Leslie G. Hickok

The Biology of the Fern Ceratopteris and Its Use as a Model System

Ceratopteris (Parkeriaceae) is a pantropical genus of annual ferns possessing several features that make it exceptionally useful as a model plant system. The uniqueness of the homosporous fern life cycle allows the study of various phenomena at both the developmentally simple haploid gametophyte stage and at the complex vascular sporophyte stage. Consequently, both cellular and whole plant studies can be carried out simultaneously in a genetically natural and stable system. In genetic approaches, the ability to screen large numbers of mutagenized spores, along with the simple haploid genetics possible with the gametophyte stage and the ability to generate complete homozygotes from a single selfing, provide a powerful means of rapidly isolating and studying mutations. Ceratopteris gametophytes are developmentally simple. However, because they are autotrophic and possess several types of differentiated cells, they offer a number of opportunities for study. Control of germination responses, pattern formation and meristem development, spermatozoid differentiation, apogamy and apospory, pheromonal controls of sexual differentiation, as well as a variety of biochemical/physiological traits can be studied using various approaches. At the same time, the sporophyte provides a typical vascular plant system for study of a variety of developmental, genetic or biochemical/physiological phenomena. Recent and ongoing improvements in culture techniques, along with increased use of molecular approaches, will substantially expand the use of Ceratopteris as a model plant system.

The Programming of Sexual Phenotype in the Homosporous Fern Ceratopteris richardii

Genetically identical Ceratopteris gametophytes are either hermaphroditic or male. The determinant of sex type is antheridiogen (ACE), a pheromone that is secreted by the hermaphrodite and promotes male development of sexually undetermined gametophytes. The hormone abscisic acid (ABA) blocks the ACE response. The morphological and physiological changes in developing wildtype and mutant hermaphroditic1 (her1) gametophytes have been characterized and used to define six discrete stages of gametophyte development. Stage 1 begins when the spore is inoculated on culture medium. During this stage, the spore imbibes water but the spore wall remains intact and exposure to ACE will not promote male development, probably because of levels of ABA in the spore that are sufficiently high to block the ACE response. During stage 2, 3-4 d after spore inoculation, the spore wall cracks. Exposure of the two- to three-celled gametophyte to ACE during stage 2 is necessary for initiating a male program of expression. If not exposed to ACE at stage 2, the gametophyte initiates a hermaphroditic program of expression that cannot be reversed by exposure to ACE at later stages. During stage 3, 4-5 d after inoculation, the gametophyte consists of a uniserate protonema of three to five cells with one to three rhizoids. Gametophytes must be continuously exposed to exogenous ACE from stage 2 through stage 3 to develop as males. At stage 4, 5-6 d after inoculation, bidimensional growth of the prothallus begins. Although the male and hermaphroditic prothalli are morphologically indistinguishable at this stage, gametophytes begin to secrete ACE. At stage 5, beginning 6-7 d after spore inoculation, the male and hermaphroditic morphologies become distinct and mature sexually by stage 6. While the hermaphroditic program of expression is stable once initiated, the male program of expression, if initiated at stage 2, is reversible by the withdrawal of ACE or by exposure to ABA, indicating that ACE is required for both the initiation and maintenance of the male program of expression in Ceratopteris.

A Novel Method for Surface-Sterilizing and Sowing Fern Spores

Numerous methods are available for the sterilization of fern spores (see Dyer, 1979). Many of these methods are inefficient in terms of time and spore loss and require bulky equipment such as a centrifuge or vacuum filtration units. We present an easy and effective method for spore sterilization that requires no specialized tools or equipment, other than a sterile area. We have used the method presented here routinely to sterilize Ceratopteris richardii Brongn. spores. In addition, this technique has worked with spores of Lygodium, Pteridium, and other species of Ceratopteris (unpublished data). The basic innovation in this method involves the removal of liquid from a spore suspension in a conical centrifuge tube by means of a Pasteur pipet while leaving the spores in place. This method requires the following supplies: 1) sterile Pasteur pipets (Fisherbrand? borosilicate glass, 53/4 inches) with square-cut ends that are not cracked or chipped; 2) sterile pipet bulbs (natural rubber dropper bulb); and 3) sterile conical centrifuge tubes (Pyrex? No. 8060, 15 ml). To accomplish spore removal, insert a pipet with attached bulb into a conical centrifuge tube that contains a liquid and spores and suspend the spores in solution by bubbling air into the liquid. While continuing to bubble air, gently, but securely, seat the pipet tip onto the base of centrifuge tube. A slight rotation of the pipet may assist seating, but excess downward or lateral force will crush the pipet tip. Squeeze the pipet bulb to force additional air out of pipet, then slowly release the bulb to aspirate liquid into the pipet; most spores should collect at the base of the centrifuge tube and not be drawn into the pipet. This procedure has been incorporated into the following generalized spore sterilization and sowing method. Spore preparation.-These procedures can be performed outside of a sterile area. Transfer spores into a conical centrifuge tube and presoak them with distilled water for 16 hours. We routinely dry-sterilize (120?C, 2 hours) centrifuge tubes to eliminate cross-contamination of spore stocks and minimize contamination from the tube. If the spore source is exceptionally dirty or contaminated, wetted spores may be transferred with a Pasteur pipet to a clean sterile centrifuge tube immediately prior to sterilization. This significantly reduces the amount of coarse debris that may harbor and shield contaminants during sterilization. We prepare and sterilize from 2 to 250 mg of Ceratopteris spores per tube, depending on the desired sowing density and number of cultures to be sown. Spore sterilization.-All procedures for spore sterilization and spore sowing should be performed in a sterile area using standard sterile technique. All materials that come into contact with spores should be sterilized. To avoid contamination, use a clean sterile pipet and pipet bulb for each step. To sterilize spores, remove the presoak solution using the method described

