Keywords

These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

3.1 Background

While collection of tissues in liquid nitrogen and −80 °C storage may be highly suitable for most plant species, it can be impractical in some developing countries owing to the expense and difficulty in procuring liquid nitrogen. The provision of continuous power supplies for ultralow (−80 °C) freezers may also be difficult and costly. Lyophilization, or freeze drying, is an alternative approach that results in tissue samples that can be stored at room temperature for many months prior to the isolation of DNA. This has been used to produce high quality genomic DNA suitable for high throughput TILLING assays (Till et al. 2004). Lyophilization circumvents the need for continual −80 °C storage, but commercial lyophilizers are also expensive. An alternative method is described in this chapter. Tissue is collected and stored in silica gel (Chase and Hills 1991; Liston et al. 1990). This removes water from tissues, and in many cases the dried material is stable at room temperature for weeks to months before the isolation of DNA. The exact length of time that dried tissue can be stored and still yield suitable quantities and quality of genomic DNA should be determined empirically. Other factors such as stress-induced accumulation of phenolic compounds may also limit the utility and shelf-life of the material. This is likely to vary between species and genotypes (Savolainen et al. 1995).

3.2 Materials

Materials needed for the desiccation of plant tissues at room temperature are listed in Table 3.1.

Table 3.1 Materials for collection, storage, and desiccation of plant tissues
Full size table

3.3 Methods

  1. 1.

    Label envelopes for tissue storage. Tissue desiccation works best when it is stored in porous materials. Paper envelopes, tea bags, or kimwipes work well.

  2. 2.

    The material should be cut to roughly the same length as the collection envelope to facilitate desiccation (Fig. 3.1, left panel).

    Fig 3.1
    figure 1

    Leaf tissue is collected and placed in an envelope (left panel). The length of the tissue should equal the length of the envelope. Silica gel with color indicator turns white when fully hydrated (right panel). Only gel with an orange color should be used

    Full size image
  3. 3.

    Immediately upon collection, place the envelopes containing the leaf material into a container containing silica gel. Seal the container with Parafilm to limit the effects of atmospheric humidity. The ratio of silica gel to tissue should be no less than 10:1 by weight (Weising et al. 2005). Orange silica gel has a moisture indicator. When fully dehydrated and ready for use, it is orange; when fully hydrated, the silica gel turns white (Fig. 3.1, right panel). The silica gel can be dehydrated by heating at a high temperature (over 80 °C) until the color returns to orange and may be re-used many times.

  4. 4.

    Incubate the material with silica gel for at least 48 h at room temperature (RT). The tissue is suitable for DNA extraction when brittle. Incubate for additional time if necessary. The tissue can be stored for long periods (>1 month) in silica gel at RT. It is suggested that you perform the tests in your own laboratory to determine the maximal amount of time that tissue can be stored under these conditions.