actually occurred. Gene expression may be due not to stable integration of a transgene, but
due to transient expression (Bellucci et al. 2003; Wenck et al. 2003). For annual, sexually
reproducing species, genetic segregation of the transgene in T 2 progeny will provide
additional evidence for Mendelian inheritance (Birch 1997; Potrykus 1991). The original
transgenic plant is called the T 0 generation. Suitable controls such as nontransformed,
wild-type plants and plasmid should be included where appropriate.
11.3 Initial Screens: Putative Transgenic Plants
Initial screens to identifyputative transformantsshould be simple, economical, and high-
throughput. Biotechnologists use “putative” in an optimistic way—the transgenic events
remain putative until significant evidence mounts from definitive molecular analyses.
Screens are designed to minimize detection of false positives later, thereby decreasing
the number of samples to be analyzed by the labor-intensive gel blots. Putative transfor-
mants are usually identified as those that withstand selection agents and are positive with
polymerase chain reaction (PCR) and/or ELISAs. Selectable and scorable markers (see
Chapter 10) are also helpful in initial screens of transgenic plants. None of these screens
are typically publishable in the scientific literature without accompanying in-depth molecu-
lar and phenotypic data.
11.3.1 Screens on Selection Media
Transformed plants regenerated from callus or afterin plantatransformation of germinating
seedlings are termed T 0 plants. T 0 plants are alwayshemizygous—meaning that there is a
copy of the transgene at a novel locus in the plant genome—the DNA gets integrated into
one chromosome but not the homolog at the same locus. T 0 plants produce T 1 seeds, which,
in turn, develop into T 1 plants that carry the transgene in either a hemizygous, homozygous
positive, or homozygous negative state in an expected 2 : 1 : 1 Mendelian ratio if there is a
single-copy insertion of the transgene. Similarly, transformed seed produced after trans-
forming T 0 ovaries using the floral dip method (see the previous chapter) are T 1 seeds,
which will be hemizygous for the transgene. In this case, the T 2 generation is the generation
in which there will be segregation and recessive phenotypes will be unmasked.
Screening transgenic seeds can be accomplished by aseptically plating seed on solidified
media with negative selection agents such as kanamycin, hygromycin, or phosphothricin in
Petri plates to identify plants that withstand the selection agent (Weigel and Glazebrook
2002). Surviving plants are transplanted to pots with potting mix to grow to maturity for
sample and seed collection. Initially, it may be necessary to determine the minimum
lethal dose of selection agent that kills 100% (LD 100 ) of wild-type plants but allows trans-
formed plants to survive. Nontransformed plants may become chlorotic, or bleached, or fail
to develop roots (Fig. 11.1). For comparative purposes, wild-type samples should be plated
on media without selection agent. In some species, optimal concentrations of selection
agent may be cultivar-specific.
Let’s imagine that an individual T 0 transgenic plant is self-pollinated to produce T 1 seed.
The T 0 plant will have the transgene present in the hemizygous condition. The T 1 seeds and
plants will segregate in a 3 : 1 phenotypic ratio for the transgene if there is asingle insertion
of the transgene. Single-insert transgenic events are desirable because the Mendelian inheri-
tance is simple, and expression patterns are often more predictable than with multiinsert
11.3. INITIAL SCREENS: PUTATIVE TRANSGENIC PLANTS 277