Reinhold Brettschneider

Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories

A number of PCR-based techniques can be used to detect polymorphisms in plants. For their wide-scale usage in germplasm characterisation and breeding it is important that these marker technologies can be exchanged between laboratories, which in turn requires that they can be standardised to yield reproducible results, so that direct collation and comparison of the data are possible. This article describes a network experiment involving several European laboratories, in which the reproducibility of three popular molecular marker techniques was examined: random-amplified fragment length polymorphism (RAPD), amplified fragment length polymorphism (AFLP) and sequence-tagged microsatellites (SSR). For each technique, an optimal system was chosen, which had been standardised and routinely used by one laboratory. This system (genetic screening package) was distributed to different participating laboratories in the network and the results obtained compared with those of the original sender. Different experiences were gained in this exchange experiment with the different techniques. RAPDs proved difficult to reproduce. For AFLPs, a single-band difference was observed in one track, whilst SSR alleles were amplified by all laboratories, but small differences in their sizing were obtained.

Regeneration of transgenic, microspore-derived, fertile barley

We have developed a system for the biolistic transformation of barley using freshly-isolated microspores as the target tissue. Independent transformation events led, on average, to the recovery of one plant per 1×107 bombarded microspores. Putative transformants have been regenerated using phosphinothricin as a selective agent. R0 plants have been transferred to soil approximately 2 months after bombardment. Integration of the marker genes bar and uidA has been confirmed by Southern analysis. The marker genes are inherited in all progeny plants confirming the expected homozygous nature of the R0 plants.

Molecular characterisation of two novel maize LRR receptor-like kinases, which belong to the SERK gene family

Abstract. Genes encoding two novel members of the leucine-rich repeat receptor-like kinase (LRR-RLK) superfamily have been isolated from maize (Zea mays L.). These genes have been named ZmSERK1 and ZmSERK2 since features such as a putative leucine zipper (ZIP) and five leucine rich repeats in the extracellular domain, a proline-rich region (SPP) just upstream of the transmembrane domain and a C-terminal extension (C) after the kinase domain identify them as members of the SERK (omatic mbryogenesis eceptor-like inase) family. ZmSERK1 and ZmSERK2 are single-copy genes and show 79% identity among each other in their nucleotide sequences. They share a conserved intron/exon structure with other members of the SERK family. In the maize genome, ZmSERK1 maps to position 76.9 on chromosome arm 10L and ZmSERK2 to position 143.5 on chromosome arm 5L, in regions generally not involved in duplications. ZmSERK1 is preferentially expressed in male and female reproductive tissues with strongest expression in microspores. In contrast, ZmSERK2 expression is relatively uniform in all tissues investigated. Both genes are expressed in embryogenic and non-embryogenic callus cultures.

Stable transformation of barley via PEG-induced direct DNA uptake into protoplasts

SummaryProtoplasts isolated from a barley cell suspension (cv Dissa) were transformed with plasmid DNA containing the neomycinphosphotransferase II (NPT) and β-glucuronidase (GUS) genes, using polyethyleneglycol (PEG) to induce DNA uptake. Transformed microcalli were selected in media containing G418 sulphate. NPT activity was detected in all antibiotic-resistant cell lines, but not all NPT-positive cell lines had GUS activity. Southern analysis confirmed the presence of sequences homologous to the APT and GUS genes in DNA of G418-resistant callus.

Efficient transformation of scutellar tissue of immature maize embryos

Abstract  An efficient transformation system for maize was established by improving transformation conditions for the particle bombardment of the scutellar tissue of immature embryos. Particle bombardment was carried out using constructs containing the pat gene as the selection marker and a PDS 1000/He gun (Biorad). Transformation parameters, such as the amount of gold particles used per bombardment, particle velocity, preculture time of the scutellum prior to bombardment and osmotic treatment of the target tissue before and after bombardment, were analysed. Fertile transgenic regenerants of the maize inbred lines H99, A188 and Pa91 and the crosses A188×H99 and Pa91×H99 were selected on Basta-containing medium. The transformation frequency was between 2% and 4%. A total of 29 transgenic plant lines was obtained and verified with Southern blot analysis. All of the transgenic plants were fertile and set seeds. The R1 progeny of single plants was analysed. A Mendelian segregation of the transgenes was observed for all of the transformants tested. For 1 candidate, stable inheritance and stable expression of the transgenes were followed up to the R4 generation.

Comparative Analysis of Different DNA Extraction Protocols: A Fast, Universal Maxi-Preparation of High Quality Plant DNA for Genetic Evaluation and Phylogenetic Studies

Four DNA extraction protocols were compared for ability to produce DNA from the leaves or needles of several species: oak, elm, pine, fir, poplar and maize (fresh materials) and rhododendron (silica dried or frozen material). With the exception of maize and poplar, the species are known to be difficult for DNA extraction. Two protocols represented classical procedures for lysis and purification, and the other two were a combination of classical lysis followed by anion exchange chromatography. The DNA obtained from all procedures was quantified and tested by PCR and Southern hybridisation.Test results indicated superiority of one of the four protocols; a combination of CTAB lysis followed by anion exchange chromatography which enabled DNA extraction from all seven species. A second protocol also produced DNA from leaves or needles of all species investigated and was well suited for PCR applications but not Southern hybridisations. The remaining protocols produced DNA from some but not all species tested.Abbreviations: CTAB, hexadecyltrimethylammonium bromide; EtOH, Ethanol; TBE, tris-borate-EDTA.

