This review examines how real-time PCR can be used to determine copy number and zygosity in transgenic plants. Distinguishing between plants that harbor one and two copies of a transgene or are hemizygous and homozygous requires the ability to routinely distinguish twofold differences, a detection difference which approaches the resolution of PCR-based quantification methods. After explaining the basic principles, especially the threshold cycle (Ct value) as the basic measuring unit in real-time PCR, we introduce three quantitation methods currently in use. While the absolute and relative standard curve approaches are qualitative methods that distinguish high-copy from low-copy transformants, the comparative (2(-DeltaDeltaCt)) method with double-dye oligonucleotides (TaqMan probes) is able to detect twofold differences. In order to obtain reliable results, Ct values for an amplicon should be below 25 and the standard deviation below 0.3. Although real-time PCR can deliver exact copy number determinations, the procedure is not fail-safe. Therefore, real-time PCR should to be viewed as complementary to--rather than as a replacement of--other methods such as Southern analysis, but it is particularly useful as a preliminary screening tool for estimating copy numbers of a large number of transformants.

In its natural habitat, Astragalus bisulcatus can accumulate up to 0.65% (w/w) selenium (Se) in its shoot dry weight. X-ray absorption spectroscopy has been used to examine the selenium biochemistry of A. bisulcatus. High concentrations of the nonprotein amino acid Se-methylseleno-cysteine (Cys) are present in young leaves of A. bisulcatus, but in more mature leaves, the Se-methylseleno-Cys concentration is lower, and selenate predominates. Seleno-Cys methyltransferase is the enzyme responsible for the biosynthesis of Se-methylseleno-Cys from seleno-Cys and S-methyl-methionine. Seleno-Cys methyltransferase is found to be expressed in A. bisulcatus leaves of all ages, and thus the biosynthesis of Se-methylselenoCys in older leaves is limited earlier in the metabolic pathway, probably by an inability to chemically reduce selenate. A comparative study of sulfur (S) and Se in A. bisulcatus using x-ray absorption spectroscopy indicates similar trends for oxidized and reduced Se and S species, but also indicates that the proportions of these differ significantly. These results also indicate that sulfate and selenate reduction are developmentally correlated, and they suggest important differences between S and Se biochemistries.Many selenium (Se) compounds are toxic to mammals at high concentrations, but Se is also an essential micronutrient, and low doses have been implicated in cancer prevention (Clark et al., 1996; Combs et al., 1997). Not all diets provide adequate Se, and an obvious and inexpensive way to provide Se may be to engineer food plants to accumulate higher levels of the element (Ip et al., 1994). The Se hyperaccumulator Astragalus species, such as Astragalus bisulcatus, may be an excellent source of genetic material from which to isolate genes to develop such plants. In the wild, A. bisulcatus can accumulate Se levels of up to 0.65% (w/w) dry weight in the shoots (Byers, 1936), predominantly as Se-methylseleno-Cys (Trelease et al., 1960), and similar results are readily obtained in plants grown hydroponically in the laboratory (Orser et al., 1999). Understanding Se uptake in A. bisulcatus might also allow the development of highly effective cultivars for phytoremediation (Salt et al., 1998).A critical step in the biotransformation of selenate is the initial two-electron reduction to selenite. Hyperaccumulating plants might achieve this in at least three different ways: by substituting selenate into the sulfate reduction pathway (reduction by ATP sulfurylase/adenyl sulfate (APS) reductase; Shrift, 1969;Setya et al., 1996), by substituting selenate into the nitrate uptake pathway (microbial nitrate reductases can reduce selenate; Sabaty et al., 2001), or by a specific selenate reductase. For nonhyperaccumulating plants, there is good evidence that selenate reduction occurs via substitution for sulfate in the ATP sulfurylase/APS reductase system, and that this is the ratelimiting step in selenate transformation (Shrift, 1969;Shaw and Anderson, 1974; Burnell, 1981; Pilon-Smits et al., 1999). In these spe...

Genotypes of Nicotiana attenuata collected from Utah and Arizona were transformed with 17 different vectors (14 unpublished vectors based on 3 new backbone vectors) using an Agrobacterium-mediated procedure to functionally analyze genes important for plant-insect interactions. None of the 51 T1-T3 transgenic Utah lines analyzed by the flow cytometry were tetraploid, as opposed to 18 of 33 transgenic Arizona lines (55%). Analysis of T0 regenerants transformed with the same vector carrying an inverted repeat (IR) N. attenuata pro-systemin construct confirmed the genotype dependency of tetraploidization: none of the 23 transgenic Utah lines were tetraploid but 31 (72%) of 43 transgenic Arizonas were tetraploid. We tested the hypothesis that the differences in polysomaty of the explant tissues accounted for genotype dependency of tetraploid formation by measuring polysomaty levels in different seedling tissues. Hypocotyls, cotyledons, and roots of Utah and Arizona genotypes contained similar percentages of 4C nuclei (61 and 60; 7 and 5; and 58 and 61%, respectively). Since we used hypocotyls as explant sources and the nonoccurrence of tetraploid Utah transformants does not correspond to the high percentage of 4C nuclei in Utah hypocotyls, we can rule out a direct relationship between tetraploid formation and polysomaty level. We hypothesize that the difference between the Utah and Arizona genotypes results from the failure of polyploid Utah callus to regenerate into fully competent plants. We propose that future work on post-transformation polyploidy concentrate on the processes that occur during callus formation and plant regeneration from callus.

Background: After transformation, plants that are homozygous and contain one copy of the transgene are typically selected for further study. If real-time PCR is to be used to determine copy number and zygosity, it must be able to distinguish hemizygous from homozygous and one-copy from two-copy plants. That is, it must be able to detect two-fold differences.