Sonication assisted <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation enhances the transformation efficiency in flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

A sonication-assisted, Agrobacterium-mediated, co-cultivation technique was used in an attempt to increase the transformation efficiency of flax. Hypocotyls and cotyledons excised from about 10-day-old flax seedlings grown in vitro were placed into a 10 mM MgSO₄ solution, and inoculated with an A. tumefaciens vector bearing the mgfp5-ER gene driven by the CaMV 35S promoter. The explants were subjected to pulses of ultrasound delivered by a sonicator apparatus (35 kHz) for 0–150 s and co-cultivated for 2 h at 27°C. The dried hypocotyls and cotyledons were grown on a selective MS medium to promote shoot regeneration. An electron microscopic study showed that the sonication treatment resulted in thousands of microwounds on and below the surface of the explants. A stereo microscope Leica MZ 12 equipped with a GFP adaptor was used to assess the infection and transformation of plant tissues in real time. After only 48 h and for at least 30 days after bacteria elimination, signs of transgene expression could be seen as a bright fluorescence. Our results show that treatment with ultrasound facilitates an enhanced uptake of plasmid DNA into the cells of flax hypocotyls and cotyledons and that its efficiency depends on the duration of the treatment and the frequency used. SAAT could be a promising tool for enhancing transformation efficiency in flax.     

Variation of γ-glucuronidase activity in cauliflower plants with <Emphasis Type="Italic">gus</Emphasis> gene introduced by <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis> mediated transformation

In this paper we show the effect of leaf tissue sampling on estimation of β-glucuronidase activity. Pieces of leaves taken from Agrobacterium-mediated transformed T₀ plants of cauliflower were sampled and the GUS activity was measured fluorometrically. Whole leaf tissue and samples of small pieces representing various leaf zones were compared. A great variation of GUS activity within leaf was observed, for which coefficient of variation reached up to 70%. The GUS activity was nearly symmetrical for the left and right side of a leaf blade, with the highest values along the top and middle parts of a leaf blade edge. The relible and repeatable estimation of GUS activity was obtained only if a whole leaf tissue, except the midrib, was used, which allow to reduce the variation to about 10%.     

Electron microscopy and X-ray microanalysis as tools for fine localization of the β-glucuronidase activity in transgenic plants harbouring the GUS reporter gene

Summary The GUS reporter gene encoding -glucuronidase is very useful in various domains of plant genetic engineering. A method for ultrastructural detection of its activity was developed using 35S-GUS transgenic tobacco root tips. Short glutaraldehyde prefixation at 4°C preserved up to 70% enzyme activity and was followed by brief incubation in X-Glu, strong postfixations, then quick dehydration at low temperature before resin embedding. In these conditions, transgenic cells were well preserved and displayed electron dense indigo precipitates with a crystalline structure as shown by electron diffraction. Due to other dense structures in the tissues, controls of the nature of the reaction product (diX-indigo) were necessary. A first control was carried out by means of X-ray microanalysis in order to check the presence of bromine. Other controls, including incubated non-transformed tissues, non-incubated or boiled transgenic roots as well as transgenic samples incubated with the specific -glucuronidase inhibitor, D-saccharic acid-1,4-lactone, were also carried out. The discussion points out the potential uses but also the limits of the method, non-specific localizations of the diX-indigo microcrystals being possible.Abbreviations X-Glu 5-bromo-4-chloro-3-indolyl--D-glucuronic acid - diX-indigo 5,5-dibromo-4,4-dichloro-indigo - MUG methyl umbelliferyl glucuronide - 4-MU 4-methyl umbelliferone - EDTA ethylene diamine tetraacetic acid disodium salt - PB phosphate buffer - CaMV cauliflower mosaic virus