events. Therefore, if 100 T 1 millet seeds segregating for a single insertion are plated onto
tissue culture media containing a selection agent, 3 : 1 segregation might appear as approxi-
mately 75 of the seedlings appearing as the ones in Figure 11.1 left, and the approximately
25 nontransgenic segregants appearing as those in Figure 11.1 right. Note that the word
“approximate” is used here since by chance the actual numbers can vary. Because of this
variation, multiple replicate plates would be used and a statistical test, such as the chi-
squared (x^2 ) test would be employed to see if the observed results do not differ from the
expected result of 3 : 1 segregation.
11.3.2 Polymerase Chain Reaction
Polymerase chain reaction(PCR) (Mullis 1990; Saiki et al. 1986) is a highly sensitive
method that can be used to screen for genes of interest (GOIs) or selectable markers
(SMs) using relatively crude DNA extracts. It is the amplification of a DNA sequence in
a microcentrifuge tube with two primers that are complementary to the 5^0 and 3^0 ends of
the DNA sequence and Taq DNA polymerase from the thermophile bacteriumThermus
aquaticus. Through repetitive heating and cooling cycles, the DNA sequence between
the two primers is amplified (Reece 2004). PCR results are invalid if positive and negative
controls are omitted or do not work.
Figure 11.1.Comparison of wild-type millet seedlings grown on media with and without selection
agent. Wild-type millet seedlings grown on MS media with 0 mg/L geneticin (left) versus wild-type
millet seedlings grown on MS media with 60 mg/L geneticin (right). Seedlings grown on geneticin
failed to develop roots and are more bleached in appearance.
278 TRANSGENIC PLANT ANALYSIS