Single gene mutants tolerant to NaCl in the fern Ceratopteris: Characterization and genetic analysis

Abstract NaCl-tolerant mutants of the fern Ceratopteris richardii Brongn. were isolated using a whole plant selection procedure in vitro. Two mutants, HaN10 and HaN16, showed enhanced spore germination and gametophytic growth compared to the wild type in the presence of 75–150 mM NaCl. Genetic analysis indicated a single nuclear gene as the basis of inheritance in both mutants. These mutants provide a means of examining mechanisms of osmoregulation and the development of salt tolerance in higher plants.

Assessment of gene copy number in the homosporous ferns Ceratopteris thalictroides and C. richardii (Parkeriaceae) by restriction fragment length polymorphisms

Homosporous ferns are generally considered polyploid due to high chromosome numbers, but genetically diploid since the expression of isozymes is generally controlled by a single locus. Gene silencing over evolutionary time is one means by which this apparent contradiction can be explained. A prediction of this hypothesis is that silenced gene sequences still reside in the genomes of homosporous ferns. We examined the genomes ofCeratopteris richardii andC. thalictroides for sequences which are similar to expressed gene sequences. Genomic DNA blots hybridized withC. richardii cDNA clones showed that the majority of these clones detected multiple fragments, suggesting that most gene-like sequences are duplicated inCeratopteris. Hybridization signal intensity often varied between fragments of the same size between accessions, sometimes dramatically, which indicates that not all sequences are equivalent, and may represent the products of silenced genes. Observed reciprocal differences in intensity could be due to reciprocally silenced genes. In addition, an unusual segregation pattern for one locus followed by one probe may indicate homeologous chromosome pairing and segregation.

INCHOATE SPECIATION IN CERATOPTERIS : AN ANALYSIS OF THE SYNTHESIZED HYBRID C. RICHARDII X C. PTERIDOIDES

Interspecific hybridization in the homosporous ferns usually results in the formation of sterile diploid or triploid hybrids. Sterility in these instances is the result of extreme differences in chromosome homologies between the parental taxa and the subsequent lack of normal chromosome pairing in the hybrids. Hickok and Klekowski (1973) have demonstrated that this type of hybridization is accompanied usually by the meiotic production of unreduced spores by the sterile hybrid; these spores have the potential to produce polyploid derivatives of the hybrid (allopolyploids) that have the ability to reproduce through normal sexual processes. Through this process, reticulate evolution of the type documented by Manton and Walker (1953) and Walker (1955, 1961) inDryopteris and by Wagner (1954) in Asplenium can be seen to be a major feature in the evolution of homosporous ferns. A notable exception to this production of sterile hybrids and fertile polyploid offspring in the ferns was documented by Walker (1958) in Pteris where hybridization between P. quadriaurita and P. multiaurita resulted in the formation of completely fertile F1 diploid hybrids that exhibited complete bivalent formation at meiotic prophase. The subsequent production of F2 segregates and backcross hybrids to parental types resulted in the production of a hybrid swarm in which all of the taxonomic

The Fern Ceratopteris richardii as a Lower Plant Model System for Studying the Genetic Regulation of Plant Photomorphogenesis

