Klaus Eimert

Phytohormone-regulated beta-amylase gene expression in rice.

The expression of β-amylase genes in rice (Oryza sativa) and its regulation by phytohormones gibberellic acid (GA) and abscisic acid (ABA) were examined. Upon germination β-amylase is synthesizedde novo in aleurone cells and (GA) is not required. Exogenous addition of GA does not enhance the β-amylase activity, while ABA inhibits the β-amylase activity, mRNA accumulation, and the germination of rice seeds. GA can reverse ABA inhibition of β-amylase expression, but not ABA inhibition of seed germination. Such regulation represents a new interaction of ABA and GA.

Transformation of cauliflower (Brassica oleracea L. var. botrytis)- an experimental survey

The paper compares different approaches for the genetic transformation of cauliflower (Agrobacterium-mediated, PEG-mediated and/or electroporation). Transient expression of the neomycin phosphotransferase II (NPTII) gene could be detected after direct gene transfer. Stable transformation was achieved using both Agrobacterium-mediated and direct gene transfer. Expression as well as incorporation of the NPTII sequence could be demonstrated.

Molecular phylogeny of banana cultivars from Thailand based on HAT-RAPD markers

Musa acuminata Colla (AA genomes) and Musa balbisiana Colla (BB genomes) are the wild progenitors of the cultivated banana, they are highly variable in Thailand. The genetic system is relatively unknown and complicated due to interspecific hybridization, heterozygosity and polyploidy, which are common in most clones. These factors make identification of closely related banana cultivars difficult, especially when sterile. The high annealing temperature-random amplified polymorphic DNA (RAPD) technique was used to estimate the genetic relationship between 22 selected banana cultivars, utilizing 14 random primers. Phylogenetic relationship was determined by unweighted pair group method with arithmetical averages cluster analysis. The dendrogram constructed from the similarity data showed that all the 22 cultivars analysed were closely related with a narrow genetic base. There were sufficient RAPD polymorphisms that were collectively useful in distinguishing the cultivars. The dendrogram grouped all the AA, BB, AAA, AAB and ABB genomes into a major cluster. Several subgroups are recognized within the major clade. As expected, Ensete glauca Roxb. (Musaceae) and Strelitzia reginae Banks (Strelitziaceae) were clearly differentiated from the analysed edible bananas. Our study showed that RAPD markers are sufficiently abundant to classify and readily dissect genetic differences between the closely related Musa germplasm and provide a basis for the selection of parents for improvement of this germplasm.

KNOX overexpression in transgenic Kohleria (Gesneriaceae) prolongs the activity of proximal leaf blastozones and drastically alters segment fate

KNOX (knotted1-like homeobox) genes have a widely conserved role in the generation of dissected leaves. Ectopic KNOX activity in leaves in various angiosperm lineages causes leaf form changes that can elucidate how the configuration of leaf development evolved. We present an analysis of leaf morphology and morphogenesis in transgenic Kohleria lines overexpressing a heterologous KNOX gene. Kohleria, like many members of Gesneriaceae, has simple-serrated leaves with pinnate venation. KNOX overexpression causes prolonged segment proliferation in proximal, but not distal, parts of leaf blades. Elaborate dissected segments reiterate the zonation of the whole leaf, with organogenic activity persisting between a distal maturation zone and a proximal intercalary elongation zone. The architecture of vascular bundles is severely altered, with a reduced midvein and a more palmate venation. The initial establishment of organogenically competent primordial margins (marginal blastozones) and the onset of tissue differentiation in early stages of leaf development were similar in wild-type and KNOX overexpressing lines. However, leaves overexpressing KNOX often failed to fully mature, and persistent marginal blastozones were found at the base of blades in mature portions of the shoot. We conclude that KNOX-mediated perpetuation of marginal blastozones in Kohleria is sufficient to induce a set of processes that result in highly dissected leaflets, which are unusual in this plant family. Spatial confinement of blastozones between an early maturing tip and a late elongating petiole zone reflects the presence of distinct maturation processes that limit the ability of the leaf margins to respond to ectopic KNOX gene expression.

