The FAST technique: a simplified Agrobacterium-based transformation method for transient gene expression analysis in seedlings of Arabidopsis and other plant species

Background  

Plant genome sequencing has resulted in the identification of a large number of uncharacterized genes. To investigate these unknown gene functions, several transient transformation systems have been developed as quick and convenient alternatives to the lengthy transgenic assay. These transient assays include biolistic bombardment, protoplast transfection and Agrobacterium-mediated transient transformation, each having advantages and disadvantages depending on the research purposes.     

Early diagnosis of ploidy status in doubled haploid production of maize by stomata length and flow cytometry measurements

Reliable discrimination of haploid (H), doubled haploid (DH) and crossing (C) plants in early growth stages could streamline DH production in maize. By detecting in early growth stages undesirable sterile H plants and undesirable heterozygous C plants, a large proportion of resources required for DH production could be saved. The goal of this study was to evaluate the effectiveness of early classification of plants in growth stages V3‐V4 as H, DH or C in the context of DH production using flow cytometry and stomata length. As the reference classification, we used a field score based on plant phenotype commonly applied in DH production and research. Our results show that identification of misclassified C seeds is possible because the overlap in distributions of stomata length between H&DH and C plants is small and the association between flow cytometry and the reference field score is high. In contrast, overlap between H and DH distributions is substantial. Consequently, the main application we see for these classification methods in early growth stages is the identification of C seedlings.     

Effects of ecological compensation meadows on arthropod diversity in adjacent intensively managed grassland

An important goal of ecological compensation areas (ECAs) is to increase biodiversity in adjacent intensively managed farmland and the agricultural landscape at large. We tested whether this goal can be achieved in the case of the agri-environmental restoration scheme implemented for Swiss grassland using five large arthropod taxa (bees, true bugs, orthopterans, ground beetles and spiders) representing different ecological and functional groups. The species richness and abundance of all groups and species, respectively, was measured along 100 m transects from ECA-meadows into the adjacent intensively managed grassland at 24 sites. Species richness of all arthropod taxa except ground beetles, and the abundance of 63% of the 234 arthropod species sampled with at least five individuals were higher in ECA-meadows than in their surroundings, while the total abundance of spiders and ground beetles was higher in intensively managed meadows. The abundance of 8% of these species were only increased in the ECA-meadows themselves (“stenotopic” species) but 40% had increased abundance both in the ECA-meadows and the adjacent grassland, declining exponentially with increasing distance from ECA-meadows (“edge species”). The 90%-decay distances for these edge species differed among taxonomic groups (117 ± 18 m for true bugs, 137 ± 24 m for spiders, 152 ± 34 m for bees, 167 ± 5.7 m for orthopterans, 185 ± 34 m for ground beetles; mean ±1 standard error) and independent of taxonomic group were larger for large-sized or predacious species than for small-sized or phytophagous species. Because the average distance between neighbouring ECA-meadows in Swiss grassland is only 73 ± 4 m, the current agri-environment scheme very likely enhances arthropod diversity and possibly associated ecosystem services in the Swiss agricultural landscape at large.     

Growth- and breed-related changes of marbling characteristics in cattle

The objective of this study was to investigate the growth- and breed-related changes of marbling characteristics in cattle. Four cattle breeds with different growth impetus and muscularity were reared and slaughtered under experimental conditions. German Angus, as a typical beef cattle; Galloway, as a smaller, environmentally resistant beef cattle; Holstein-Friesian, as a dairy-type cattle; and double-muscled Belgian Blue, as an extreme type for muscle growth, were used. These 4 breeds were expected to have differences in muscle development and i.m. fat deposition. Between 5 and 15 bulls of each breed were slaughtered at 2, 4, 6, 12, or 24 mo of age. Marbling characteristics were determined and classified in LM and semitendinosus muscle by computerized image analysis. Among breeds, differences appeared in the quantity, structure, and distribution of the marbling flecks in both muscles. The deposition of fat in the double-muscled Belgian Blue bulls remained substantially inferior to that of the other breeds, up to the age of 24 mo. Marbling in German Angus bulls particularly showed larger (P < 0.05) marbling fleck areas. Galloway cattle had the greatest (P < 0.05) number and the most regular (P < 0.05) distribution of the marbling flecks in young animals. Furthermore, for marbling characteristics in Holstein-Friesian animals, a great number and slightly finer structure were observed compared with the other breeds investigated. Postnatal growth-related changes of marbling in LM were characterized by as much as a 40-fold increase in the number of marbling flecks from 2 to 24 mo of age but also by up to a 4-fold enlargement in the area of the marbling flecks. The structure of marbling flecks was determined by 2 development trends. On the one hand, the marbling flecks became larger (P < 0.05), and the structure became coarser, which was reflected by an increasing (P < 0.01) proportion of long marbling flecks as well as an increasing (P < 0.01) maximum skeleton line length. On the other hand, continually new small, round marbling flecks appeared. This caused a decrease (P < 0.01) in the proportion of the 3 largest marbling fleck areas. The distribution of the marbling flecks became more regular (P < 0.05) with increasing proportion and number of marbling flecks. The results suggest that hyperplasia of adipocytes plays an important role in marbling during growth of muscle in cattle.     

