Explant Disinfection

Explant surface disinfection is a delicate and indispensable procedure, because every subsequent step in culture must take place under sterile conditions. An optimal disinfection protocol is a compromise between the minimisation of surface microorganisms and the avoidance of tissue damage by the disinfectant. For mistletoe, these requirements can be fulfilled by treating explants in a solution of either 1% (w/ v) sodium hypochlorite for 15 min, or 0.1-0.2% (w/v) mercuric chloride for 12 or 20-25 min. Mistletoe leaf explants are generally more sensitive to surface sterilisation than stem explants.

When tissue-culturing V. album, explant browning can be an serious problem: explants develop necrotic-like areas which occasionally lead to explant decline and death, thus negatively affecting callus induction. However, depending on the disinfection protocol and the composition of the induction medium, a callus tissue can be formed on 4-12% of the declined explants and proliferate further. These callus tissues retain a black-brownish colour even after repeated subcultures.

The addition of ascorbic acid (at a concentration of 10 mg/1) to the culture medium greatly reduced the frequency of the necrotic symptoms, allowing thus for an increased callus induction (13-45% of cultured explants, depending on medium composition and explant source). These calli obtain a pink-whitish colour (Fig. 1A) and demonstrate a higher proliferation rate (expressed as callus fresh and dry weight) than the black ones (Fig. 1B). The formation of creamy-pink callus tissues has been observed by Fukui et al. (1990) as well.

Figure 1 A. Pink-whitish callus tissues induced from mistletoe leaf expiants (arrow indicates seed-like structures). B. Callus tissue (black-brownish type) covered with filamentous protrusions. C. Mistletoe callus with globular somatic embryos. D. A mistletoe protoplast isolated from leaf tissue.

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