In the course of DNA profiling of grapevine cultivars using microsatellite loci we have occasionally observed more than two alleles at a locus in some individuals and have identified periclinal chimerism as the source of such anomalies. This phenomenon in long-lived clonally propagated crops, such as grapevine, which contains historically ancient cultivars, may have a role in clonal differences and affect cultivar identification and pedigree analysis. Here we show that when the two cell layers of a periclinal chimera, Pinot Meunier, are separated by passage through somatic embryogenesis the regenerated plants not only have distinct DNA profiles which are different from those of the parent plant but also have novel phenotypes. Recovery of these phenotypes indicates that additional genetic differences can exist between the two cell layers and that the Pinot Meunier phenotype is due to the interaction of genetically distinct cell layers. It appears that grapevine chimerism can not only modify phenotype but can also impact on grapevine improvement as both genetic transformation and conventional breeding strategies separate mutations in the L1 and L2 cell layers.
The secondary metabolite amygdalin is a cyanogenic diglucoside that at high concentrations is associated with intense bitterness in seeds of the Rosaceae, including kernels of almond (Prunus dulcis (Mill.), syn. Prunus amygdalus D. A. Webb Batsch). Amygdalin is a glucoside of prunasin, itself a glucoside of R-mandelonitrile (a cyanohydrin). Here we report the isolation of an almond enzyme (UGT85A19) that stereo-selectively glucosylates R-mandelonitrile to produce prunasin. In a survey of developing kernels from seven bitter and 11 non-bitter genotypes with polyclonal antibody raised to UGT85A19, the enzyme was found to accumulate to higher levels in the bitter types in later development. This differential accumulation of UGT85A19 is associated with more than three-fold greater mandelonitrile glucosyltransferase activity in bitter kernels compared with non-bitter types, and transcriptional regulation was demonstrated using quantitative-PCR analysis. UGT85A19 and its encoding transcript were most concentrated in the testa (seed coat) of the kernel compared with the embryo, and prunasin and amygdalin were differentially compartmentalised in these tissues. Prunasin was confined to the testa and amygdalin was confined to the embryo. These results are consistent with the seed coat being an important site of synthesis of prunasin as a precursor of amygdalin accumulation in the kernel. The presence of UGT85A19 in the kernel and other tissues of both bitter and non-bitter types indicates that its expression is unlikely to be a control point for amygdalin accumulation and suggests additional roles for the enzyme in almond metabolism.
The recently developed microprojectile method for gene transfer to intact cells has been successfully used to transform plant species including some which previously resisted attempts using Agrobacterium and protoplast mediated techniques. In addition, microprojectile bombardment has already proved uniquely suitable for other applications including direct transformation of organelle genomes and rapid assessment of transient expression of genetic constructs introduced into cells of intact tissues. Here we describe various microprojectile acceleration devices and the steps necessary to develop an effective microprojectile mediated transformation system for any plant species. We ernphasise the need to optimise the delivery of DNA into cells, and to tailor strategies for generating stably transformed plants based on the nature of the target tissue, behaviour in tissue culture, and available marker genes. Patterns of cotransformation and coexpression of introduced genes in stable nuclear transformants generated with microprojectiles are summarised, and other applications including organelle transformation are briefly described. We mention technical limitations to the application of microprojectile-mediated gene transfer which need to be overcome if the method is to achieve its full potential as a near-universal gene transfer technique with exciting applications in basic plant molecular biology and practical plant improvement.
A microprojectile accelerator has been constructed and used to bombard cultured sugarcane tissues with GUS reporter gene constructs. Design features useful to minimise target tissue damage and variation between shots are described. Transient expression of GUS occurred in pEmuGN-bombarded cells of nonregenerable suspension culture as well as in regenerable embryogenic callus of commercial sugarcane cultivar 463, and in suspension cultures capable of regeneration to plants. Parameters yielding transient GUS expression in up to 1055 cells per bombardment in homogeneous suspension cultures of sugarcane have been established with a mean of 206 expressing cells per bombardment over a series of 8 independent experiments. Approximately 4% of these transiently expressing cells continued to express GUS for extended periods, indicating probable stable transformation of intact cells of the commercial sugarcane cultivar. Microprojectile bombardment appears the most promising of the available gene transfer techniques for practical genetic transformation of sugarcane because most commercial cultivars readily form regenerable callus suitable for bombardment.
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