Stability of transgenes and presence of N6 methyladenine DNA in transformed wheat cells

A number of stably transformed wheat cell lines were obtained using two different direct gene transfer techniques. When integration of the foreign genes was investigated in DNA samples taken at 10 months post-selection, complicated profiles of transgene bands were observed in Southern blot analysis. Among these, a number of common bands were identified showing similar hybridization patterns between independently transformed cell lines. This type of hybridization pattern has been a common observation and is usually interpreted as concatameric rearrangement of integrated DNA. However, when integration was reinvestigated with DNA samples taken at 30 months postselection, the hybridization pattern changed and most of the common bands had disappeared. Further analysis using a set of methylation-sensitive enzymes revealed that the DNA represented by the common bands was N⁶-adenine methylated (^m6A DNA), and there was even ^m6A DNA in some 30 month samples. Although the source of ^m6A DNA in the wheat cultures was not clearly established, the data indicate that transformation of an endophyte (e.g. a mycoplasma-like organism) may have occurred at the same time as the transformation of wheat cells. The integration pattern of undermethylated transgenes in the transformed cells became simpler and clearer after treating the DNA preparations with Dpnl, which only cuts ^m6A DNA. The implications of these data for other methods of inoculating cereals with DNA are discussed.     

Tissue organisation,pollen receptivity and pollen tube guidance in normal and mutant stigmas of the grass Pennisetum typhoides (Burm.) Stapf et Hubb.

Summary The normal stigma of Pennisetum typhoides is twin-branched, each branch bearing unbranched trichomes. As is general among the grasses, the papillate cells of the trichomes possess a discontinuous cuticle with overlying protein and polysaccharide secretions. These adaptations for pollen capture and hydration are absent from the stigma axes. Pollen tubes emerging from grains received on the trichomes are guided into the axes with the tips directed towards the ovary by the architecture of the basal cell complex. There are no defined transmitting tracts in the stigma axes, and further passage is through intercellular spaces of a tissue of elongated cells between the epidermis and the central vascular strands. In the mutant tr, tr, the stigmas are twin-branched, but lack trichomes. However, the principal adaptations of the trichome cells for the capture and hydration of pollen are expressed in the epidermal cells of the branches, which have permeable cuticles and the characteristic surface secretions. Pollen tubes emerging from grains germinating on the branches enter between the files of epidermal cells, or at their ends. In the absence of the guidance provided by trichome structure in the normal stigma, they pass indifferently either towards or away from the ovary. The implications of the comparison between the normal and mutant genotypes for understanding the requirements for pollen capture, germination and pollen-tube guidance in the grasses are discussed.     

Molecular and physical organization of highly repetitive,undermethylated DNA from Pennisetum glaucum

A HaeIIl monomer of a repetitive DNA family from Pennisetum glaucum (L.) R. Br. cv. Massue has been cloned and characterized. The repeat is 137 bp long and is organized in head-to-tail orientation in tandem arrays. The HaeIII monomer contains 55% A+T residues. The distribution of this highly repetitive sequence in different Pennisetum species and in other cereals was investigated. The HaeIII satellite is present in all Pennisetum species investigated but absent from other genera examined. In situ hybridization revealed a centromeric localization of this sequence on all seven chromosome pairs and indicated chromosome-specific differences in copy number. Methylation was investigated by comparative restriction enzyme analysis (Msp/HpaII) which showed a greater extent of methylation of the internal C of the enzyme recognition site 5′-CCGG. A South-Western analysis, using an anti-methylcytosine antibody to examine the methylation status in P. glaucum confirmed that the sequence is not highly methylated.