Photomorphogenetic research on fern gametophytes has provided important insights about pigment localization, ionic currents, and signal transduction. The first part of this article characterizes how light affects various aspects of gametophyte growth, such as spore germination, filamentous growth, and prothallial growth, in the three principal fern species used for photomorphogenetic research, namely, Adiantum capillus-veneris, Ceratopteris richardii, and Onoclea sensibilis. Although each species offers particular advantages for investigating certain photoresponses, we conclude that the process of spore germination in Ceratopteris is especially conducive to the selection of photomorphogenetic mutants. The second part describes the available Ceratopteris mutants isolated from three different selection schemes. Dark-germinating 1 (dkg1) exhibits the unique phenotype of reversed photoregulation of spore germination. Four other mutants, which are provisionally assigned the names of germ 1-4, are characterized in terms of germination percentages and prothallial growth in darkness, red light, and blue light. Germ 1 and 2 are impaired in their ability to respond to blue light, with the mutated genes apparently encoding signal transduction proteins that act close to the blue light photoreceptor. Germ 3 and 4 exhibit a de-etiolated phenotype in which the gametophytes grown in complete darkness resemble broader prothalli exposed to continuous light. Thus, the Ceratopteris mutants isolated to date complement, and perhaps extend, the range of comparable mutants in Arabidopsis seedling growth.

Selection of a mutation conferring high NaCl tolerance to gametophytes of Ceratopteris

SummarySpores from a weakly salt tolerant strain of Ceratopteris richardii containing the mutation stl1 were irradiated and sown on nutrient medium supplemented with 200 mM NaCl. A single highly salt tolerant gametophyte was recovered and selfed to generate a homozygous sporophyte. Spores from this strain, 10α23, were used to document the sexual transmission of the trait and to monitor the inheritance of tolerance in crosses to both the wild type and to the parental salt tolerant strain. Genetic analysis showed the 10α23 strain to possess both the original stl1 mutation and an additional semi-dominant nuclear mutation, stl2, that individually conferred a high level of tolerance to gametophytes. In combination, both mutations had additive effects. Tolerance was also evident in sporophytes, but at a lower level than in gametophytes.

Control of Sexual Development in Gametophytes of Ceratopteris richardii: Antheridiogen and Abscisic Acid

The species-specific chemical messenger, antheridiogen, mediates the differentiation of male gametophytes in the fern Ceratopteris richardii Brongn. For different genetic strains, characteristic frequencies of sexual gametophytes primarily depend upon the relative sensitivity of gametophytes to antheridiogen. Exogenous supplementation with abscisic acid inhibits this antheridiogen response in sensitive strains of C. richardii. To further clarify the basis of the antheridiogen sensitivity, we examined the responses of gametophytes to antheridiogen and abscisic acid in three strains with distinct sensitivities to these agents. Depending upon strain and sexual phenotype, abscisic acid inhibited male morphology, inhibited antheridia production, and reduced gametophytic growth. An inverse relationship of antheridiogen and abscisic acid sensitivity indicated that endogenous levels of abscisic acid may contribute to the antheridiogen sensitivity of individual gametophytes. Even though abscisic acid contents of spores and young gametophytes did not correspond to the relative sensitivities of strains to antheridiogen, concentrations in mature spores and sexually indeterminate gametophytes were sufficient to contribute substantially to a constraint of antheridiogen responses.

PHOTOBIOLOGICAL CHARACTERIZATION OF A SPORE GERMINATION MUTANT dkgl WITH REVERSED PHOTOREGULATION IN THE FERN Ceratopteris richardii

Abstract— This paper describes the mutant dkgl in the fern Ceratopteris richardii, which shows rapid germination in darkness but is markedly inhibited by white light. Action spectra plotted at 10 nm intervals from 400 to 800 nm are presented for germination responses of wild‐type and mutant spores to photon flux densities of 0.004, 0.04 and 0.4 jtmol/mVs. The action spectra for wild‐type spores exhibit a sharp phytochrome‐mediated peak at 660 nm, a broad peak from 670 to 740 nm resulting from an apparent high irradiance response and no germination below 560 nm. In the corresponding action spectra for mutant spores, the blue region displays rather complex fine structure with prominent minima at 450 and 470 nm, which suggests that cryptochrome is unaltered in these spores. The region from 550 to 640 nm shows the greatest inhibition of spore germination, but this region exhibits no obvious fine structure, which argues rather strongly against the possibility of a unique photoreceptor being active in mutant spores. The mutant spectra resemble the wild‐type spectra in the region from 650 to 800 nm, and thus phytochrome seems normal in the mutant spores. The dkgl mutation appears to act late in the phytochrome transduction pathway where a hypothetical coupling protein may regulate the light‐sensitive step in spore germination.