Experiment 2: Asymbiotic Seed Germination and Development of Orchids



In Experiment 1, you learned about the various stages involved in in vitro asexual propagation from woody plant axillary shoot cultures. In general, asexual approaches are most frquently used in micropropagtion. It should be noted that some workers (e.g. Arditti, 1992) prefer to restrict the use of the term "micropropagation" to asexual methods only (in contrast to its use in the title of this experiment). At any rate, in vitro sexual propagation (embryo culture) is very important in the routine commercial propagation of orchid hybrids and some species (e.g. Paphiopedilum sp.). Embryo culture is also important as a tool for modern plant breeding of non-orchid horticultural and agronomic crops (wide crosses).

Orchid seeds are extremely small, ranging from 0.25 mm to 1.2 mm long, and 0.9 mm to 0.75 mm wide. They weigh only 0.3 to 14 mg per seed. The seed consists of a seed coat and rudimentary embryo of 80 to 300 undifferentiated cells (Arditti, 1992).

Under natural conditions (in soil, in the case of terrestrial orchids or on the bark of a mother tree in the case of epiphytes), these relatively minute orchid seeds have insufficient stored food reserves directly available to the embryo to support seed germination. Germination in nature is entirely dependent on the formation of a mycorrhizal association with an appropriate fungus, and hence is referred to as symbiotic germination. By digesting soil organic matter such as cellulose the fungus makes soluble sugars available to the embryo (Arditti, 1992).

In the laboratory, however, it is possible to germinate orchid seeds without a mycorrhizal fungus by providing simple carbohydrates (sugar) and other nutrients in a nutrient medium. This process is known as asybiotic germination was first demonstrated by Dr. Lewis Knudson (Professor and Chairman of the Department of Botany at Cornell University, 1911-52), in a paper which he published in 1922 (Knudson, 1922). This and subsequent papers described the method and the recipe for the still commonly used Knudson C nutrient medium. Knudson's breakthrough made possible modern orchid breeding and mass production on a much larger scale.

This experiment is composed of 3 parts. In the first part you will learn how to sterilize orchids, collected from dehisced seed pods, and sow them on nutrient agar. Due to their relatively slow growth (even under ideal conditions) you have time during the course to observe the early stages of germination, but insufficient time to observe seedling development for transplanting out of culture. Therefore, in the 2nd part of this experiment, orchid seedlings, sown approximately 2 months before this lab will be available for you to observe and subculture (reflasking). Finally, in the third part of the experiment, orchid seedlings sown approximately 6.5 months ago and subcultured approximately 4.5 months ago will be made available for you to transplant from the tissue culture vessels to a pot.

Although many orchid species or hybrids could be used for this experiment, the large white flowered Phalaenopsis hybrid or Dendrobium antennatum have been chosen because both are easily flowered in the greenhouse (for seed production) and because both germinate and grow relatively rapidly in vitro (for orchids, that is).

Summary (Klecha, 1999)

Orchid Seeds

Symbiotic Association With Mycorrhizal Fungi (Bechtel et al, 1992)

Asymbiotic Micropropagation Time Line and Sequence (Chu, 1995)

  1. Pollination (+0 months, T=0)
  2. Mature yellow stage capsules harvested (+ 5 months, T=5)
  3. Subculture seedlings onto fresh media (+2 months, T=7)
  4. Outplanting into Spagnum Moss (+4.5 months, T=11.5)
  5. Acclimatization (+0-4 weeks, T=11.5-12.5)

Propagation Phalaenopsis hybrid and Dendrobium antennatum (Chu,1995)

Laboratory Purpose