Sonication assisted Agrobacterium-mediated transformation enhances the transformation efficiency in flax (Linum usitatissimum L.)

Abstract

A sonication-assisted, Agrobacteriummediated, co-cultivation technique was used in an attempt to increase the transformation efficiency of flax. Hypocotyls and cotyledons excised from about 10-dayold flax seedlings grown in vitro were placed into a 10 mM MgSO 4 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.

Explant position effect on gene transformation to flax (Linum usitatissimum L.) via Agrobacterium tumefaciens

“…However, the recovery of transgenic flax plants (13,16). When hypocotyl segments were directly inoculated, transgenic shoot frequency was extremely low due to the plant's defense mechanism caused by the pathogenic attack by the Agrobacterium infection (17).…”

Sonication-assisted Agrobacterium-mediated transformation of the ACC gene to interfere the production of ethylene in spring Dendrobium cv. ‘Sanya’

“…However, our preliminary experiments demonstrated that ultrasound treatment and a subse quent immersion in A. tumefaciens suspension for inoculation was more effective than treatment with ultrasound concurrently with A. tumefaciens inocula tion.. We suggest that ultrasound treatment forms microwounds on the surface of PLBs and facilitates Agrobacterium infection. The ultrasound treatment was successfully employed to enhance transformation efficiency in several other plant species [7][8][9][10][11][12]. The impact of different ultrasonic power on transgenic 'Sanya' requires further study.…”

“…Pathak and Hamzah [6] found that in chickpeas the transformation efficiency was more than twice higher with SAAT treatment than at simple Agrobacterium transformation without soni cation. Subsequently, the ultrasound treatment was successfully employed to enhance transformation effi ciency in several plant species, both by direct DNA transfer to protoplasts and tissue cultures and by co cultivation with vector bacteria [7][8][9][10][11][12].…”

Studies on genetic transformation of Theobroma cacao L.: evaluation of different polyamines and antibiotics on somatic embryogenesis and the efficiency of uidA gene transfer by Agrobacterium tumefaciens

“…It is important to note that no GUS expression was detected in the absence of SAAT, using the ordinary ''dipping'' method, demonstrating the relative recalcitrance of cacao to the practices of Agrobacteriummediated transformation. SAAT has been proved to be an efficient system of T-DNA delivery to cells in a number of plants (Santarém et al 1998;Tang et al 2001;Zaragozá et al 2004;Beranová et al 2008;de Oliveira et al 2009), especially those that are typically more recalcitrant to Agrobacteriummediated transformation (Trick and Finer 1997). This method involves subjecting the plant tissue to brief periods of ultrasound in the presence of Agrobacterium.…”

“…It creates microwounding released from the cavitation of microbubbles causing minute visible wounds within and on the tissue (Gaba et al 2006). Such phenomena allow better access and infection of plant cells by Agrobacterium (Beranová et al 2008). In addition, the wounded tissue often produces inducers of the T-DNA transfer process, caused by the secretion of more phenolic compounds, enhancing the accessibility of putative cell wall binding factor to the Agrobacterium during transformation (Stachel et al 1985).…”