Thomas R. Warne

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.

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 In addition, this technique has worked with spores of Lygodium, Pteridium, and other species of Ceratopteris (unpublished data).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.

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.

The Analysis of Genetically and Physiologically Complex Traits Using Ceratopteris: A Case Study of NaCl-Tolerant Mutants

Genetic and physiological complexities associated with salt tolerance in plants have limited progress in the analysis of specific factors responsible for the salt-tolerant phenotype. We have used the homosporous fern Ceratopteris richardii as a model plant to investigate the physiological basis of salinity tolerance by selecting single gene mutants that confer tolerance in the gametophyte generation. The unique genetic system of homosporous ferns permits the generation of mutants in a genetic background nearly isogenic to the wildtype, such that comparative studies with the wildtype can identify specific physiological responses associated with salt tolerance. One of these mutations, stl2, confers a high level of tolerance to

CERATOPTERIS: AN IDEAL MODEL SYSTEM FOR TEACHING PLANT BIOLOGY

{\rm Na}^{+}\ ({\rm I}_{50}\approx 175\ {\rm mM\ {\rm NaCl)}}

CERATOPTERIS RICHARDII: APPLICATIONS FOR EXPERIMENTAL PLANT BIOLOGY'

and generates a complex suite of related phenotypes. For example, in addition to Na+ tolerance, stl2 exhibits tolerance to Mg2+ salts, sensitivity to supplemented K+, higher K+-dependent efflux of K+, altered responses to Ca2+ supplementation and moderate tolerance to osmotic stresses. Based upon its physiological attributes, we have proposed that the mechanism of action for this mutation involves an enhanced influx of K+ and higher selectivity for K+ over Na+ in a K+ channel. The direct and indirect consequences of this alteration can account for NaCl tolerance and the other phenotypes evident in stl2. The complex set of phenotypic responses from such a single gene mutation illustrates the potential for even more extreme pleiotropy in multigenic salt-tolerant strains.

Teaching Preservice Science Teachers How to Do Science: Responses to the Research Experience

We have successfully utilized Ceratopteris richardii as a pedagogical tool in introductory and upper-division investigative exercises and in independent research projects. Undoubtedly, this is one of the simplest and easiest to implement biological systems in educational applications. Plants, especially gametophytes, are readily maintained in culture and can be used as a means to explore a wide array of biological phenomena, research tools, and techniques. This fern affords the advantage of rapid growth, especially for studies of growth regulation, development, and genetics. It enables experimentation on single cells and subsequent manipulation of differentiation into multicellular organisms. Hundreds of individuals may be maintained in a single petri dish, thus facilitating observation of population dynamics and genetics.