445 bp and 235 bp for RGA and Lr21 genes, respectively. The amplified products were cloned, sequenced and submitted to the GenBank. The nucleotide sequences of the amplified fragments were aligned with their corresponding genes using the BLAST. The expressions of the two genes in the infected and healthy plants were studied using RT-PCR. The RGA expression was induced and detected by RT-PCR, which is up-regulated by fungal infection. The Lr21 expression was detected on both healthy and infected plants, although the expression was higher in infected plants

Molecular and serological studies on a plant virus affecting strawberry

Viruses form a major threat to the strawberry industry in Egypt causing severe economic losses. Rapid and simple methods for the detection of the major strawberry viruses are absent due to the lack of sensitive diagnostic tools. Plants showing virus-like symptoms [VLS] were collected from the field and subjected to indirect enzyme-linked immunosorbent assay [I-ELISA] tests using the polypeptide CP3 antiserum specific for whitefly transmitting geminivirus [WTG] in addition to Tomato yellow leaf curl geminivirus [TYLCV] polyclonal antiserum. In addition, plants were subjected to PCR as a molecular diagnosis test for further confirmation. Experiments proved that the virus could be transmitted mechanically, by viruliferous whiteflies and by grafting. Inoculated strawberry plants with viruliferous whiteflies showed curling and upward cup shape of the leaves. Primers specific for whitefly transmitted geminivirus were used in PCR diagnosis of the inoculated plants. Based on the positive molecular and serological diagnosis results, we concluded that the virus belongs to WTGs. PCR was also carried out for the inoculated plants using primers specific for TYLCV, however negative amplification was obtained indicating that the virus under this study is not a TYLCV. Electron microscopy of purified virus preparation showed the presence of geminate virus particles about 18x20 nm. Antiserum was raised against the purified virus and used for indirect-ELISA to measure the antigenicity of the raised antibodies. Western blot analysis was also used for confirmation of the specificity of the raised antiserum. The isolated virus was given the name strawberry leaf curl geminivirus [StLCV] and it represents the first record of WTG that infect strawberry plants in Egypt