Agrobacterium-mediated transformation of oat (Avena sativa L.) cultivars via immature embryo and leaf explants

Abstract

This paper reports on the successful Agrobacterium-mediated transformation of oat, and on some factors influencing this process. In the first step of the experiments, three cultivars, two types of explant, and three combinations of strain/vectors, which were successfully used for transformation of other cereals were tested. Transgenic plants were obtained from the immature embryos of cvs. Bajka, Slawko and Akt and from leaf base explants of cv. Bajka after transformation with A. thumefaciens strain LBA4404(pTOK233). The highest transformation rate (12.3%) was obtained for immature embryos of cv. Bajka. About 79% of the selected plants proved to be transgenic; however, only 14.3% of the T 0 plants and 27.5% of the T 1 showed GUS expression. Cell competence of both types of explant differed in terms of their transformation ability and transgene expression. The next step of the study was to test the suitability for oat transformation of the pGreen binary vector combined with different selection cassettes: nptII or bar under the nos or 35S promoter. Transgenic plants were selected in combinations transformed with nos::nptII, 35S::nptII and nos::bar. The highest transformation efficiency (5.3%) was obtained for cv. Akt transformed with nos::nptII. A detailed analysis of the T 0 plants selected from a given callus line and their progeny revealed that they were the mixture of transgenic, chimeric-transgenic and non-transgenic individuals. Southern blot analysis of T 0 and T 1 showed simple integration pattern with the low copy number of the introduced transgenes.

Efficient Agrobacterium-mediated transformation of Pennisetum glaucum (L.) R. Br. using shoot apices as explant source

“…Several workers reported the optimum cell density for Agrobacterium-infection in O.D. \ 1 [Zhao et al (2000) in sorghum; Kumria et al (2001) in rice; Sarker and Biswas (2002) in wheat; Gasparis et al (2008) in oat] while several reported O.D. = 1 as best suited for Agrobacterium-infection [Kumar et al (2005a) in rice; Shrawat et al (2007) in barley; Yookongkaew et al (2007) in rice].…”

Cotyledon with Hypocotyl Segment as an Explant for the Production of Transgenic Citrullus vulgaris Schrad (Watermelon) Mediated by Agrobacterium tumefaciens

“…EHA101 was also reported to be the effective vector for Agrobacterium transformation of Sesbania drummondii cotyledonary nodes and Petunia hybrida leaves (Padmanabhan andSahi, 2009: Thirukkumaran et al, 2009). However in oat, Boehmeria nivea and Astragalus racemosus, it was found that LBA4404 was a preferred strain (Gasparis et al, 2008;Ma et al, 2009;Darlington et al, 2009).…”

Genetic transformation of major cereal crops

“…A TF as high as 12.3% was obtained for cultivar Bajka with the LBA4404/pTOK233 system and kanamycin selection. Southern analysis on selected events revealed that 1-3 transgene copies occurred in the IE-derived transgenic plants (Gasparis et al, 2008).…”

“…Two types of explants (IEs and leaf base segments) from three oat cultivars (Bajka, Slawko, and Akt) were evaluated with the superbinary system LBA4404/ pTOK233 and 50 mg/L kanamycin as selective agent and the standard binary system AGL1/pGreen and 2 mg/L PPT (Gasparis et al, 2008). The TF was estimated as the percentage of selected, independent callus lines (each from one explant) that gave rise to at least one fertile transgenic plant.…”