Ceraratopteris Richardii Research Paper

Ceratopteris richardii: The effects of increasing spore density in detecting higher percentages of sexually expressed gametes.

Abstract:

The Ceratopteris genus is a model plant system in allowing the study of life and reproductive cycles of spores, allowing for spore to gamete cellular and physical observation. The triangle water fern, Ceratopteris richardii, provided for visual identification of this transition. Spores of different densities were inoculated, cultured, and observed in germination and sexual expression. We estimated higher spore densities increase percentages of sexually expressed gametes, male and hermaphrodite; and concluded that spore density has a direct effect on germination and sexual expression numbers. The ability to observe the spore to gamete transition leads to opportunity in manipulation of gametes for biological studies
related to vascular plants. Introduction: The model use of the homosporous Ceratopteris richardii (C-Fern) provides for high accessibility and understanding of its life and reproductive cycles, both haploid and diploid (2, 3.) The life and reproductive cycles of the sporophyte are easily observable under a compound microscope; and volume harvesting and culturing of spores for experimental studies are relatively easy due to the quick life cycle of the Ceratopteris at 120 days or less (4.) Spore life and reproductive cycle summary consists of spore germination, gametophyte differentiation, pheromone antheridiogen sexual differentiation, embryonic development, through to spore maturation (Fig. 1.) Male or hermaphrodite gametes, of the Ceratopteris, are determined by the pheromone antheridiogen or ACE. This pheromone is responsible for stimulating the determination of male gametes and is secreted from the hermaphrodite gametes. This allows for a ratio of males to hermaphrodite gametes to vary depending on the spore density (Fig 3, 4) (5.)
We estimated that higher spore densities will generate higher amounts of gametes and sexually expressed gametes, several spore densities were cultured to ascertain the percentages of gamete production. The higher the spore density, the increased amount in the germination rate of gametes; allowing for volume culturing, harvesting, and manipulation of gametes for sexual expression in biological studies (2.) Materials and Methods:

The initial week, six different densities of C-fern spore medium were inoculated into six separate Petri dishes, labeled A-F, with A containing the highest C-fern spore density. Upon inoculation, each of the Petri dishes were sampled for spore density by using a dissecting microscope, and spore densities were recorded in Table 1. This procedure was repeated during several laboratory sessions. The second week, it was discovered that there was contamination exposure within the source vials of spore density medium, after identifying evidence of contamination within the inoculated Petri dishes. The third week specific matched groups of Petri dishes, identified as having no visual indication of the contamination, were then collected and analyzed for the experiment. The C-fern spores were observed and visually identified, using a compound microscope at 100 times magnification, to identify the developing gametophytes. The dissection microscope was then used to visually identify the number of germinating gametophytes, and their numbers were then recorded into Table 1. Upon the fourth week, the gametophytes were visually identified for variances in sexual development and expression, using a compound microscope at 400 times magnification. Individual quantities of both mature hermaphroditic gametophytes and mature male gametophytes were then recorded in Table 1. The recorded data was entered into Microsoft Office Excel, and an ANOVA: single factor was completed to determine any significant changes.
Results:

Ceratopteris genus, producing the largest spores of any homosporous fern, has been proven with accessibility in understanding reproductive pathways for vascular plants (2, 3.) Ceratopteris richardii gametophyte germination produces sexually expressed gametes within a matter of 10-12 days after inoculation (2.) Visual indication of sexual expression was achieved using a compound microscope at 100 times, beginning the fourth week (Table 1.) Higher percentages of both mature male and mature hermaphrodite gametes were also evident the beginning of the fourth week.
Increases in spore density from the source sporophyte density medium vials showed an increase spore to gamete cycling in the inoculated samples (Table 2.) Studies have determined that the ratio of sexual expression within cultures is dependant upon the spore densities at inoculation (2.) The total calculation of spores for experimental purpose was 577 spores determined within six each 23 cm2 Petri dishes, generating a total of 738 gametes, resulting in 66 combined mature sexually expressive male and hermaphrodite gametes .
Discussion: Indications of sexual ratio to spore density at inoculation for this experiment were higher in samples inoculated with the higher spore density (Table 1.) The higher source spore density medium A-F produced a higher percentage of gametes and sexually expressive gametes, implying a positive hypothesis of greater chance for accumulation of sexually expressive gametes per spore density inoculations and for future biological studies. The Genus, Ceratopteris, has the ability to produce large spores, and is a model system in displaying reproductive capabilities for experimentation purposes (2, 3.) A study in gene silencing using reverse genetic analysis of gene function in C-fern gametophytes resulted in successful sexual expression through manipulation of the pheromone ACE, concluding that DNA structures are sufficient enough to induce gene silencing in gametophytes, and is useful as an capable genetic tool for screening the reproductive functions of the gametes. In comparison of the study of the Ceratopteris as a model system with this particular experiment, it is supported that the Ceratopteris genus is strongly accessible for a variety of experimental studies including determination and manipulation of reproductive expression experimentation (3.) Influences on data such as contamination of source spore density vials and errors in the data collection could have had an effect on the results. Future experiments should conduct pre-trials to check for contamination prior to conducting actual experiments, repeated quantity tallies of spore and gamete per samples should be conducted as a calculation error deterrent.