Direct gene transfer in potato: A comparison of particle bombardment of leaf explants and PEG-mediated transformation of protoplasts

Abstract

Direct gene transfer methods in potato would facilitate the transfer of multiple genes and the manipulation of metabolic pathways in this species. In this study, up to 1.8 transformation events per shot (=0.5 per bombarded leaf) and 67.2 events per million protoplasts treated were obtained with particle bombardment and PEG-mediated direct DNA uptake, respectively. Limited disassociation of both HPT and GUS genes appeared to occur during the process of integration in only 19% of transformants. A large number of transformed potato plants with transgene expression at levels comparable to Agrobacterium-mediated transformation was obtained. High levels of GUS expression were only obtained in lines derived from PEG treatment. No correlation between the number of gene insertions and gene expression levels was found, suggesting that multiple insertions may have little or no effect on transgene expression.

Transformation of potato via Agrobacterium coated microparticle bombardment

“…Biolistic gene transfer, a method by which foreign genes may be introduced into cells via high velocity microprojectiles (Klein et al 1988) has been used in transforming monocots such as orchids (Suwanaketchanatit et al 2007) and dicots such as soybean (El-Shemy et al 2007), but has only recently been used in potato. Up to 0.5 transformants per leaf shot were achieved using particle bombardment (Craig et al 2005), almost two orders of magnitude higher than the first report in potato .…”

“…We then tested the integration of the three genes using Agrobacterium-biolistic mediated transformation of potato with the strain LBA4404 carrying plasmid pVDH396 (hpt-ipt-uidA). Selection and transformation carried out using the same conditions for biolistic transformants (Craig et al 2005), yielded 9 hygromycin resistant lines from 20 shots ( Table 1). All hygromycin resistant lines were tested in numerous PCRs for the presence of ipt, uidA and hpt.…”

“…For PCR analysis, total plant DNA from wild type and hygromycin resistant calli or shoots was isolated using the DNeasy ® Plant Mini Kit (Qiagen, Valencia, CA, USA), following the kit instructions. The presence of uidA and hpt was confirmed by PCR with primer pairs described previously (Craig et al 2005) generating 365 and 508 bp products respectively. Ipt was screened for with the following primer pair: ipt forward 5'-TCAACCGGAAGCGGACGACC-3', reverse 5'-GCC ATGTTGTTTGCTAGCCA-3' product size 355 bp.…”

“…Ipt was screened for with the following primer pair: ipt forward 5'-TCAACCGGAAGCGGACGACC-3', reverse 5'-GCC ATGTTGTTTGCTAGCCA-3' product size 355 bp. The thermal cycling programs for uidA and hpt were as described (Craig et al 2005). The thermal cycling program for ipt primers was set at 94 °C for 5 min, followed by 35 cycles at 1 min at 94 °C, 30 s at 60 °C and 1 min at 72 °C with a final extension at 72 °C for 7 min.…”

Morphological attributes and production components of potato cv. Baronesa and its transformed genotype

“…Research on the genetic modification of different potato cultivars aiming to increase disease resistance and to improve the nutritional and industrial quality has been described in several papers (ANDERSSON et al, 2003;CRAIG et al, 2005;FIGUEIRA FILHO et al, 1994;LÓPEZ;CHAPARRO, 2007;ROMANO et al, 2001;TORRES et al, 2000).…”