Entomopathogenic nematodes for the management of Agrotis ipsilon: effect of instar, nematode species and nematode production method

BACKGROUND: Previous laboratory studies have indicated the potential of some entomopathogenic nematode (EPN) species for the control of larvae of the black cutworm (BCW). To determine the most promising EPN species and the most susceptible BCW stages, a more in‐depth evaluation of seven EPN species against different BCW instars was carried out, the efficacies of in vitro‐ and in vivo‐produced EPNs were compared and the suitability of BCW instars for EPN reproduction was examined. RESULTS: Heterorhabditis megidis was the most virulent species, irrespective of larval stage in small arenas, followed most often by H. bacteriophora. In pots with grass, Steinernema carpocapsae tended to be the most virulent species, followed by H. bacteriophora, H. megidis and S. riobrave. Fourth and/or fifth instars were the most susceptible stages to most EPN species, and pupae the least susceptible. Furthermore, H. bacteriophora, H. megidis and S. carpocapsae successfully reproduced in fifth and sixth instars and pupae. In vivo‐produced H. megidis and S. carpocapsae controlled fifth instars better than the corresponding in vitro‐produced products; production method did not affect H. bacteriophora and S. riobrave efficacy. CONCLUSIONS: Several in vitro‐produced commercial EPN strains were highly virulent to BCW and warrant further testing under field conditions, along with some in vivo‐produced strains. Copyright © 2011 Society of Chemical Industry     

Variation and covariation for Gibberella ear rot resistance and agronomic traits in testcrosses of doubled haploid maize lines

Gibberella ear rot (GER) of maize caused by Fusarium graminearum reduces grain yield and leads to contamination of the grains with deoxynivalenol (DON), a mycotoxin that adversely affects the health of humans and animals. The objectives of this study were to (1) analyze means and genotypic variances for line per se performance (LP) and testcross performance (TP) of doubled haploid (DH) lines for GER severity and DON concentration as well as for some agronomic traits, (2) examine correlations among these traits, (3) validate QTL for resistance detected in previous studies for LP and their effect on TP and (4) investigate the relative efficiency of indirect selection (RE) for LP to improve TP. Testcross progenies of 94 DH lines originating from four flint populations were developed using a susceptible dent tester as pollinator. Artificial inoculations with F. graminearum led to appreciable disease development. Average TP for GER severity and DON concentration were lower than the mean mid-parent values of the tester and DH lines, indicating mid-parent heterosis for resistance. Genotypic variation for resistance was significant for LP and TP. Genotypic correlations between LP and TP were low and resistance QTL for LP had no significant effects on TP. Accordingly, RE for resistance was low, suggesting to allocate resources mostly to the evaluation of testcrosses. Correlations of resistance to GER and DON contamination with grain yield (measured under non-inoculated conditions) were not significant, indicating that selection for resistance and higher grain yield can be carried out simultaneously.     

Development of in vivo haploid inducers for tropical maize breeding programs

Lack of adapted haploid inducers currently impedes adoption of the doubled haploid technology in tropical maize breeding programs. Our objective was to generate inducers with improved adaptation to tropical conditions. We developed segregating generations from crosses between temperate inducers having haploid induction rates (HIR) of 8–10 % and tropical CIMMYT maize lines (CML; HIR = 0 %) and evaluated these for HIR and agronomic performance under tropical lowland field conditions. The applied pedigree breeding scheme comprising mass selection on individual F₂ plants for highly heritable and visually scorable traits, followed by family-based selection for HIR and other agronomic characteristics in advanced selfing and backcross (BC) generations seems highly suitable for breeding improved haploid inducers with adaptation to different agroecologies. The most advanced tropical inducer candidates (TIC) combine HIR of up to 10 % with improved pollen production, disease resistance, and plant vigor compared to temperate inducers under tropical conditions. Agronomic characteristics were significantly improved in the BC to CML compared to BC to inducers, while mean HIR of both populations were similar, indicating that backcrossing to the adapted parent was suitable to improve adaptation of new inducers without sacrificing high HIR. When screening random open-pollinated maize accessions, HIR of up to 3 % were observed, suggesting that novel genetic variation may be present in maize accessions that could be exploited to improve HIR in maize. In conclusion, tropical inducer development proceeds well, but evaluation of TIC in multi-environment trials needs to be completed before large-scale dissemination can commence.