UV-inducible transient expression in parsley protoplasts identifies regulatory cis-elements of a chimeric Antirrhinum majus chalcone synthase gene

It was shown previously that parsley protoplasts retain differential responsiveness to external stimuli, e.g. UV light. This opened the way for the development of transient expression assays to identify DNA sequences acting as regulatory cis‐elements in the transcriptional activation of UV‐inducible plant genes. The chalcone synthase (chs) gene of Antirrhinum majus (Snapdragon) is inducible by visible and by UV light. We demonstrate that the kinetics of light induction of a chimeric A.majus chs‐nptII gene observed during transient expression in the parsley protoplasts reflect the kinetics of UV‐induced CHS expression in A.majus seedlings in vivo. We define three regulatory sequence regions, each differing qualitatively and quantitatively in their effects on gene expression. Immediately upstream of the TATA box (‐34) a sequence, with coordinates −39 to −197, functions as an orientation independent UV‐light responsive element. The complete 1.1 kb promoter or three tandem copies of this element are capable of rendering a heterologous minimal promoter UV responsive. The next upstream region (‐197 to −357) contains sequences that do not by themselves cause UV‐induced expression, but specifically potentiate the level of UV‐induced expression when combined with the TATA‐box proximal UV‐responsive element. A third element (‐661 to −564) has the properties of a general enhancer since it increases the level of both uninduced and UV‐induced expression.

An Essential Pentatricopeptide Repeat Protein Facilitates 5′ Maturation and Translation Initiation of rps3 mRNA in Maize Mitochondria

This work finds that a maize PPR protein (MPPR6) localized to the mitochondria is directly involved in 5′ maturation and translation initiation of rps3 mRNA. This dual role supports a general principle of action for PPR proteins in RNA processing and translation. Pentatricopeptide repeat (PPR) proteins are members of one of the largest nucleus-encoded protein families in plants. Here, we describe the previously uncharacterized maize (Zea mays) PPR gene, MPPR6, which was isolated from a Mutator-induced collection of maize kernel mutants by a cDNA-based forward genetic approach. Identification of a second mutant allele and cosegregation analysis confirmed correlation with the mutant phenotype. Histological investigations revealed that the mutation coincides with abnormities in the transfer cell layer, retardation of embryo development, and a considerable reduction of starch level. The function of MPPR6 is conserved across a wide phylogenetic distance as revealed by heterologous complementation of the Arabidopsis thaliana mutant in the orthologous APPR6 gene. MPPR6 appeared to be exclusively present in mitochondria. RNA coimmunoprecipitation and in vitro binding studies revealed a specific physical interaction of MPPR6 with the 5′ untranslated region of ribosomal protein S3 (rps3) mRNA. Mapping of transcript termini showed specifically extended rps3 5′ ends in the mppr6 mutant. Considerable reduction of mitochondrial translation was observed, indicating loss of RPS3 function. This is consistent with the appearance of truncated RPS3 protein lacking the N terminus in mppr6. Our results suggest that MPPR6 is directly involved in 5′ maturation and translation initiation of rps3 mRNA.

Fertile, transgenicTriticale ( ×Triticosecale Wittmack)

Fertile transgenicTriticale ( ×Triticosecale Wittmack) plants expressing theβ-glucuronidase (uidA) and phosphinothricin acetyltransferase (bar) genes were obtained after microprojectile bombardment of scutellar tissue with the plasmid pDB1 containing theuidA gene under the control of the actin-1 promoter (Act1) from rice and the selectable marker genebar under the control of the CaMV 35S promoter. From 465 bombarded scutella about 4000 plantlets were regenerated; 300 plants survived the selection. These regenerants were screened for enzyme activity by the histological GUS assay and by spraying the plants with a herbicide (Basta). Twenty-five regenerants showed GUS activity and survived repeated Basta spraying. Southern blot analysis showed the presence of both marker genes introduced into the genome of analysed plants.All transgenic plants were fertile. They were grown to maturity and set seed. Pollen and progeny analyses provided evidence for inheritance of the introduced genes to the next generation.

TRANSPARENT LEAF AREA1 encodes a secreted proteolipid required for anther maturation, morphogenesis, and differentiation during leaf development in maize.

We report the identification and functional analysis of TRANSPARENT LEAF AREA1 (TLA1), a maize (Zea mays) gene representing a novel class of secreted, extremely hydrophobic peptides (proteolipids) with a C-terminal Caax box–like motif. ZmTLA1 encodes 27 amino acid residues and is most strongly expressed in the egg cell and microspores. Lower transcript amounts were detected during vegetative development. Transgenic maize expressing an antisense transcript displayed a variety of phenotypes. The most visible phenotypes were dwarfism and transparent leaf areas resulting from defective morphogenesis of mesophyll, bundle sheath, stomatal, and epidermal cells during leaf development. Incomplete cell walls were observed, indicating a defect of cytokinesis. The accumulation of gerontoplasts was probably a secondary effect caused by defects of leaf cell morphogenesis. A defect of anther maturation was observed in ∼30% of the plants displaying the tla phenotype. Male sterility was mainly caused by incomplete disintegration of the tapetal cell layers and tetrad callose as 90% of the microspores developed into functional pollen. Overexpression of ZmTLA1 seemed to have a lethal effect both in maize and Arabidopsis thaliana. Development of primary roots, root hairs, primary leaves, and chloroplasts was suppressed in Arabidopsis seedlings expressing an inducible ZmTLA1–green fluorescent protein (GFP) fusion protein. GFP signals were exclusively detected in cell walls. Based on our observations, we suggest that the ZmTLA1 peptide represents a class of novel plant morphogens required for the development and maturation of leaf and reproductive tissues.