A Summary of Research on Ornamental Ferns in Tissue Cultures
By Kara Mosovsky
This website provides a summary of some basic facts about ferns, their modes of propagation, their tissue and gametophyte cultures, and the formation of their sporophytes. The information on this page is summarized from a publication written by Bertrand H. Fernandez and M. A. Revilla.
All ferns belong to the family of organisms known as Pteridophyta. This family is over 300 million years old and contains roughly 12,000 different species of ferns today. Their are two main phases of a fern life cycle: the gametophyte and the sporophyte. Immediately upon germination of a spore, the organism is officially a gametophyte. A fern in the gametophyte phase is small in size, its cells are haploid (contain one set of chromosomes) and it progresses to maturity through stages of appearance and morphology. Although the gametophyte stage of a fern's life cycle is short-lived. The three main stages of development of a gametophyte are filamentous, spatulate, and heart shaped. When the fern reaches the heart shaped morphology the fern produces sex cells (gametes) for sexual reproduction. Each mature gametophyte has male sex organs (antheridia) and female sex organs (archegonia) which are located in the protected center of the heart shaped gametophyte. The sperm (antherozoids) fertilize the egg and give rise to the sporophyte generation of the fern.
Life Cycle of a Fern
Image created by Kara Mosovsky
In a fern's life cycle the sporophyte is larger than the gametophyte in size, its cells are diploid (contain two sets of chromosomes), and it releases spores for asexual reproduction. The spores are released from sporangia, tiny clusters of openings on the back of each frond of a fern. The sporophyte phase is the longest lasting stage and its morphological appearance is most closely related to what people imagine a fern looking like.
Naturally Growing Ferns in the Sporophyte Stage of their Life Cycles
5photo courtesy of http://plants.usda.gov/cgi_bin/topics.cgi?earl=plant_profile.cgi&symbol=ADCA&photoID=adca_002_ahp.tif
Modes of Propagation
Besides the sexual mode of propagation involving the union of an antherozoid and an egg within the gametophyte and the growth of a sporophyte from a spore, ferns can also be propagated by means of a vegetative method known as either in vitro or tissue culture. Although this method is reliable and effective, no genetic variability is produced through the vegetative method of propagation. The vegetative method is frequently used in mass producing ornamental and decorative ferns. Small pieces of rhizomes can be taken from a parent and planted separately. This rhizome will then grow into a new plant of identical genetic makeup of the parent. Frond tips, root buds, and bulbils are just a few other parts of a fern that can be separated or cut from a parent plant to propagate a new plant.
Although sterile technique and expensive lab equipment can be more successful to generate ferns by vegetative propagation for study or profit, vegetative propagation can also be done at home. Plant lovers everywhere can now begin their own family of ferns.
Cultures of Spores
Tissue cultures are widely used because they effectively grow spores in a partially sterile or completely sterile environment. Sand banks are partially sterile while Petri dishes plated with agar slabs are completely sterile. A sterile environment in this sense means that there is no contamination from foreign spores and no bacteria, molds, or fungi to grow on or overtake the tissue cultures. Before the tissue is set into the sand bank or Petri dish, the plant itself is sterilized. Mostly sodium hypochlorite is used to sterilized ferns but the concentration of the chemicals and the duration of its exposure to the plant differ between fern species and the organ of the fern which is being sterilized. Generally, the tissue is simply dipped into the sterilization liquid. If spores need to be sterilized, they can be suspended in a solution of sodium hypochlorite. These spores are then retrieved by centrifugation.
Different species of ferns differ in regard to the temperature that the tissue cultures will have optimum growth in, the nitrogen source that the ferns will utilize, and the mineral content the ferns need. Carbohydrates are another important aspect of the media that is used in tissue cultures because carbs aid in the growth and development of gametophytes. Also, light and pH are important factors in gametophyte germination and growth. Most ferns cannot germinate or grow in darkness or in media with a basic pH.
Cultures of Gametophytes
In nature the gametophyte's survival is dependent on a its environment. Conditions, whether too wet or too dry, can greatly influence the health of the gametophyte. In in vitro cultures however, a controlled environment can allow different types of media to successfully yield healthy gametophytes. The pH of the media, the nutrients available to the spore, and the amount and type of light that the spore is exposed to are just some of the many factors that influence the growth of the prothallium.
Many different types of nutrients are employed by gametophytes in in vitro cultures. Ammonia is one chemical that is often added to in vitro cultures. For heterotrophic plants ammonia serves as a rich supplier of nitrogen, an element which is needed for cellular and enzyme function. Another important nutrient for gametophyte growth is sucrose. Plants in in vitro cultures obtain their carbon from this sugar more than any other source.
Each and every species of fern has a certain set of requirements for optimum growth. Different fern species vary in the amount and type of nutrients they require. For example, ammonia inhibits growth in gametophytes for Osmunda regalis, but promotes growth of gametophytes for Pteris ensiformis and Blechnum spicant. Also, different species of ferns require different concentrations of sucrose in their growing media. For example, in in vitro cultures of Osmunda regalis no sucrose is required by the plant to grow. Although normally heterotrophic in nature, this independent growth illustrates that this specific species of fern is autotrophic in nature when grown in cultures.
Light, pH, and the physical state of the growing media are still other factors that are required in varying amounts for different species of fern gametophytes. While most prothallium require ample light for growth and development the heart shaped structure of a Osmunda regalis gametophyte can still develop without the presence of light. In fact, the growth of the gametophyte in the species Asplenium nidus is favored by environmental conditions of less light. While some in vitro cultured ferns only thrive in a medium with a specific pH others can tolerate extreme fluctuations in pH. The species Asplenium nidus and Blechnum spicant, for example, can survive when its cultured medium undergoes 4.5 units of change to its pH. The physical state of the growing medium also influenced the growth of the gametophyte. More solid cultures (ie. cultures supplemented with more agar) were less effective than more liquid cultures for increasing gametophyte growth.
The Formation of Sporophytes
Again, different species of ferns vary in different aspects of their formations. The sporophyte can be formed either relatively quickly or over a long period of time for different species of ferns. The life cycle of the gametophyte can last between one month and 6-8 months depending on the species in question. The length of the gametophyte's life cycle is not the only variance between species of sporophytes. In some species such as Asplenium nidus and Dryopteris affinis, the gametophyte stops growing after it starts to form the sporophyte. Other species grow simultaneously.
Both sexual and asexual forms of reproduction occur in ferns during sporophyte formation, however sexual reproduction is by far the most common method of sporophyte formation. In sexual reproduction, the sporophyte is produced when one female and one male sex cell fuse together. The two sex cells can either be from the same organism or from different organisms, but in nature, crosses involving a single sex cell from two different organisms is favored. These intergametophytic crosses between individuals creates more genetic variability in the population. Sporophytes can also be produced through asexual reproduction. In "obligate apogamy" sexual fusion of sex cells is not utilized to create a sporophyte. Tissue cultures and in vitro growing techniques can be used to asexually reproduce ferns.
Close-up of a Fern Sporophyte
6photo courtesy of http://plants.usda.gov/cgi_bin/topics.cgi?earl=plant_profile.cgi&symbol=ADPE&photoID=adpe_002_ahp.tif
4) original summary article by H. Fernandez and M. A. Revilla (see below)
This webpage is the summary of In Vitro Culture of Ornamental Ferns written by H. Fernandez and M.A. Revilla in Plant Cell, Tissue and Organ Culture. Vol. 73 pages 1-13, 2003