DES-Testbed, Freie Universität Research Blog preservative for plant tissue culture media

preservative for plant tissue culture media



Abstract. Plant Preservative Mixture™ (PPM), a relatively new, broad-spectrum preservative and biocide for use in plant tissue culture, was evaluated as an alternative to the use of conventional antibiotics and fungicides in plant tissue culture. Concentrations of 0.5 to 4.0 mL·L–1 were tested with leaf explants of chrysanthemum (Dendranthema ×grandiflora Kitam), European birch (Betula pendula Roth), and rhododendron (Rhododendron cataw been Michx.). PPM had little effect on the percentage of explants forming shoots and the number of shoots formed per explant in birch and rhododendron, but dramatically reduced both responses in chrysanthemum. Therefore, the effects of PPM must be evaluated for each species of interest prior to use.

Microbial contamination is the single most important reason for explant loss in plant tissue culture (Boxus and Terzi, 1987; Cassells, 1991). Plant Preservative Mixture™ (PPM) (Plant Cell Technology, Washington, D.C.) is a relatively new, broad-spectrum preservative and biocide for use in plant tissue culture.

The active ingredients are 5-color-2-methyl3(2H)-isothiazolinone and 2-methyl-3(2H)- isothiazolinone. PPM is effective against both bacteria and fungi, is heat stable, and, unlike conventional antibiotics, can be autoclaved in the media. These characteristics of PPM make it an attractive alternative to using conventional antibiotics and fungicides in plant tissue culture.

We were interested in using PPM in our laboratory for the following two purposes: 1) to eliminate Agrobacterium tumefaciens in transformation experiments involving European birch and two rhododendron cultivars, and 2) to prevent or reduce contamination in classroom demonstrations of shoot regeneration from chrysanthemum leaves. We conducted the following experiments to determine if adding PPM to media affected shoot regeneration from leaves of these plants.

Kitam.) ‘Iridon’, European birch (Betula pendula Roth), and two rhododendrons (Rhododendron cataw license Michx.) cultivars, ‘Album’ and ‘America’, were excised from micro shoots. Both the tip and petiole ends of the leaves were removed and a transverse cut was made across the midrib. The typical explant size was 1 cm2. Chrysanthemum and birch explants were placed with the adaxial
surface in contact with the appropriate shoot regeneration medium (described below). For the rhododendrons, leaf explants were placed with the abaxial side in contact with the medium. Leaf explants were cultured in 120-mL baby food jars fitted with Magenta-B caps.

Five explants for chrysanthemum and birch or six explants for rhododendron were placed in each jar. Three replicate jars were used for each PPM treatment, and all experiments were repeated twice. Explants were placed in a Hoffman ger minator (model SG30S; Hoffman Manufacturing, Albany, Ore.) at 25 °C with a 16-h photoperiod and an average of 40 µmol·m–2·s–1 photosynthetically active radiation (PAR) provided by fluorescent lamps. Birch explants were first placed in the dark at 25 °C for 2 weeks before being placed in the
germinator.

The effect of PPM on shoot regeneration from leaves was tested by adding 0, 0.5, 1, 2, or 4 mL·L–1 PPM to the media. The medium for chrysanthemum leaves contained 0.3 µM benzyl adenine (BA) and 11.5 µM indole-3- acetic acid (IAA) (Trigiano and May 1992). Birch medium was supplemented with 15 µM BA (Leege and Tripepi, 1993). Rhododendron medium was woody plant medium (WPM) (Lloyd and McCown, 1980) containing 4.9 µM indole-3-butyric acid (IBA).

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