Aslihan Temel

GENETIC AND EPIGENETIC VARIATIONS IN BARLEY CALLI CULTURES

ABSTRACT Genetic and epigenetic stability of calli were investigated with RAPD and CRED-RA techniques, respectively. Mature embryos of barley (Hordeum vulgare cv. Zafer-160) were cultured in callus induction MS (Murashige and Skoog) medium supplemented with 1 mg/L 2,4-D and maintained on same medium for 24 weeks. During RAPD analyses 16 primers of tested 20 random primers, produced 103 amplification products. Twenty-five of them were common in both tissues however 24 were mature embryo and 54 were calli specific. Three primers, which produced best amplification products during RAPD analyses, were used for CRED-RA analyses and they produced 17 amplification products. There were 8 different amplification products. Culture conditions cause genetic variations and there are also evident cytosine methylation alterations.

Variations in BARE-1 insertion patterns in barley callus cultures.

The stability of aging barley calli was investigated with the barley retroelement 1 (BARE-1) retrotransposon specific inter-retrotransposon amplified polymorphism (IRAP) technique. Mature embryos of barley (Hordeum vulgare cv. Zafer-160) were cultured on callus induction MS medium supplemented with 3 mg/L 2,4-D and maintained on the same medium for 60 days. Ten IRAP primers were used in 25 different combinations. The similarity index between 30-day-old and 45-day-old calli was 84%; however, the similarity index between mature embryos and 45-day-old calli was 75%. These culture conditions caused BARE-1 retrotransposon alterations to appear as different band profiles. This is the first report of the use of the IRAP technique in barley in an investigation of callus development.

Effects of epirubicin on barley seedlings

Epirubicin (EPI) is one of the anthracycline antibiotics, which is used in cancer chemotherapy. It inhibits DNA and RNA synthesis and causes cell death by DNA cleavage and production of free radicals. In this study, phytotoxicity of EPI was investigated on root and shoot growth, antioxidant enzymes and retrotransposons’ movements in 10- and 20-day-old barley seedlings. Mature embryos of barley were germinated on Murashige and Skoog medium supplemented with 250 and 500 μg/ml EPI. Our results showed that EPI treatment significantly inhibited shoot and root growth when compared with control group. Treatment with 250 and 500 μg/ml of EPI reduced shoot length in the 10-day-old plants by approximately 1.5- and 2-fold, respectively; the same treatments reduced total root length by 2- and 4-folds, respectively. However, the shoot and root lengths of 20-day-old plants were observed to be more affected by EPI-treatment. A 500-μg/ml concentration decreased total protein levels and peroxidase (EC 1.11.1.11) activity and increased superoxide dismutase (EC 1.15.1.1) and catalase (EC 1.11.1.6) activities. To investigate the effect of EPI on the movements of BARE-1, SUKKULA and BAGY2 retrotransposons, inter-retrotransposon amplified polymorphism technique was performed. While some polymorphic polymerase chain reaction bands were observed for BARE-1, no polymorphism was identified in SUKKULA and BAGY2 movements. To our knowledge, this is the first report showing phytotoxic effects of EPI on plant germination and retrotransposons’ movements.

Genetic and epigenetic effects of salinity on in vitro growth of barley

Morphological, physiological and molecular changes were investigated in in vitro salt-stressed barley (Hordeum vulgare L. cv. Tokak). Mature embryos were cultured in Murashige and Skoog medium containing 0 (control), 50 and 100 mM NaCl for 20 days. Both concentrations inhibited shoot growth, decreased fresh weight and protein content, and increased SOD (EC 1.15.1.1) activity in a dose-dependent manner. The lower concentration increased root growth. Salinity caused nucleotide variations in roots, but did not affect shoot DNAs. The higher concentration caused methylation changes, mainly hypermethylation in shoots. This is the first study on genetic and epigenetic effects of salinity in barley.

Analysis of retrotransposition and DNA methylation in barley callus culture.

Mature barley (Hordeum vulgare cv. Zafer-160) embryos were cultured on callus induction medium (MS+ 4 mg l-1 Dicamba) for 30 days and embryogenic calli were transferred onto regeneration medium (MS+ 0.5 mg l-1 trans-zeatin riboside). Callus induction percentage was 67.2%; embryogenic callus induction percentage was 42.3% and their regeneration percentage was 63.8%. Retrotransposon movements and methylation alterations in 15-day-old, 30-day-old calli and 4-day-old barley seedling (control) were investigated with Inter-Retrotransposon Amplified Polymorphism (IRAP) and Methylation-Sensitive Restriction Fingerprinting (MSRF), respectively. IRAP patterns were quite monomorphic however MSRF indicated increase in cytosine methylation during callus formation. Changes in retroelement movements and methylation alterations were evaluated and discussed in the light of literature.

Transposon Activity in Plant Genomes

Transposable elements (TEs) were first discovered in maize plants. However, they exist in all plant species investigated so far. Although plants with small genomes have smaller transposon percentages, plants with large genomes have high transposon percentages. For example,Arabidopsis thaliana has a genome size of 125 Mb, which comprises 14 % transposons, and theHordeum vulgare genome (5300 Mb) has 80 %. TEs are classified into two major groups based on their transposition mechanism. Class I elements are characterized by DNA sequences with homology to reverse transcriptase, and they are often referred to as retroelements, retrotransposons, or retrovirus-like elements. Retrotransposons function by a copy-and-paste transposition mechanism. Class II TEs (DNA transposons) move by a cut-and-paste mechanism. TEs affect the genome dynamics of plants by regulation of gene expression and chromosomal mutations (such as duplications, insertions/deletions, and structural variations). Transposition rates among generations are about 10−3 to 10−4, which is a higher rate than spontaneous mutations. All TEs in a cell are named as transposomes, and transposomics is a new area to work with transposomes. Although some bioinformatics software has recently been developed for the annotation of TEs in sequenced genomes, there are very few computational tools strictly dedicated to the identification of active TEs using genome-wide approaches. In this review article, after a brief introduction and review of the transposable elements, we discuss the effects of TEs in plant gene expression and evolution, and also present our recent research data on barley retrotransposons.

Cytotoxic effects of metaphase-arresting methods in barley

Metaphase-arresting agents amiprophos-methyl (APM), colchicine (COL) and cell cycle-synchronization (CCS) with APM and hydroxyurea (HU) were tested for growth, metaphase index and cytogenetic abnomalities in barley (Hordeum vulgare cv. Bornova-92). Seeds were germinated for 2 days and then seedlings were treated with 8 μM (2.4 mg/L) APM for 2 h or 1.25 mM (0.5 g/L) COL or synchronized (CCS) with 1.25 mM (95 mg/L) hydroxyurea for 18 h and with 4 μM (1.2 mg/L) APM for 2 h. APM and CCS caused metaphase indices 12.57 and 38.82% respectively. COL also arrested metaphase (14.10%) but also resulted in nuclear aberrations (11.15%). After removal of APM and CCS, cells were released to grow and divide. However, COL caused irreversible effects on cell division and growth and meanwhile was shown to be effective for micronucleus formation.