Direct creation of marker-free tobacco plants from agroinfiltrated leaf discs

Abstract

Agroinfiltration is employed as a fast way to directly create marker-free transgenic tobacco plants. As an example for the efficiency of the method, Agrobacterium cells harboring a marker-free vector coding for beta-glucuronidase (GUS) were infiltrated into the leaf discs of Nicotiana tabacum, which were then used as explants for marker-free plant regeneration by tissue culture. Through GUS staining, a large number of small calli were shown to be stably transformed on the treated leaf discs at 17 days after agroinfiltration. Most importantly, after continuous culture of the leaf discs until shoot regeneration, about 15% of the regenerants were proven to be transformants by polymerase chain reaction (PCR) analysis.

Agroinfiltration of intact leaves as a method for the transient and stable transformation of saponin producing Maesa lanceolata

“…In contrast, a number of plant cells could be regenerated and stably transformed by agroinfiltration. Similar to our findings, using agroinfiltration in the creation of marker-free tobacco plants was also more successful than using the immersion technique (Jia et al 2007;Kopertekh and Schiemann 2005). We speculate that agroinfiltration has an advantage over immersion because in agroinfiltrated tissue more plant cells are accessible to Agrobacterium adherence and interaction.…”

Selectable marker-free transgenic orange plants recovered under non-selective conditions and through PCR analysis of all regenerants

“…A second PCR analysis was performed at this point. No escape shoot was recovered due to false-positive results in the first PCR analysis, an issue that may count up to more than 30% in the genetic transformation procedures of other species, such as tobacco (Jia et al 2007;Li et al 2009). Putative transgenic sweet orange plants were analyzed later by Southern blot to confirm the insertion of the complete sgfp module and integration of the transgene in different loci within the genome.…”

“…Using the same system, Permingeat et al (2003) obtained 2.2% SM-free transgenic wheat plants after bombarding embryogenic calli derived from immature embryos. More recently, Jia et al (2007) reported the production of marker-free tobacco plants through shoot analysis by GUS staining and PCR and the benefits of agroinfiltration to achieve a higher transformation efficiency (up to 15%) than after explant-Agrobacterium co-cultivation. Li et al (2009) also demonstrated the recovery of a large number of transgenic tobacco plants under non-selective conditions (2.2-2.8% transformation efficiency) and addressed several important issues associated with this system, such as the incidence of chimerism or escapes, transgene inheritance, and factors affecting transformation efficiency.…”

Strategies for developing marker-free transgenic plants

“…In initial research, marker-free transgenic plants were obtained using a non-selected approach in vegetative propagating crop potato and tobacco [57,58]. In these studies, explants were infected with A. tumefaciens harboring a binary vector without a selectable marker gene, either by soaking in the Agrobacterium solution for a few minutes as in potato or by agroinfiltration as in tobacco.…”