As evident from the above-mentioned exceptional transmission of paternal plastid genes in Chlamydomonas and the discussion of paternal vs. biparental inheritance in gymnosperms, there is a grey zone between uniparental inheritance and biparental inheritance. In most instances, the conclusion that a given species transmits its plastids uniparentally is based on the phenotypic analysis of at most a few thousand progeny plants from reciprocal crosses (see 4.2). Failure to detect variegated seedlings is usually interpreted as uniparental mode of inheritance. However, in this approach, occasional plastid transmission from the other parent goes undetected if it occurs only at a very low level. How strict maternal plastid inheritance can be has been a highly controversial issue, particularly in the context of the level of transgene containment provided by plastid transformation technology (see below). Paternal leakage, the low-level paternal transmission of plastids in species believed to inherit their plastids maternally, is known to occur at least in some plant species (Avni and Edelman 1991; Medgyesy et al. 1986; Horlow et al. 1990; Wang et al. 2004). A large-scale genetic study in foxtail millet, Setaria italica, employed crosses between male-sterile yellow- or green-leafed herbicide susceptible lines (as maternal parent) and a line with chloroplast-inherited atrazine resistance as pollen donor (Wang et al. 2004). Assaying more than 780,000 hybrid offspring for atrazine resistance as it would be caused by paternally transmitted plastid genomes, paternal leakage was detected at a frequency of 3x10-4. Unfortunately, similarly reliable quantitative data in other plant species are largely lacking. It seems reasonable to suspect that the rate of paternal leakage can be very different in species representing the different subtypes of maternal inheritance (Fig. 2; Table 2), but this remains to be established experimentally.
The laborious and time-consuming genetic analyses required to establish low-level paternal leakage make it desirable to develop faster assays suitable to assess a species' potential to occasionally transmit paternal plastids via pollen. A rapid screening method that has been widely used employs staining of pollen with the DNA fluorochrome DAPI to identify plastid DNA in generative cells (Corriveau and Coleman 1988; Zhang et al. 2003). DAPI stains intensely plastid nucleoids which then can be readily detected by fluorescence microscopy. Absence of stain-able plastid DNA from generative and sperm cells was taken as evidence for strictly maternal inheritance, whereas species with detectable ptDNA in generative and/or sperm cells were classified as potentially capable of occasional or regular biparental plastid transmission (which, however, does not mean that these species indeed display biparental plastid transmission: species of the Triticum type regularly have plastids in their sperm cells, but yet transmit their plastids maternally; Fig. 2 and 4.1.3). The latter was the case for roughly one fifth of the species investigated (Corriveau and Coleman 1988). Generally, how reliable DAPI staining of pollen grains can predict paternal leakage will require confirmation by rigorous genetic analysis.
Finally, it seems possible that environmental factors influence the rate of occasional paternal plastid transmission. Experimental evidence for this has been obtained already in Chlamydomonas where exposure of mt+ gametes to UV light increases the rate of occasional paternal chloroplast DNA transmission (Hagemann 2004). Whether or not abiotic stress conditions also affect plastid inheritance in higher plants (which is mechanistically very different from Chlamydomonas; Table 2) remains to be investigated.
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