Isolation and culture of protoplasts of pigeonpea (Cajanus cajan L.)

Summary High yields of protoplasts were obtained from leaves of aseptically grown plants and calli originated from different explants, in several cultivars of Cajanus cajan L. The protoplasts divided to form cell clusters in modified KM 8p medium and developed to protocolonies after dilution with liquid Caboche's medium within three to four weeks of culture. The protocolonies proliferated to form green calli on solid Caboche's medium. No shoots or plants were obtained.Abbreviations BAP 6-benzylaminopurine - NAA -napthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - Kin kinetin - Zea zeatin - Adn S adenine sulphate - GA ₃ gibberellic acid     

Mechanical cues in cellular signalling and communication

Multicellular organisms comprise an organized array of individual cells surrounded by a meshwork of biomolecules and fluids. Cells have evolved various ways to communicate with each other, so that they can exchange information and thus fulfil their specified and unique functions. At the same time, cells are also physical entities that are subjected to a variety of local and global mechanical cues arising in the microenvironment. Cells are equipped with several different mechanisms to sense the physical properties of the microenvironment and the mechanical forces arising within it. These mechanical cues can elicit a variety of responses that have been shown to play a crucial role in vivo. In this review, we discuss the current views and understanding of cell mechanics and demonstrate the emerging evidence of the interplay between physiological mechanical cues and cell-cell communication pathways.     

Lox-dependent gene expression in transgenic plants obtained via Agrobacterium-mediated transformation

Lox sites of the Cre/lox recombination system from bacteriophage P1 were analyzed for their ability to affect on transgene expression when inserted upstream from a gene coding sequence adjacent to the right border (RB) of T-DNA. Wild and mutated types of lox sites were tested for their effect upon bar gene expression in plants obtained via Agrobacterium-mediated and biolistic transformation methods. Lox-mediated expression of bar gene, recognized by resistance of transgenic plants to PPT, occurred only in plants obtained via Agrobacterium-mediated transformation. RT-PCR analysis confirms that PPT-resistant phenotype of transgenic plants obtained via Agrobacterium-mediated transformation was caused by activation of bar gene. The plasmid with promoterless gus gene together with the lox site adjacent to the RB was constructed and transferred to Nicotiana tabacum as well. Transgenic plants exhibited GUS activity and expression of gus gene was detected in plant leaves. Expression of bar gene from the vectors containing lox site near RB allowed recovery of numerous PPT-resistant transformants of such important crops as Beta vulgaris, Brassica napus, Lactuca sativa and Solanum tuberosum. Our results demonstrate that the lox site sequence adjacent to the RB can be used to control bar gene expression in transgenic plants.     

Maternal genomic variability of the wild boar (Sus scrofa) reveals the uniqueness of East‐Caucasian and Central Italian populations

The phylogeography of the European wild boar was mainly determined by postglacial recolonization patterns from Mediterranean refugia after the last ice age. Here we present the first analysis of SNP polymorphism within the complete mtDNA genome of West Russian (n = 8), European (n = 64), and North African (n = 5) wild boar. Our analyses provided evidence of unique lineages in the East‐Caucasian (Dagestan) region and in Central Italy. A phylogenetic analysis revealed that these lineages are basal to the other European mtDNA sequences. We also show close connection between the Western Siberian and Eastern European populations. Also, the North African samples were clustered with the Iberian population. Phylogenetic trees and migration modeling revealed a high proximity of Dagestan sequences to those of Central Italy and suggested possible gene flow between Western Asia and Southern Europe which was not directly related to Northern and Central European lineages. Our results support the presence of old maternal lineages in two Southern glacial refugia (i.e., Caucasus and the Italian peninsula), as a legacy of an ancient wave of colonization of Southern Europe from an Eastern origin.     

Comparison of several Nicotiana species as hosts for high-scale Agrobacterium-mediated transient expression

Agrobacterium-mediated transient expression may be regarded as a promising method for inexpensive large-scale production of recombinant proteins. We optimized the protocol of transient expression in Nicotiana benthamiana and compared six Australian species of Nicotiana as hosts for transient expression. The transient expression of GFP under 35S CaMV promoter was observed in all species tested, although the GFP content in leaves of N. benthamiana, N. exigua, and N. excelsior was significantly higher (3.8, 3.7, and 2.0% TSP, respectively). Usage of viral-based expression system resulted in considerable increase of GFP accumulation in N. excelsior and N. benthamiana (63.5 and 16.2% TSP, respectively). We displayed that N. excelsior has the best characteristics in regard to biomass yield as well as GFP accumulation level for both types of the expression cassettes tested.     

