A yeast cadmium factor 1 (YCF1) is a member of the ATP-binding cassette (ABC) transporter family associated with multi-drug resistance, and it is localized at the vacuolar membrane in Saccharomyces cerevisiae. To determine ability to increase heavy metal tolerance and accumulation, YCF1 was introduced into Brassica juncea plants by Agrobacterium-mediated genetic transformation. YCF1 gene presence in transgenic plants was demonstrated by polymerase chain reaction (PCR). Reverse transcriptase-PCR analysis confirmed YCF1 gene expression in the transgenic plants, but the degree of YCF1 expression varied among the lines. YCF1 overexpression in B. juncea conferred enhanced tolerance to cadmium (Cd [II]) and lead (Pb[II]) stress. Transgenic B. juncea seedlings showed 1.3-to 1.6-fold tolerance to Cd stress and 1.2-to 1.4-fold tolerance to Pb stress compared to wild type (WT) plants (per gram fresh weight). Most importantly, the shoot tissues of transgenic seedlings contained about 1.5-to 2-fold higher Cd(II) and Pb(II) levels than those of WT, demonstrating significantly increased accumulation of both Cd(II) and Pb(II) in transgenic plants.

Molecular cytogenetic analyses using fluorescence in situ hybridization (FISH) and genomic in situ hybridization (GISH) were carried out to elucidate inter-specific relationships among wild Lilium species distributed in Korea. FISH revealed four to eight 45S rRNA gene loci, which are located on chromosomes 1-7, 10, and 11 among the different species. In contrast, the 5S rRNA gene locus was conserved on the long arm of chromosome 3, occasionally with two adjacent sites on the same chromosome arm in a few species. The 5S rDNA site was located adjacent to the 45S rDNA site in only three species, Lilium distichum, Lilium hansonii, and Lilium tsingtauense. GISH analysis using genomic DNA probes detected strong hybridization of genomes between diploid and triploid Lilium lancifolium species, demonstrating that triploid plants were derived from diploid L. lancifolium and not from Lilium maximowiczii. Phylogenetic analysis of the ITS and NTS sequences supported the cytogenetic data as well as Comber's classification of the genus Lilium.

AtATM3, a member of the ATP-binding cassette transporter family, is localized at the mitochondrial membrane of Arabidopsis thaliana and is involved in the biogenesis of Fe-S clusters and iron homeostasis in plants.Through Agrobacterium-mediated genetic transformation, the AtATM3 gene driven by the cauliflower mosaic virus 35S promoter (CaMV35S) was introduced into Brassica juncea (Indian mustard), a plant species suitable for phytoremediation, with the aim of improving heavy metal tolerance and accumulation in plants. The presence of the AtATM3 gene in transgenic plants was confirmed by polymerase chain reaction (PCR). Reverse transcriptase-PCR analysis confirmed AtATM3 expression in transgenic plants, but the level of AtATM3 expression varied between lines. AtATM3 overexpression in B. juncea conferred enhanced tolerance to cadmium [Cd(II)] and lead [Pb (II)] stresses. Importantly, the shoot tissues of transgenic seedlings contained about 1.5-to 2.5-fold higher Cd(II) and Pb(II) levels than wild type (WT) seedlings, demonstrating significantly-increased accumulation of both Cd(II) and Pb(II) in transgenic plants. The enhanced capacity of heavy metal tolerance and accumulation by AtATM3 transgenic plants was attributed to higher BjGSHII (B. juncea glutathione synthetase II) and BjPCS1 (phytochelatin synthase 1) expression levels induced by AtATM3 overexpression. In addition, AtATM3 overexpression regulated the expression of several metal transporters in the transgenic B. juncea plants under heavy metal stress conditions. Therefore, AtATM3 transgenic plants are more tolerant of and can accumulate more heavy metals to enhance phytoremediation of contaminated soils.

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Internal transcribed spacers (ITS) of nuclear ribosomal DNA cistrons were studied in wild Lilium species native to Korea and compared with those species in the genus distributed in other countries to investigate the phylogenetic relationships among Lilium species. Sequence analysis data of Lilium plants collected from China and Japan were also included to study the inter-and intra-specific relationships in the genus. Lilium species formed a monophyletic clade and were separated in two clusters that belonged to the section Sinomartagon and Martagon. The phylogenetic relationships of Lilium species inferred from the ITS sequences using the maximum likelihood method were highly congruent with previous studies of cytogenetic evidence and classification (Comber, 1949) based on the morphological characters. The length of ITS1 and ITS2 regions in Lilium species native to Korea was uniform, however, nucleotide polymorphisms were noted. A region with a conserved sequence was observed from position 330-340 among all the species. Comparison of the sequence analysis of Lilium accessions native to different countries revealed the presence of six insertions/deletions (indels) and 121 substitutions (transitions and transversions) in both regions of ITS1 and ITS2. Indels were responsible for variation of ITS length in different accessions. The sequences of Lilium species collected from each country had less variations than those of species collected from different countries. Sequences variations in both ITS regions suggested that they could be used for assaying genetic diversity in Lilium at the inter-and intra-specific level.Additional key words: lily, nuclear ribosomal DNA, phylogeny, sequence polymorphism Hort. Environ. Biotechnol. 52(5):502-510. 2011.The ribosomal RNA genes (45S rDNA) of higher eukaryotes are part of repeat units arranged in tandem arrays within the nucleolar organizer regions (NORs), which typically are about 10 kb (Heslop-Harrison, 2000). Each repeat unit of 45S rDNA consists of individual 18S, 5.8S, and 26S rRNA genes, which are separated from the next repeat unit by the intergenic spacer region (IGS) (Apples and Honeycutt, 1986). Within each repeat, the coding regions of these three genes are separated by internal transcribed spacers (ITS) that flank the 5.8S rRNA gene (Gründler et al., 1991;Wanzenböck et al., 1997).The coding regions of these rRNA genes show a little sequence divergence between closely related species, whereas the ITS regions show higher rate of divergence that is useful for inferring phylogenies of closely related species and populations in a given species. The length and sequence of ITS regions are believed to be fast evolving and, therefore, may be variable. In angiosperms, ITS sequences vary in length from approximately 500-700 bp (Baldwin et al., 1995). Because of their relatively high rate of mutations, ITS regions are useful for studying inter-or intra-specific genetic variations (Delgado-Salinas et al.