Roland Bilang

Microprojectile-mediated transient and integrative transformation of rice embryogenic suspension cells: effects of osmotic cell conditioning and of the physical configuration of plasmid DNA

Abstract Microprojectile-mediated transient and integrative transformation frequencies in rice (Oryza sativa cv. Taipei 309) embryogenic suspension cells were studied as a function of various parameters. Mannitol at concentrations of 0.5 and 0.6 m was best for osmotic preconditioning of the cells for transient, but not for integrative transformation, for which sucrose yielded the best and most reliable results. Denaturation of the transforming plasmid DNA prior to bombardment improved transient and integrative transformation frequencies two to three fold. Delivery of double-stranded plasmids in linear form had no effect on transient transformation when compared to supercoiled plasmid DNA, but led to an overall two fold increase in integrative transformation frequency. This shows that optimized protocols for generating transgenic plants should not be based exclusively on transient gene expression assays.

Single-stranded DNA as a recombination substrate in plants as assessed by stable and transient recombination assays.

Two separate assays, one that requires stable integration of recombination products and one that does not, were employed to elucidate the role of single-stranded DNA in extrachromosomal homologous recombination in Nicotiana tabacum. Both assays revealed that single-stranded DNA in linear and in circular forms was an efficient substrate for recombination, provided that the cotransformed recombination substrates were of complementary sequence, so that direct annealing was possible. Recombination was inefficient when both single-stranded recombination partners contained homologous regions of identical sequence and generation of a double-stranded DNA was required prior to heteroduplex formation. These results indicate that direct annealing of single strands is an important initial step for intermolecular recombination in tobacco cells. Annealed cotransformed single-stranded molecules yielded intermediates that could be further processed by either continuous or discontinuous second-strand synthesis. The type of intermediate had no influence on the recombination efficiency. Double-stranded circles were unable to recombine efficiently either with each other or with single-stranded DNA. Our results suggest that a helicase activity is involved in the initial steps of double-stranded DNA recombination which unwinds duplex molecules at the site of double-strand breaks.

Direct gene transfer to protoplasts of Arabidopsis thaliana

We performed a series of direct gene transfer experiments with protoplasts of Arabidopsis thaliana ecotype Zürich. An average of more than 100 transformants were selected per 1066 treated protoplasts. Stable transformation was confirmed by integration of the marker gene into high molecular weight DNA and by its genetic transmission to subsequent offspring generations.

Gene transfer to inflorescence and flower meristems using ballistic micro-targeting

Direct gene transfer to floral meristems could contribute to cell-fate mapping, to the study of flower-specific genes and promoters, and to the production of transgenic gametes via the transformation of sporogenic tissues. Despite the wide potential of its applications, direct gene transfer to floral meristems has not been achieved so far because of the lack of suitable technology. We show in this paper that ballistic micro-targeting is the technique of choice for this purpose, and in this way, we were able to transfer genes efficiently into excised wheat immature spikes. Particle size was adjusted for optimal penetration into the L1 and L2 cell layers of the spikes with limited cell damage. Spikes at different developmental stages were shot either with a plasmid containing two genes involved in anthocyanin biosynthesis or with a plasmid bearing the uidA (β-glucuronidase) gene. The transient expression of these marker genes was observed in the different developmental stages tested and in cells of both the L1 and the L2 layers. The transient expression of the uidA gene was significantly increased when the sucrose concentration in the culture medium was increased from 0.06 to 0.52 M. At the highest concentration, 100% of the targeted spikes expressed the uidA gene, with an average of 69 blue cells per spike. Twelve days after microtargeting, multicellular sectors showing transgene expression and containing up to 17 cells were found in 85% of the shot immature inflorescences. This indicated that targeted cells survived particle bombardment. Sectors were found in primordia of both vegetative and reproductive organs.

Arabidopsis thaliana: protocol for plant regeneration from protoplasts.

We report a protocol for plant regeneration from leaf protoplasts of Arabidopsis thaliana ecotype Zürich. The protocol has been established in 1988 and has since been in routine use in our laboratory. Whereas recovery of proliferating protoplast-derived clones is routine, the success in plant regeneration from protoplast-derived clones is highly variable. In the hands of one of us (H.K.) average shoot regeneration frequency (% of calli regenerating at least one shoot) was ca. 60% and average plant regeneration frequency (% of calli yielding fertile plants in soil) was ca. 40%.

PEG-mediated direct gene transfer and electroporation

For many years of genetic manipulation in plants, direct uptake of naked DNA by plant protoplasts has been the sole alternative to Agrobacterium tumefaciens-mediated gene transfer. The first experiments demonstrating direct gene transfer included the delivery of isolated plasmid DNA to protoplasts of petunia and tobacco in the presence of poly-L-ornithine or polyethylene glycol (PEG) [1–4]. During the following years, protoplast transformation mediated by PEG [5] or electroporation [6] was substantially simplified and their efficiency in model systems was increased by several orders of magnitude (reviewed by Paszkowski et al. [7]).

Lincomycin Treatment: A Simple Method to Differentiate Primary and Processed Transcripts in Rice (Oryza sativa L.) Chloroplasts

Visualizing full-length primary transcripts is helpful in identifying transcription initiation sites and mapping promoter regions of plastid genes and operons. Detection of primary unprocessed transcripts from certain regions of the plastid genome is difficult, and sometimes impossible, because of their rapid and extensive processing. We tested the effect of lincomycin, a prokaryotic protein synthesis inhibiter, on in vivo RNA processing activities in different types of rice plastid. Steady-state levels of RNA produced from the region of the rice plastid genome that includes thetrnV and 16s rRNA genes were analysed by using an RNase protection assay. Results show that sublethal lincomycin levels inhibit RNA processing in leaf chloroplasts and allow the accumulation of primary transcripts, easily distinguishable from processed and processing intermediates. These features were used to identify regions of the 16r andtrnV transcription start sites. This is the first report of the use of lincomycin for mapping plastidic transcripts.