فصلنامه ژنتیک نوین
سال هشتم شماره 1 (پیاپی 32، بهار 1392)

he high efficiency of gene transfer to maize (Zea mays L.) using a standard binary vector is used widely in studies of genetic engineering and functional Genomics. The objective of the present study was to enhance efficiency of gene transfer to maize varieties using heat shock and hormones. To do this, embryo induction and plant regeneration from immature embryos of B73 and Mo17 cultivars immature embryos (1.5- 2 mm) were infected with Agrobacterium tumefaciens strain AGLO1 harboring pCAMBIA3301 binary vector containing gus gene. The immature embryos were treated by T1 (46 °C for 3 min, on ice for 1 min, centrifugation in 20000 g at 4 °C for 30 min) and T2 (without T1 treatment) treatments before inoculation with Agrobacterium. Moreover, the effects of Dithiothreitol (DTT) and Pluronic acid F68 (F68) detergents were studied on transformation efficiency by adding them to culture media. The results showed that heat shock and centrifugation treatments improved slightly genetic transformation of B73 but not on Mo17. The addition of DTT or F68 to co-culture medium led to a dramatic increase in T-DNA transfer to the B73, while there was no effects on Mo17 transformation. In B73, depending on the type of treatment, 18-5% of the immature embryos were produced resistant calli to phosphinotricin (PPT). Histochemical GUS assay on 8 weeks calli resistant to PPT and PCR analysis confirmed the transferring and expression of gus gene. These analyses showed 18% transformation frequency in T1+F68 treatment in comparation with 0% in T2-Detergents. Based on our results, the treatment T1+ F68 was suggested an efficient methodology to transfer genes by Agrobacterium to maize cultivar B73.

oth DNA genomic fragment and open reading frame (ORF) encoding a Trx h, called VvCxxS2, were isolated and cloned from grape (Vitis vinifera L.) berry tissue. The DNA genomic fragment of VvCxxS2 possesses two introns at positions identical to the Trx h genes from grape and higher plant. The exon-intron junctions obey the rule GT-AG, as in higher plant genes. The intron position analysis in the chlamydomonas reinhardtii and vertebrates Trx genes revealed that two introns of chlamydomonas and one intron from vertebrates Trx genes are located at the same positions with introns of higher plants Trxs h and f genes, strongly suggesting a common origin for green algae, higher plants Trxs h and f, and vertebrates. The intron position analysis in the Trx o genes showed that these genes have been subjected a late evolution and in contrast to Trxs h and f, contain a different pattern of evolution. The introns position in Trxs m, p, x, and y was also analyzed. These genes possess one intron between the regions encoding the transit peptide and the mature protein, and suggest a recent entry of other introns during late evolution. Totally, obtained results suggest that Trxs f, h, and o have a eukaryotic origin whereas Trxs m, x, y, and p are related to prokaryotic Trxs, indicating different patterns of evolution.