Fern gametophytes are ideal model systems for study of the mechanisms of hotomorphogenesis from the standpoint of physiology, photobiology, and cell biology (Wada, 2003, 2007;Kanegae and Wada, 2006). Positive aspects of the fern system include the following.
(1) Spores can be preserved at room temperature and they germinate under appropriate conditions within about a week in many species, becoming gametophytes that grow rapidly, at least in their critical early stages.
(2) Gametophytes are nutritionally autonomous, facilitating ease of cultivation.
(3) Gametophytes are not enclosed by other tissue, so that observation, light irradiation, and experimental manipulation are readily performed.
(4) Each developmental step can be controlled synchronously because gametophytes are highly sensitive to light. Each step in development is completely dependent on light; indeed, without light, development does not proceed.
Since the nineteenth century, especially in Germany, fern gametophytes have been used (see Dyer, 1979a)tostudy photo-physiological phenomena, such as light dependent spore germination (Mohr, 1956a), differentiation from one-dimensional protonemata to two-dimensional prothalli (Mohr, 1956b), and intracellular dichroic orientation of phytochrome (Etzold, 1965). Even though fern gametophytes are very good materials for the study of both photobiology and cell biology, only a few laboratories use them presently, probably for the following reasons.
(1) Although mutants can be obtained easily by phenomenological screening (gametophytes are haplophase), making crosses for genetic studies is difficult and time consuming.
(2) The biochemistry is also challenging because collecting enough gametophyte tissue for biochemical analyses is difficult.
(3) Molecular biological techniques are not yet established (e.g., stable transformation
is not available, although transient gene expression is possible).
(4) Most ferns are not commercially valuable plants, although some species, such as Osmunda japonica, Pteridium aquilinum,and Matteuccia struthiopteris,are edible and obtainable commercially in eastern Asia, or are used as ornamental plants, or for cleaning soil polluted by heavy metals including arsenic (Ma et al., 2001).
Nevertheless, fern gametophytes have structural and physiological characteristics that seed plants do not have, making them more tractable systems for studying many phenomena that are common to ferns and seed plants. For example, we have analyzed factors controlling the pre-prophase band (PPB) formation and its disruption (Murata and Wada, 1989b, 1991a, 1991b, 1992)(Figure 1.1). The PPB is recognized as a factor controlling the attachment site of newly synthesized cell plates to mother cell walls (Mineyuki, 1999). It appears before prophase of
thenuclear division cycle at the future site of cell plate fusion to the mother cell wall, but disappears before cell plate formation. The kind of information remaining at the PPB region has long been a mystery, as have the factors that determine the future cell plate attachment site and disrupt the PPB. To study this issue physiologically, Murata and Wada (1989b, 1991b, 1992)useda long protonemal cell cultured under red light in which cell division occurred at
40–60 µmfrom the tip where the division site is pre-determined by the PPB. During the period when the PPB was polymerizing, protonemal cells with a premature PPB were centrifuged to reposition the nucleus. A new PPB formed at the new nuclear site, distant from the original position, and then cell division occurred, suggesting that the nucleus must be close to the PPB polymerization site. In these cells the first PPB at the apical part did not de-polymerize even after cell division occurred, but if a dividing nucleus was returned to the former PPB
site, the PPB de-polymerized. This result indicates that PPB de-polymerization requires a nucleus and/or surrounding cytoplasm. Experiments such as these could not be done using seed plant cells because long cells like protonemal cells are not found in seed plants, except in some special cases such as cambium cells, where cell division occurs periclinally, making them inappropriate for the experiment. Experiments using long protonemal cells were also performed to study the recovery of a nucleus elongated by cell centrifugation (Wunsch and
Wada, 1989; Wunsch et al.,1989).
To analyze the physiological characteristics at each step of the developmental process or during transitions from one step to another of photobiological responses in fern gametophytes, various tools and special techniques have been developed. These include microbeam irradiators to stimulate only a small part of a cell and identify the photoreceptive site, i.e. the localization of photoreceptor molecules mediating a target phenomenon. The first machine was constructed in 1978 (Wada and Furuya, 1978)(Figure 1.2). Current microbeam projectors are now in their fourth or fifth generation, and are equipped with
various accessories depending on their purpose (Iino et al., 1990;Yatsuhashi and Wada, 1990).
This chapter will focus on recent analyses performed mostly by my laboratory group using Adiantum capillus-veneris.I also include some results that have not been published but are based on a synthesis of nearly 40 years of my experience with fern gametophytes. Our knowledge, mostly obtained from A. capillus-veneris, assumes that this species follows a pattern of development that is typical of most ferns. However, because of the large diversity in species and gametophytes, numerical data such as the growth rate of protonemata mentioned here may or may not be applicable to other fern species. For more information refer to books byDyer(1979b)and Raghavan (1989)andthefollowing reviews: Wada and Kadota (1989), Wada and Sugai (1994), Kanegae and Wada (2006), and Wada (2007).