Marie B. Connett

A biolistic approach for the transfer and expression of a gusA. reporter gene in embryogenic cultures of Pinus radiata.

SummaryThe biolistic® particle delivery system was used for the delivery of DNA into embryogenic tissue culture cells of Pinus radiata D. Don. Several experiments with varying parameters were performed to increase the delivery efficiency. Six different controlling elements were cloned upstream of the ß-glucuronidase coding sequence (gusA reporter gene) and transient expression of the gusA reporter gene was compared three days after bombardment. The results clearly indicate a decrease in transient expression as follows: pEmu-derivatives with the ocs-enhancer-element > 2x CaMV 35S (with Kozak consensus-sequence) > 2x CaMV 35S (without Kozak consensus sequence) > CaMV 35S (with Kozak consensus-sequence) > CaMV 35S (without Kozak consensus sequence). Time course experiments monitoring gusA expression showed a significant decrease in the number of blue spots 10–14 days after bombardment. A few blue clumps however, were still detected 35 days after shooting. Embryo initials expressing the gusA gene in all cells were also detected. The results suggest that it will be possible to develop a reliable biolistic protocol for stable integration of genes into Pinus radiata embryogenic cultures which are capable of plant regeneration.

Genetic variation in antarctic populations of the moss Sarconeurum glaciale

Abstract Sixty-six isolates of the moss Sarconeurum glaciale were collected from sites in continental Antarctica at Ross Island, southern Victoria Land and the Vestfold Hills. Genetic variation within and among the populations was estimated using isozymes and random amplified polymorphic DNA (RAPD) technology. Isozyme results only reproducibly showed variation between the populations with one enzyme; RAPDs indicated significantly higher levels of genetic variability within and among the Vestfold Hills samples than in the Ross Sea region samples. A dendrogram produced from the RAPD bands suggested that the Ross Island and southern Victoria Land samples form one population, and those from the Vestfold Hills form a separate and more variable population, possibly resulting from separate colonisation events on the continent.

Mechanisms of maternal inheritance of plastids and mitochondria: Developmental and ultrastructural evidence

SummaryA number of maternally inherited characters are now known to be associated with mitochondria or chloroplasts, which contain small genomes segregating separately from that of the nucleus. The reason often given for maternal inheritance of plastid-associated characters in plants is the absence of plastids in the generative cell of pollen following an unequal mitosis (Vaughn, 1980). However, fine ultrastructural studies have not established “exclusion” as the sole mechanism for maternal inheritance; in many cases, other mechanisms may be operating. Three lines of evidence concerning the mechanism of maternal inheritance will be discussed:First, while it is true that thorough fine ultrastructural studies have failed to find plastids in generative cells of many seed plants (Cass and Karas, 1975), similar studies in some seed plants have found plastids or structures taken to be plastids in generative cells, and a few studies using serial section electron microscopy to re-examine some plants in the first group have found plastids in generative cells and even in the sperm. Also, the exclusion model fails to account at all for maternal inheritance of mitochondria, which are found nearly universally in the generative cells and sperm which have been studied ultrastructurally.Second, maternal inheritance of plastid characters is seen in many lower plants and algae, despite the presence of plastids and mitochondria in the male gametes and their reported deposition in the zygote.Third, there is evidence for an alternative or additional mechanism which may occur in many plants: mitochondria and plastids in male gametes may be altered during development or syngamy so that, although not excluded, they are genetically and perhaps functionally debilitated, which would result in maternal inheritance. This evidence derives both from ultrastructural studies of pollen and fertilization, and from genetic and developmental analysis of algal zygotes and of embryos derived from pollen tissue culture. This mechanism is logically attractive in that it allows for the observed continuum of variation from strict uniparental inheritance in a number of plants, which cannot be explained by the “all-or-nothing” exclusion hypothesis.Indeed, it may be appropriate to think of both mechanisms as part of a continuum ranging from destruction within the zygote, to exclusion during syngamy, to pre-fertilization debilitation, to absence from male gametes and generative cells (Russell and Cass, 1981).

Cytoplasmic Male Sterility in Petunia

A maternally-inherited male sterile phenotype is known in many plant genera, including Petunia (Laser and Lersten, 1972; Hanson and Conde, 1985). As all seed on a cytoplasmic male sterile (CMS) plant must result from cross-pollination, the trait has attracted commercial interest for hybrid seed production. Problems with exploitation of the CMS trait, however, include its lack of natural occurrence in certain important crop species as well as unwanted phenotypic “side effects” of the CMS cytoplasm in combination with particular nuclear backgrounds.

Micropropagation of Forest Trees

Micropropagation as a delivery system for producing plants for the clonal propagation of genetically superior and genetically transformed forest trees is reviewed. Recent advances in the micropropagation of material from juvenile and mature trees produced via either the organogenesis or embryogenesis pathways, field evaluation of micropropagules, genetic transformation, and automation of micropropagation systems for forest trees are discussed. Micropropagation of juvenile trees from immature embryos, mature embryos and young seedling explants has been successful, with major advancements recently in embryogenesis and in large-scale clonal production via organogenesis. Field trials of trees grown from micropropagules of juvenile trees planted in the early 1980s showed advanced maturation characteristics in some cases; some of the factors involved in this have now become apparent. Micropropagation of material from mature trees has also had some major breakthroughs: reliable techniques have been developed for plantlet formation from mature buds, and embryogenesis from male reproductive tissues has been demonstrated for a few hardwood species. Some advances have been made in the genetic transformation of forest trees for transient expression, particularly with the use of DNA-coated microprojectiles (“biolistic” methods). The simultaneous development of better regeneration systems is essential for stable transformation. There is still much to be researched on the long-term stability of the transforming DNA and identification of genes of major interest to forest managers and researchers for transformation. Automated systems to reduce the cost of micropropagated trees are being developed for both organogenesis and embryogenesis systems.

Molecular studies of cytoplasmic male sterility in Petunia

A large number of different mutations has been shown to give rise to male sterility in over 175 species of plants, and references are fast going out of date as more are being mapped all the time (Regan and Moffatt 1990). In over 140 species of angiosperms, certain male sterility mutations are inherited with the cytoplasmic genomes rather than following the Mendelian inheritance patterns of typical nuclear genes (Laser and Lersten 1972). Cytoplasmic Male Sterility (CMS) is a maternally inherited trait which results in the abortion of pollen development and thereby the loss of male fertility in an otherwise hermaphroditic plant. The trait is economically important because it is used commercially for the production of hybrid seed in some agriculturally significant cultivated species such as maize. Unfortunately the CMS phenotype has been associated with undesirable effects in some plants where it was used extensively in the breeding germplasm, such as susceptibility to southern corn leaf blight in maize associated with type T CMS, which wiped out 15% of the total U.S. maize crop in 1970 (reviewed in Leaver 1992). Thus, due to its economic importance, CMS has been a subject for molecular study in a number of species, such as brassicas, rice, maize, sunflower, and beans (Hanson and Conde 1985). Interestingly, though the chloroplast genome is also maternally inherited, CMS has been found to be associated with the mitochondrial genome in every species in which its coding location has been determined Thus, study of the CMS phenomenon has also led us to some fascinating insights into the structure and function of plant mitochondrial genomes.