Agrobacterium tumefaciens-mediated transformation of Festuca arundinacea (Schreb.) and Lolium multiflorum (Lam.)

Abstract

Agrobacterium tumefaciens strain LBA4404 carrying plasmid pTOK233 encoding the hygromycin resistance (hph) and beta-glucuronidase (uidA) genes has been used to transform two agronomic grass species: tall fescue (Festuca arundinacea) and Italian ryegrass (Lolium multiflorum). Embryogenic cell suspension colonies or young embryogenic calli were co-cultured with Agrobacterium in the presence of acetosyringone. Colonies were grown under hygromycin selection with cefotaxime and surviving colonies plated on embryogenesis media. Eight Lolium (six independent lines) and two Festuca plants (independent lines) were regenerated and established in soil. All plants were hygromycin-resistant, but histochemical determination of GUS activity showed that only one Festuca plant and one Lolium plant expressed GUS. Three GUS-negative transgenic L. multiflorum and the two F. arundinacea plants were vernalised and allowed to flower. All three Lolium plants were male- and female-fertile, but the Festuca plants failed to produce seed. Progeny analysis of L. multiflorum showed a 24-68% inheritance of the hph and uidA genes in the three lines with no significant difference between paternal and maternal gene transmission. However, significant differences were noted between the paternal and maternal expression of hygromycin resistance.

Genotypic variation of Agrobacterium-mediated transformation of Italian ryegrass

“…Finally, regenerated putative transgenic shoots were separated and transferred onto rooting medium containing half strength MS medium supplemented with 30 g/L sucrose, 2 g/L Gelrite, 250 mg/L cefotaxime, and 50 mg/L hygromycin. While, in the earlier study (Bettany et al 2003), putative transgenic calli were selected with relatively higher concentration of hygromycin (75 mg/L), the present study we found that selection of putative transformant callus with higher than 50 mg/L hygromycin caused a severe inhibition of shoot regeneration from the Hm-resistant calli. In addition, it has been reported that continuous selection of low concentration of hygromycin produced higher number of transgenic plants without escape (Dalton et al 1995).…”

“…It is also demonstrated that transgenic Italian ryegrass could be produced by the use of the easily available pCAMBIA1301 vectors. The transformation efficiency was comparable with other Agrobacteriummediated transformation systems in grass species such as, Italian ryegrass (Bettany et al 2003), perennial ryegrass (Wu et al 2005) and orchardgrass (Lee et al 2006). …”

“…Advantages of Agrobacterium-mediation of genetic transformation, including the ability to transfer large pieces of DNA with minimal rearrangement, low experimental costs, and integration of relatively low numbers of transgene copies, have been described elsewhere (Hiei et al 1994). To the best our knowledge, there have only been a few reports regarding Agrobacterium-mediated transformation (Bettany et al 2003), which recovered a total of eight transgenic plants wherein only one showed GUS expression. In addition, up to now, there was no comparative evaluation of the responses of Italian ryegrass cultivars in Agrobacteriummediated transformation system.…”

“…Genetic transformation may be an important contribution to the improvement of forage grasses. However, the efficiency with which Italian ryegrass cultivars are transformed is very low (Bettany et al 2003). In this study, we have reported an improved transformation protocol for seven cultivars of Korean Italian ryegrass by use of mature seed-derived calli as explants.…”

“…A wide range of factors, however, such as plant genotype, explants type and age, cocultivation media and period, can influence the gene transfer efficiency from Agrobacterium to plant cells (Uranbey et al 2005;Ahsan et al 2007). Although, Agrobacterium-mediated transformation in Italian ryegrass was introduced in 2003 (Bettany et al 2003), it still does HPT: hygromycin phosphotransferase X-gluc: 5-bromo-4-chloro-3-indolyl-b-glucuronide not yield consistent results in respect of genotypic response and transformation frequency. The absence of highly efficient genotype independent regeneration and transformation systems is a major obstacle to the application of gene transfer technology to economically important Italian ryegrass cultivars.…”