Light strongly promotes gene transfer from Agrobacterium tumefaciens to plant cells

Abstract Light conditions during Agrobacterium-based plant transformation, the most routinely used method in plant genetic engineering, differ widely and, to our knowledge, have not been studied systematically in relation to transformation efficiency. Here, light effects were examined in two already optimized transformation procedures: coculture of Agrobacterium tumefaciens with callus from two genotypes of the crop plant Phaseolus acutifolius (tepary bean) and coculture of root segments from two ecotypes of Arabidopsis thaliana. Except for the light conditions during coculture, all steps followed established procedures. Coculture was done either under continuous darkness, under a commonly used photoperiod of 16 h light/8 h darkness or under continuous light. b-glucuronidase (GUS) production due to the transient expression of an intron-containing uidA gene in the binary vector was used to evaluate T-DNA transfer. In all situations, uidA expression correlated highly and positively with the light period used during coculture; it was inhibited severely by darkness and enhanced more under continuous light than under a 16 h light/8 h dark photoperiod. The promotive effect of light was observed with Agrobacterium strains harboring either a nopaline-, an octopine-or an agropine/succinamopine-type nononcogenic helper Ti plasmid. The observed positive effect of light has obvious implications for developing and improving transient and stable transformation protocols, specifically those involving dark coculture conditions

Electroporation-mediated DNA delivery to seedling tissues of Phaseolus vulgaris L. (common bean)

DNA was delivered to intact embryonic axes of the legume Phaseolus vulgaris L. through electroporation. Expression of the beta-glucuronidase reporter gene was observed in hypocotyl and epicotyl tissue in a spot-like manner. Transgene expression was high when a single pulse of 260 ms at a field strength of 225 V . cm(-1) was applied but could be achieved within a wide range of electrical conditions. Linearization of plasmid DNA greatly enhanced transient expression levels. The procedure was successful for embryonic axes of all tested P. vulgaris cultivars, for similar explants of several large-seeded leguminous species, as well as for some other tissues of P. vulgaris

Plant regeneration from callus in a range of Phaseolus acutifolius A. Gray genotypes

The ability of a range of Phaseolus acutifolius A. Gray genotypes to regenerate shoots from callus was investigated. Shoots and viable plants were obtained from green nodular callus derived from buds and pedicels. Seven of 12 genotypes tested produced morphologically normal shoots. Regenerants of six genotypes were established in the greenhouse. The regeneration response reflected the taxonomic subdivision of the species in that P. acutifolius var. acutifolius was responsive whereas var. tenuifolius was not. The 'regeneration competence' trait was present in both the wild and the domesticated gene pools. The possible application of this trait for the genetic improvement of the related and economically more important species P. vulgaris is discussed

The arcelin-5 Gene of Phaseolus vulgaris Directs High Seed-Specific Expression in Transgenic Phaseolus acutifolius and Arabidopsis Plants

The regulatory sequences of many genes encoding seed storage proteins have been used to drive seed-specific expression of a variety of proteins in transgenic plants. Because the levels at which these transgene-derived proteins accumulate are generally quite low, we investigated the utility of the arcelin-5 regulatory sequences in obtaining high seed-specific expression in transgenic plants. Arcelin-5 is an abundant seed protein found in some wild common bean (Phaseolus vulgaris L.) genotypes. Seeds of Arabidopsis and Tepary bean (Phaseolus acutifolius A. Gray) plants transformed with arcelin-5 gene constructs synthesized arcelin-5 to levels of 15% and 25% of the total protein content, respectively. To our knowledge, such high expression levels directed by a transgene have not been reported before. The transgenic plants also showed low plant-to-plant variation in arcelin expression. Complex transgene integration patterns, which often result in gene silencing effects, were not associated with reduced arcelin-5 expression. High transgene expression was the result of high mRNA steady-state levels and was restricted to seeds. This indicates that all requirements for high seed-specific expression are cis elements present in the cloned genomic arcelin-5 sequence and trans-acting factors that are available in Arabidopsis and Phaseolus spp., and thus probably in most dicotyledonous plants