Production and rooting behaviour of rolB-transgenic plants of grape rootstock ‘Richter 110’ (Vitis berlandieri × V. rupestris)

Genetic improvement of grape rootstocks is aimed at protection against grape phylloxera and other soil-borne pests and diseases, good rooting and graft compatibility as well as adaptability to a wide range of soil and climatic conditions. Apart from the long evaluation period required, breeding is complicated by the high heterozygosity in grapes. As an alternative to traditional crossing, gene transfer permits addition of single traits, largely without affecting the genetic background of existing valuable cultivars. Here we report on the production and rooting behaviour of transgenic grape rootstock ‘Richter 110’ carrying the Agrobacterium rhizogenes rolB gene, which is known to promote rooting. Transformation was achieved by co-cultivation of somatic embryogenic callus with Agrobacterium tumefaciens LBA4404 harbouring plasmid pHrB. The T-DNA of pHrB contains the hpt gene, conferring hygromycin resistance, and the rolB gene under control of its own promoter. PCR using transgene-specific primers verified the presence of hpt in all 36 hygromycin resistant clones selected, while only 24 clones also possessed the rolB gene. Rooting behaviour was examined in vitro, using tip, node and internode explants, and in aeroponic culture in the greenhouse, using single-node cuttings. Compared to internodes of non-transgenic ‘Richter 110’, those of rolB-transgenic clones in general showed significantly higher rooting ability and, in contrast to the former, were able to root profusely even in the absence of auxin. Cuttings of three rolB-transgenic clones in aeroponic culture produced almost twice as many primary roots as those of the non-transgenic control.

Differential gene expression during hypersensitive response in Phylloxera-resistant rootstock ‘Börner’ using custom oligonucleotide arrays

Abstract The aim of the present study was to identify genes involved in the hypersensitive response (HR) in ‘Börner’, a grapevine rootstock cultivar resistant to grape phylloxera. The HR was chemically induced in roots by an application of indol-3-acetic-acid (IAA). Comparisons between treated and untreated roots after different times of HR induction by IAA were realized by advanced custom DNA microarrays using ‘Riesling’, a phylloxera-sensitive scion, as a control. IAA induction resulted in higher numbers of differentially expressed genes in ‘Börner’ than in ‘Riesling’. In total, 27 putative HR-related genes were identified in ‘Börner’. These genes are presumably involved in the production of phytoalexins, ethylene-associated gene products, cell wall proteins and transcription factors. Thus, the present study is contributing to a better understanding of the signal transduction pathways involved in the hypersensitive reaction underlying the necrosis formation after phylloxera attack in the rootstock cultivar ‘Börner’.

Expression of the NPTII-sequence in cauliflower after injection of agrobacteria into seeds

Summary A simple procedure for gene transfer into cauliflower by macroinjection is described. Seeds of Brassica oleracea var. botrytis were injected with different Agrobacterium tumefaciens strains. The kanamycin-resistance coding NPTII region of engineered Ti-plasmid could be integrated into the plant genome. Expression as well as incorporation of the NPTII sequence could be demonstrated.

Genetic diversity within and between seedstock populations of several German autochthonous provenances and conventionally propagated nursery material of blackthorn (Prunus spinosa L.)

Genetic diversity within and between seedstocks of autochthonous provenances of common blackthorn (Prunus spinosa L.) from several locations in Germany was determined and compared with the diversity in conventionally propagated (German and Hungarian) seedstocks using a highly reproducible high-annealing-temperature random amplified polymorphic DNA (HAT-RAPD) protocol. Based on the distribution of 359 markers obtained with 11 primers we found relatively low genetic diversity in the studied autochthonous blackthorn populations (H0 0.1182–0.1333), with the majority distributed within the populations (92.22%) and only 7.78% among them. Similar levels of diversity were also found in the conventional seedstocks. Accordingly, genetic differentiation among these populations is rather low (pairwise Fst 0.0284–0.1266). In one case, we were not able to differentiate between an autochthonous population and conventional (Fst 0.0284) one. We discuss the results with respect to German conservation laws and their practical implementation.

Revisiting the provenance delineation of a widespread shrub, Frangula alnus—the role of spatial, temporal and environmental patterns

Including population genetic aspects into the selection of planting material within the framework of conservation and restoration measures is of vital importance for the long-term persistence of populations. This is especially true facing climate change since genetic diversity and the spread of potentially beneficial alleles are important for the adaptability of populations. Therefore, knowledge about genetic variability within and between populations is a critical aspect when determining provenance regions. In our study, we investigated the population genetic structure of a widespread, insect-pollinated and mainly bird-dispersed shrub species, Frangula alnus, on the basis of seven microsatellites and two chloroplast DNA markers throughout Germany. The aim was to determine the spatial, temporal and ecological processes genetically structuring populations to critically revise existing provenance regions. Therefore, we conducted analyses on different spatial scales (country-wide, regional and local) using the two different marker sets in addition to environmental variables. We detected distinct patterns on all spatial scales which indicated influences of historic recolonization processes, regional differences of seed dispersal across the landscape, as well as small-scale spatial genetic structures attributable to local dispersal processes. No relation of underlying environmental gradients such as temperature or precipitation and genetic patterns was found. We conclude that different aspects of historic and more recent gene flow shape population genetic structures and that a thorough analysis on a variety of spatial, temporal and environmental scales is necessary to appropriately select planting material for conservation and restoration measures. Correspondingly, management advice regarding provenance delineations will be provided.