Biology Reference
In-Depth Information
FIGURE 15.2
Timeline for A. thaliana transformation and subsequent recovery of fluorescent T2 seed lines
for experimentation.
The outcome of the selection process is most obvious 10-18 days after sowing.
The first set of true leaves will emerge on the antibiotic-resistant plants (botanically,
the cotyledons are embryonic leaves and not “true” leaves). Resistant plants produce
true leaves and long roots, while the nonresistant plants are stunted and do not de-
velop true leaves or long roots. Resistant plants are darker green in color in compar-
ison to the nonresistant seedlings. Resistant T1 seedlings are gently removed from
the agar plates with forceps and carefully transplanted to soil. To acclimate the seed-
lings to life on soil, the plants should be covered with a clear plastic dome for 1 week
after transplanting to maintain high humidity around the seedlings. The dome is pro-
gressively lifted away after a week. The plants are grown to maturity, allowed to set
seed, and then T2 seeds are collected for experimentation. Multiple antibiotic-
resistant T2 lines should be collected for the next screening step.
15.1.1.4
Screening T2 seed lines
At least three different transgenic lines should be used in every experiment to com-
pensate for the random insertion of the T-DNA. The construct should be transformed
into a null mutant background whenever possible, and then the T2 generation should
be tested for complementation to demonstrate the proper functioning of a transgene.
