sions to produce packets containing 64 or 128 mature sperm. Soon after they form, the sperm packets are
released into the surrounding medium and attach to the somatic cells of female spheroids. They subse-
quently make a hole in the spheroid wall and the individual sperm are released into the interior, where
they fertilize the eggs. The resulting zygote develops into a cold- and drought-resistant dormant zy-
gospore that, in culture, becomes activated only when fresh medium is added. In the meiotic division that
follows activation only one new germling is produced; the other meiotic products are polar bodies.
As with the asexual cycle, many mutants in sexual development have been isolated and character-
ized [16,23]. Somewhat surprisingly, many of these mutants mix and match the asexual, male, and female
patterns of cell division with the production of gonidia, sperm packets, and eggs: gonidia can result from
cleavages in either the male or female pattern, and sperm or eggs can result from the pattern normally seen
in the opposite sex. Thus, it is clear that there is no tight linkage between the specific cleavage pattern and
the specific type of cellular differentiation that ensues. Explanation and understanding of the controlling
factors in these processes will have to await the cloning and characterization of the relevant genes. Given
the newly available tools of molecular developmental analysis of Volvox(see later), that understanding
will probably come soon.
D. Molecular Approaches
In 1994, Volvoxfinally yielded itself up to the techniques of molecular transformation [24], and the pre-
ceding models are now being directly tested at the molecular level (see later). Early difficulties in trans-
formation in Volvoxwere probably due to the poor performance of promoters from higher eukaryotes, to
an extensive DNA methylation system, and to a very biased codon usage: these render standard selectable
markers and reporter genes unusable. Only when an endogenous gene that can be used as a selectable
marker was cloned [25] and employed did transformation succeed [24]. The gene is that for nitrate re-
ductase (nitA), which has the very useful property that one can select for both the presence and absence
of the functional gene: nitAstrains can be selected on the basis of their resistance to chlorate, and only
nitAstrains will grow on nitrate as the sole nitrogen source. Subsequently, the HUP-1gene from
Chlorella, which encodes a glucose/Hsymporter, was also found to be useful as a selectable marker in
Volvox[26], and a construct containing a bacterial blegene under the control of a Volvoxtubulin promoter
has worked to confer stable resistance to the antibiotic zeocin [27]. Transforming DNA is easily intro-
duced via particle bombardment and appears to integrate randomly into the host genome, although ho-
mologous recombination with the endogenous gene has been reported [28]. Nonselectable genes can be
introduced on separate plasmids and cotransformed with the selectable DNA. Finally, developmental
DEVELOPMENTAL GENETICS IN LOWER PLANTS 807
Figure 3 The sexual cycle of Volvox carteri. Asexual males and females (which are morphologically indis-
tinguishable from one another) respond to the sexual inducer by undergoing another round of asexual embryo-
genesis in which the patterns of asymmetric division are modified and in which the germ cells that are formed
develop not as gonidia but as sperm packets or eggs. Sperm-egg fusion results in formation of a dormant, re-
sistant zygote. When dormancy is broken by washing with fresh medium, each zygote undergoes meiosis to
form one viable germling and three polar bodies. (From Ref. 13.)