Gülruh Albayrak

Regeneration capacity of mature embryo-derived callus in barley ( Hordeum vulgare L.)

In this study, induction of regenerable callus from mature embryos in eight Turkish barley varieties was analysed by using different plant growth regulators (PGRs). Varying concentrations (0.5-4 mg l -1 ) of 2,4-dichlorophenoxyacetic acid (2,4-D) and dicamba (3,6-dichloro-o-anisic acid) were tested for callus induction from mature embryos. Highest percent of callus induction was observed in Bornova 92 variety (98.3%) on MS medium supplemented with 4 mg l -1 dicamba. Calli were transferred to regeneration media with 0.5 mg l -1 dicamba, 0.5 mg l -1 zeatin riboside (ZR) and 2 mg l -1 thidiazuron (TDZ). Low concentrations of dicamba induced multiple shoots during callus regeneration. When the effect of precultivation with 2,4-D or dicamba on the shoot induction were evaluated, lower concentrations (< 4 mg l -1 ) of auxins have been found optimal. On the regeneration medium with 0.5 mg l -1 dicamba, shoots were able to elongate up to 20 cm and shoot numbers were between 1-23 per callus. The use of ZR led to formation of short shoot buds and somatic embryos in 2 weeks period. The effect of TDZ was different from other PGRs by inducing green solid sectors on calli surfaces (Total 51 sectors/20 callus/Akhisar variety). Five plantlets have been grown from these solid cell clumps and transferred to specific media for root formation. As a result, five varieties (Süleyman Bey, Bornova 92, Vamyk Hoca, Kaya and Akhisar) tested in our study showed the potential to produce regenerable callus by using low amounts of dicamba or TDZ. The optimization process starts from culturing embryos to plantlet formation took nearly 4 weeks.

Genetic characterization of Fusarium graminearum and F. culmorum isolates from Turkey by using random-amplified polymorphic DNA.

Five Fusarium graminearum and 12 F. culmorum isolates, primarily pathogenic species of Fusarium head blight, were obtained from naturally infected wheat from various agro-ecological regions of Turkey. Genotyping of the isolates was carried out using random-amplified polymorphic DNA (RAPD). Sixty-five 10-mer oligonucleotide primers were used to amplify the RAPD markers. Among them, 50 primers produced strong and reproducible DNA amplicons. The remaining primers generated either insufficient or no amplification patterns. In total, 1200 fragments were scored, 311 of which were determined to be polymorphic and unique to the isolates. The produced RAPD markers ranged from 0.2 to 5 kb. The mean genetic similarity values of the F. graminearum and F. culmorum isolates were 61.5 and 65%, respectively. The similarity coefficient was 43 to 76.1% among F. graminearum isolates and 49 to 81.1% among F. culmorum isolates. Genetically, the most similar F. graminearum isolates were F6 and F7 (76.1%), which originated from the same agro-ecological region (Sakarya). The most similar F. culmorum isolates were F20 and F21 (81.1%), which were from different geographic regions (Bilecik and UÅŸak, respectively). Moreover, interspecific variation between the two species was determined to be 86.3 to 93.3%. Cluster analysis generated two branched groups, each containing isolates of one species, except F13 of F. culmorum. The sequencing of stable and reproducible monomorphic and polymorphic RAPD markers indicated that the Fusarium genome shared high similarity (105-625 bit scores) with the genomes of other organisms as well as with the F. graminearum reference genome.

Geneticin (G418) resistance and electroporation-mediated transformation of Fusarium graminearum and F. culmorum

Fusarium graminearum and F. culmorum are phytopathogenic species causing scab and root rot diseases in all small grain cereals worldwide including Turkey. In this study, resistance levels to geneticin (G418) of 14 F. graminearum and 24 F. culmorum isolates collected from cereals were determined. Fungal cultures were grown on potato dextrose agar medium supplemented with 0, 25, 50, 75 and 100 µg/mL of G418. Minimum inhibitory concentration was determined as 25 µg/mL. As a result, it was concluded that all isolates were highly sensitive to G418. Plasmid pFA6-kanmx4 containing geneticin resistance gene (kanmx) was introduced singly or co-electroporated with pEGFP75 plasmid, containing GFP gene, into fungal protoplast cultures obtained with lytic enzyme. Transformants were grown in media including 25 µg/mL G418. Transformation frequencies were 2.8 and 1.8 transformant per µg plasmid for F. graminearum and F. culmorum isolates, respectively. Transformation process was also confirmed by spectrofluorimetric assay. Relative fluorescence unit values in co-transformants were calculated as 1.87 ± 0.04 for F. graminearum and 2.26 ± 0.08 for F. culmorum. The results obtained from the study gave information about antibiotic resistance levels of two Fusarium species in Turkey. Moreover, it was shown that pFA6-kanmx4 plasmid was a suitable vector, which can be used in genetic manipulation studies of these two fungal species in particular suppression of endogenous and/or the expression of exogenous genes.

Amplification of Specific Genes by using RT-PCR Technique in Plants

ABSTRACT Reverse transcription polymerase chain reaction (RT-PCR) is a sensitive and powerful method for analyzing RNA. In this study, genes encoding polyphenol oxidase and glucanase enzymes were amplified by using RT-PCR technique in potato and chickpea, respectively. It was also determined that both of the genes were members of multigene family.

Expression Analysis of PKS13, FG08079.1 and PKS10 Genes in Fusarium graminearum and Fusarium culmorum

BACKGROUND Identification and quantification of mycotoxins produced by Fusarium species are important in controlling fungal diseases. OBJECTIVES Potential of zearalenone, butenolide and fusarin C production was investigated in five Fusarium graminearum and five F. culmorum isolates at molecular level. MATERIALS AND METHODS Presence of PKS13, FG08079.1 and PKS10 genes, associated with production of zearalenone, butenolide and fusarin C, respectively, were confirmed by PCR. In addition, expression levels of them together with housekeeping gene (β-tubulin) were detected by real time PCR. RESULTS PKS13 and FG08079.1 transcripts were determined in all isolates, while PKS10 specific primers failed to amplify any product, indicative of no expression. ΔΔCTCT of PKS13 was ranged between 1.79E-03-3.97E-03 and for FG08079.1 was between 0.25E-03 and 6.02E-03. The highest PKS13 expressions were 3.86E-03 in F. graminearum F9 and 3.97E-03 in F. culmorum F16. Maximum FG08079.1 expressions were calculated as 6.02E-03 and 3.81E-03 in F. graminearum 2F and F. culmorum F2, respectively. CONCLUSIONS We revealed that ten Fusarium isolates produced zearalenone and butenolide under culture conditions. However, fusarin C was not generated by them in these conditions.

Assessment of Differential Expression of Chitinase Genes Using RT-PCR in Chickpea

ABSTRACT In this study, we have found differences in chitinase gene expression between sensitive (Red chickpea) and resistant (Flip 91–4C) chickpea (Cicer arietinum L.) genotypes to Ascochyta blight using Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR) technique. Total RNAs extracted from leaves of sensitive and resistant genotypes were used for RT-PCR analysis with chitinase gene specific primer (chi) and oligo dT15 primer. Expression of chitinase genes was found to be different in the leaves of sensitive and resistant genotypes using this technique.