In vitro expression of apocarotenoid genes in Crocus sativus L.

Calli were successfully induced from style explants of Crocus sativus L. on Murashige and Skoog's medium supplemented with -naphthalene acetic acid and 6-benzylaminopurine. Then they were divided into three different types based on developmental stages and pigmentation progress in induced stigma-like structures. RT-PCR method was set up using calli in different developmental stages to detect expression levels of CsLYC, CsBCH1, CsZCD and CsUGT2 genes for apocarotenoids biosynthesis via mevalonic acid pathway in C. sativus. The results obtained from in vitro investigationof CsUGT2 expression levels in all three developmental stages were analyzed and compared with the expression levels of selected genes carried out on intact stigmas in vivo. Apparently, this gene was only expressed in the stage III of the three in vitro different SLSs developmental stages. Furthermore,the expression levels of CsLYC, CsBCH1, CsZCD were detected in stage III with fully developed SLSs and were comparable with those of in red intact stigmas

An application of kernel methods to variety identification based on SSR markers genetic fingerprinting

<p>Abstract</p> <p>Background</p> <p>In crop production systems, genetic markers are increasingly used to distinguish individuals within a larger population based on their genetic make-up. Supervised approaches cannot be applied directly to genotyping data due to the specific nature of those data which are neither continuous, nor nominal, nor ordinal but only partially ordered. Therefore, a strategy is needed to encode the polymorphism between samples such that known supervised approaches can be applied. Moreover, finding a minimal set of molecular markers that have optimal ability to discriminate, for example, between given groups of varieties, is important as the genotyping process can be costly in terms of laboratory consumables, labor, and time. This feature selection problem also needs special care due to the specific nature of the data used.</p> <p>Results</p> <p>An approach encoding SSR polymorphisms in a positive definite kernel is presented, which then allows the usage of any kernel supervised method. The polymorphism between the samples is encoded through the Nei-Li genetic distance, which is shown to define a positive definite kernel between the genotyped samples. Additionally, a greedy feature selection algorithm for selecting SSR marker kits is presented to build economical and efficient prediction models for discrimination. The algorithm is a filter method and outperforms other filter methods adapted to this setting. When combined with kernel linear discriminant analysis or kernel principal component analysis followed by linear discriminant analysis, the approach leads to very satisfactory prediction models.</p> <p>Conclusions</p> <p>The main advantage of the approach is to benefit from a flexible way to encode polymorphisms in a kernel and when combined with a feature selection algorithm resulting in a few specific markers, it leads to accurate and economical identification models based on SSR genotyping.</p

Phytobiosynthesis of gold nano-particles and comparison of two plant species (Canola and Alfalfa)

245-247Different varieties of two plant species, Canola and Alfalfa were grown at exactly same laboratory conditions and bio-reduction of Au (III) to Au (0) was studied. Subsequently, production of gold nano-particles of various morphologies and sizes were characterized. Plant seeds were grown in a culture medium that contained gold ions from KAuCl4. Gold nano-particle formation was analyzed by atomic absorption spectrometry and transmission electron microscopy. Results showed that the plants pull up gold ions from KAuCl4 and form gold particles in nano sizes due to chemical behaviour of the gold. Significant differences in the nature of nano-particles were observed when particles synthesized by these two plant species were compared. The size range of gold nano-particles synthesized by Canola was 20-128 nm, while it was 8-48 nm by Alfalfa

Classification of rice germplasm. I. Analysis using ALP and PCR-based RFLP

The potential of using a PCR-based approach to detect DNA polymorphism for rice germplasm classification was compared with that of Southern-based RFLP analysis. Thirty-five Iranian rice varieties were studied along with 2 typical Indica and 3 typical Japonica varieties. Thirteen mapped RFLP markers were used as hybridization probes against Southern blots containing digests of one restriction endonuclease; 12 of the 13 probes detected polymorphism in the varieties. Fifteen sets of oligonucleotides derived from sequences near the ends of the same probes and of two other mapped probes were used as primers for PCR amplification of total genomic DNA of the varieties. Amplicon length polymorphisms (ALPs) were detected with 6 of the 15 sets of primers. To identify additional polymorphism, the PCR products were digested with nine different restriction endonucleases recognizing 4- or 5-bp DNA sequences and analyzed by gel electrophoresis in agarose and polyacrylamide. RFLPs were detected for 11 sets of primers, due to point mutations and to addition/deletion events that were too small to be detected as ALPs. Because PCR products are easily generated and may be analyzed in detail through the use of restriction endonucleases that cut rice DNA frequently, PCR-based RFLP analysis is a useful tool for the classification of rice germplasm

