Extra precautions should be taken when assaying putative transformants produced using
Agrobacterium-mediated gene transfer as the bacteria harboring the plasmid may be a
source of nonintegrated DNA in the plant cell and produce false positives in PCR
screens.Agrobacteriumcan systemically infect grapevine, rose, and fruit trees, but there
are methods available to distinguish systemic infection (Cubero and Lopez 2005) from
transgene integration. PCR should not be attempted in young plantlets or seedlings regen-
erated directly after anAgrobacteriumtreatment without an intervening antibiotic treatment,
and even then, there might be occasionalAgrobacteriumcells living among plant tissue.
Regenerating plantlets and whole seedlings can be treated with an antibiotic such as carb-
enicillin (Cheng et al. 1997) or cefotaxime (Broothaerts et al. 2005), respectively, to mini-
mize bacterial contamination. These antibiotics killAgrobacteriumbut do not harm plant
tissue. Seed produced afterin planta(floral dip) transformation of developing inflores-
cences should be washed in a dilute sodium hypochlorite solution before plating and/or
planting (Weigel and Glazebrook 2002). T 1 plants have a much lower chance of
Agrobacteriumcontamination compared with the original putatively transformed parent,
and therefore PCR data are more reliable on DNA extracted from progeny plants.
PCR reactions are easily contaminated at setup by aerosolized DNA (Scherczinger et al.
1999); therefore, cleanroom assembly, separate from the site where DNA extractions are
performed, is ideal. Aerosol tips and dedicated pipettors are additional safeguards against
contamination. In addition, in a lab that works extensively with the same GOI, PCR can
sometimes amplify contaminating DNA.
11.3.3 Enzyme-Linked Immunosorbent Assays (ELISAs)
For some selectable markers, qualitative or semiquantitative ELISAs are simple, sensitive,
high-throughput screens (e.g., AgdiaTMNPTII assay). Relatively crude plant protein
samples are extracted, and samples are incubated in the wells of microtiter plates that are
precoated with an antibody that binds the transgenic protein (antigen), primarily through
Figure 11.2.PositiveNPTIIELISA (AgdiaTM). Duplicate samples of NPTII standards are shown in
rows on the left, and duplicate samples are shown in rows in the rest of the microtiter plate. Plant
samples that are positive for NPTII protein are shown.
11.3. INITIAL SCREENS: PUTATIVE TRANSGENIC PLANTS 279