Currently employed transformation systems require selectable marker genes
encoding antibiotic or herbicide resistance, along with the gene of interest
(GOI), to select transformed cells from among a large population of
untransformed cells. The continued presence of these selectable markers,
especially in food crops such as rice (Oryza sativa L.),
is of increasing public concern. Techniques based on DNA recombination and
Agrobacterium-mediated co-transformation with two binary
vectors in a single or two different Agrobacterium
strains, or with super-binary vectors carrying two sets of T-DNA border
sequences (twin T-DNA vectors), have been employed by researchers to produce
selectable marker-free (SMF) transgenic progeny. We have developed a double
right-border (DRB) binary vector carrying two copies of T-DNA right-border
(RB) sequences flanking a selectable marker gene, followed by a GOI and one
copy of the left border sequence. Two types of T-DNA inserts, one initiated
from the first RB containing both the selectable gene and the GOI, and the
other from the second RB containing only the GOI, were expected to be produced
and integrated into the genome. In the subsequent generation, these inserts
could segregate away from each other, allowing the selection of the progeny
with only the GOI. We tested this vector using two selectable marker genes and
successfully obtained progeny plants in which the second selectable marker
gene segregated away from the first. Using the DRB binary vector system, we
recovered SMF transgenic lines containing a rice ragged stunt virus
(RRSV)-derived synthetic resistance gene in the rice cultivars Jarrah and Xiu
Shui. Approximately 36–64% of the primary transformants of
these cultivars yielded SMF progeny. Among SMF Jarrah transgenic progeny
<50% of plants contained the RRSV transgene. Thus, we have developed
an efficient vector for producing SMF plants that allows straightforward
cloning of any GOIs in comparison with the published ‘twin T-DNA’
vectors.
Agrobacterium-mediated Genetic Transformation of Potato (Solanum tuberosum L.) var. Cardinal and Heera