A proteomic approach to analyzing drought- and salt-responsiveness in rice

The analysis of stress-responsiveness in plants is an important route to the discovery of genes conferring stress tolerance and their use in breeding programs. Proteomic analysis provides a broad view of plant responses to stress at the level of proteins. In recent years this approach has increased in sensitivity and power as a result of improvements in two-dimensional polyacrylamide gel electrophoresis (2DE), protein detection and quantification, fingerprinting and partial sequencing of proteins by mass spectrometry (MS), bioinformatics, and methods for gene isolation. 2DE provides information on changes in abundance and electrophoretic mobility of proteins, the latter reflecting post-translational modifications such as phosphorylation and free-radical cleavage. Here we review the technical aspects of proteomics and demonstrate its use in analyzing the response of rice plants to drought and salinity. More than 2000 proteins were detected reproducibly in drought-stressed and well-watered leaves. Out of >1000 proteins that were reliably quantified, 42 proteins changed significantly in abundance and/or position. We identified several leaf proteins whose abundance increased significantly during drought and declined on re-watering. The three most marked changes were seen with actin depolymerizing factor, a homologue of the S-like ribonucleases and the chloroplastic glutathione-dependent dehydroascorbate reductase. Proteomic comparisons of salt stress-tolerant and stress-sensitive genotypes revealed numerous constitutive and stress-induced differences in root proteins. Among them was caffeoyl-CoA O-methyltransferase, an enzyme of lignin biosynthesis. The abundance of ascorbate peroxidase was much higher in salt-tolerant Pokkali than in salt-sensitive IR29 in the absence of stress

Initiation and origin of stigma-like structures [SLS] on ovary and style explants of saffron in tissue culture

Saffron, made from the dried stigmas of Crocus sativus L., contains pigments and valuable aromatic compounds, and can be used in medicine and as a spice. Nowadays its production is lower than demand. Tissue culture presents an alternative biochemical tool which can be used to produce stigma-like structure (SLS) in vitro. In this study, the origin and induction of SLS formation was investigated in ovary and style explants of floral buds on MS medium supplemented with 1-naphthalene acetic acid (NAA) and 6-benzlaminopurine (BAP). SLS were directly originated through meristematic cells or indirectly in the form of colorless globular structures from parenchyma tissue. The colorless globular structures initially were conical and pale yellow color at the sharp ends; subsequently they matured into trumpet-like red stigmas with or without finger-like papillae at the margins. Light and electron microscopic observations of ultra- and semithin sections of different developmental stages of SLS showed that these structures possess two kinds of cells: (1) small cells close to parenchyma tissues and (2) large cells oriented towards the peripheral area and apparently originated from the small ones. Our results suggest that the SLS originated from internal parenchyma tissues

Enhanced resistance to two stem borers in an aromatic rice containing a synthetic cryIA(b) gene

Rice cultivars of isozyme group V include high-quality, aromatic rices that are difficult to improve by traditional methods because of the loss of quality characters upon sexual hybridization. Their low-tillering plant type predisposes them to economic loss from attack by stem borers, a group of insects to which they are susceptible. We report here the enhancement of stem borer resistance in cv. Tarom Molaii through transformation by microprojectile bombardment. Embryogenic calli derived from mature seeds were bombarded with gold particles coated with plasmid pCIB4421, carrying a synthetic truncated toxin gene based on the cryIA(b) gene from Bacillus thuringiensis, and plasmid pHygII, carrying the hygromycin phosphotransferase (hpt) selectable marker gene. Inclusion of 50 mg/l hygromycin B in culture media from bombardment through to rooting of plantlets eliminated escapes. The procedure generated three independent hpt transformants of which two also contained the cryIA(b) gene. One such line (No. 827) produced truncated (67 kDa) CryIA(b) protein equivalent to about 0.1% of total soluble protein. The cryIA(b) gene was controlled by the promoter of the maize C<SUB>4</SUB> PEP carboxylase gene and was expressed in leaf blades but was not expressed to a detectable level in dehulled mature grain. Line 827 contained about 3 copies of the cryIA(b) gene which segregated as a single dominant Mendelian locus in the second (T<SUB>1</SUB>) and third (T<SUB>2</SUB>) generations and co-segregated with enhanced resistance to first-instar larvae of striped stem borer (Chilo suppressalis) and yellow stem borer (Scirpophaga incertulas). T<SUB>2</SUB> line 827-6 homozygous for the cryIA(b) gene showed no dead hearts or whiteheads after infestation with stem borers, whereas T<SUB>2</SUB> line 827-25 lacking the gene averaged 7 dead hearts per plant and 2.25 whiteheads per plant. These results establish that transformation of high-quality rices of group V is a feasible alternative to sexual hybridization

Sequence-tagged sites and low-cost DNA marker technology for rice

Sequence-tagged sites (STSs) facilitate the conversion of a genetic map into a physical map, provide a common basis for the comparison of diverse types of mapping data, are stored and disseminated as electronic data, and are amplified from genomic DNA by polymerase chain reaction (PCR). STSs find application as DNA markers in breeding programs and germplasm management because they offer speed, convenience, reliability, and low cost in genomic analysis, but these applications are currently limited by the small number of STSs available. We report here the terminal sequencing of 354 DNA markers of the Cornell-IRRI genetic map of rice and the conversion of 100 of them into STSs by synthesis of pairs of PCR primers. PCR was used to amplify the corresponding loci from genomic DNA of IR36 (indica), Taichung 65 (japonica), and Oryza longistaminata (AA genome wild species). More than half of the RZ clones amplified DNA segments that were 0.1-2.0 kbp larger than expected, presumably because of the presence of introns. Amplicon length polymorphisms were detected between O. sativa and O. longistaminata for about one quarter of the clones. The applications of STSs are illustrated by reference to 1) DNA marker-aided selection for pyramiding of bacterial blight resistance genes, 2) breeding for gall midge resistance, 3) monitoring the inheritance of transgenes, and 4) analysis of genetic variation of AA genome wild species