Plant tissue culture technique has been improved remarkably in the last few decades. The technique is widely used in plant propagation (Pattnaik and Chand, 1997), virus elimination (Ramírez-Malagón et al., 2006; Subba Reddy and Sreenivasulu, 2011), genetic transformation (Falco et al., 2000; Taskin et al., 2003; Xue et al., 1999), germplasm preservation (Forsline et al., 1998; Gagliardi et al., 2002; Orlikowska, 1992; Reed et al., 1998; Zacchini and Agazio, 2004), and secondary metabolite production (Bourgaud et al., 2001; Cheng et al., 2008). The most commonly used culture medium is the Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) or the variant of it. The pH of the medium is generally suggested as 5.5–6.0 and not much research on it has been reported (Beyl, 2011).
Generally, the medium is adjusted to a certain pH depending on the plant species used and the purpose. For example, tetraploid black locust showed the highest proliferation rate at pH 5.6–6.0, whereas the Erqiao black locust preferred pH 6.4 (Huang and Liu, 2003). Growth and nutrient uptake of teak (Tectona grandis) seedlings in vitro was optimum on a medium at pH of 6.0 (Hong and Yin, 2010; Zhou et al., 2009). The pH of the medium is also responsible for the occurrence of vitrification (a phenomenon of plantlets appearing turgid, glaucous, and watery), which has been shown on Prunus salicina within pH 5.4 to 5.8 (Ding et al., 2008). In the case of apple, the medium pH regulated root formation of the microcuttings of the cultivars Gala and Triple Red Delicious by affecting indole-3-butyric acid (IBA) uptake (Harbage and Stimart, 1996; Harbage et al., 1998), and for cultivar Jork9, better rooting occurred at pH 5.3 (Klerk et al., 2008).
However, not all plant species are that sensitive to pH variation of the medium within a certain range. For instance, the proliferation and growth of Oncidium plantlets did not show significant difference with variable pH from 5.2 to 6.5 (Cui et al., 2004). Likewise, plantlets of Plantago almogravensis and Plantago algarbiensis performed equally with variation of medium pH from 4.50 to 5.75 (Martins et al., 2011). Although optimal medium pH varied from plant species to species, in most cases, it is adjusted to the range of 5.0–6.0 (Li, 2002).
Adjusting medium pH is normally done by adding diluted HCl or NaOH solution. In many cases, repeated adjustment by adding HCl and NaOH in turn is needed to get an exact pH value. It is quite understandable that adding these chemicals changes the ion concentration which might have adverse effects on plant growth. The objectives for this study were 1) to analyze the changes of the ion concentration in medium after adjustment of pH, 2) to assess the effect of medium pH range on apple subculture proliferation, rooting, and adventitious bud regeneration from leaves, and 3) to determine the necessity of pH adjustment while preparing medium for apple tissue culture.
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