Tunneling nanotubes: a new route for the exchange of components between animal cells

Recently, highly sensitive nanotubular structures mediating membrane continuity between mammalian cells have been discovered. With respect to their peculiar architecture, these membrane channels were termed tunneling nanotubes (TNTs). TNTs could form de novo between animal cells leading to the generation of complex cellular networks. They have been shown to facilitate the intercellular transfer of organelles as well as, on a limited scale, of membrane components and cytoplasmic molecules. It has been proposed that TNTs represent a novel and general biological principle of cell-to-cell communication and it becomes increasingly apparent that they fulfill important functions in the physiological processes of multicellular organisms.     

The ability of protein tyrosine phosphatase SHP-1 to suppress NFkappaB can be inhibited by dominant negative mutant of SIRPalpha

In contrast with hematopoietic cells and fibroblasts, which express mainly one form of protein tyrosine phosphatase (PTP) SHP-1 or SHP-2, epithelial cells like A431, HeLa, and 293 express both forms of PTP. These two PTP regulate NFkappaB activity differently; SHP-1 inhibits and SHP-2 stimulates NFkappaB activation. In epithelial cells the process of NFkappaB activation depends on the combination of two PTP activities. The activity of PTP SHP-1 dominates in this tandem according to our data. The signal regulatory protein (SIRPalpha) is the adapter and the substrate of PTP SHP-1 and SHP-2. We investigated the role of SIRPalpha and its dominant negative mutant in PTP activities in 293 cells. The overexpression of wild-type SIRPalpha suppresses the activities of both PTP, but has a stronger effect on PTP SHP-2, especially when this protein is overexpressed in 293 cells. In contrast with wild-type SIRPalpha, its dominant negative mutant acts predominantly against PTP SHP-1, and can be detected in the complex with PTP SHP-1. The expression of dominant negative mutant of SIRPalpha has an effect similar to the expression of dominant negative PTP SHP-1 in the process of NFkappaB activation.     

Activity of the corn Spm transposon system in transgenic plants Orychophragmus violaceus (L.) O.E. Schulz obtained by both direct transfer of DNA to protoplasts and agrobacterial transformation of root explants

Transposon mediated insertional mutagenesis is one of the approaches for the unique gene cloning. A wild species of Cruciferae family Orychophragmus violaceus (L.) O.E. Schulz, which is of interest for practical breeding as a donor of improved plant oil, was an object of the investigation. Plasmid construction used in the experiments included selective NPT II gene, reported GUS gene serving as an excision marker, structural BAR gene located within the dSpm element and Spm transposase. The GUS gene of this plasmid had not his own promoter and became functional only after Spm-transposition. Transformed Orychophragmus violaceus (L.) O.E. Schulz. plants were obtained by direct mesophyll protoplast transformation as well as Agrobacterium tumefaciens-mediated root explant transformation. Gene transfer and the transposition event were confirmed by the GUS activity and the PCR analysis. Relative transformation efficiency using protoplasts was 5.8%.     

Obtaining and analysis of intergeneric somatic hybrids between Brassica napus and "albino" line of Orychophragmus violaceus

The Orychophragmus violaceus chlorophylldefective line of "albino" type has been obtained by spectinomycin treatment. Somatic hybridization between Orychophragmus violaceus and Brassica napus was performed by fusion of green mesophyll protoplasts of rape and callus protoplasts of the O. violaceus "albino" line. Near two hundred of regenerant plants were selected according to the regeneration type and ability to become green, and were determined as hybrids. Chloroplast DNA in selected hybrids was identical to rape chlDNA, which was confirmed by the PCR-RFLP analysis of plastid DNA fragments. Fragments of hybrid mitochondrial DNA analyzed by the PCR-RFLP analysis were identical to fragments of O. violaceus. The nuclear genome of the majority of hybrids was represented by the O. violaceus genome, which was demonstrated by analyses of isoenzymes, DNA telomeric sequences, ribosomal and satellite DNAs, and the RAPD analysis. The cytogenetic analysis of a number of lines has shown variability in the number of chromosomes in the obtained lines.     

Analysis of nuclear and mitochondrial genomes of transplastomic Salpiglossis sinuata plants obtained by transfer of transformed plastids from N. tabacum (+S. sinuata) cybrid

New model system of plastid transformation has been proposed using a wild representative of Solanaceae family--S. sinuata. Earlier obtained cybrid plants N. tabacum (+ S. sinuata) were used for transformation experiments by PEG treatment of protoplasts with aadA gene that confers resistance to spectinomycin. Transformed S. sinuata plastome was transferred from N. tabacum (+ S. sinuata) cybrid to S. sinuata wild type plants by somatic hybridization. Molecular analysis of nuclear and mitochondrial DNA has been performed.