Micropropagation can rapidly increase numbers of stock plants, particularly for new cultivars that are available in limited quantities. Because of their genetic stability and potential for high proliferation, single- and multiple-node shoot pieces are commonly used as explants for in vitro propagation of apple (De Klerk, 1992). Efficient propagation using this method depends on rapid shoot development and elongation following establishment. However, many important apple rootstock cultivars exhibit a lack of shoot elongation during the proliferation phase with the foliage appearing as a rosette (Aklan et al., 1997; Geng et al., 2015; Lane and McDougald, 1982; Pua et al., 1983; Webster and Jones, 1991). A prolonged period in sterile culture can promote shoot growth, but adds to the duration and cost of micropropagation. Lack of shoot growth during the proliferation stage limits the entire propagation process, but causal factors remain unknown. Identifying methods for rapid and efficient proliferation during initial phases of culturing is needed to increase propagation efficiency.
Shoot growth during the proliferation stage is more rapid when explants are collected in spring compared with later dates in summer or fall (Hutchinson, 1984; Kane, 2005; Modgil et al., 1999). The cause of the reduction of in vitro shoot growth with summer and fall collection dates is not clear, but may be due to the onset of dormancy (Borkowska, 1986; De Klerk, 1992), which is typically overcome by exposing micropropagated plants to low (<7 °C), nonfreezing temperatures (Howard and Oehl, 1981; Isutsa et al., 1998; Kavanagh et al., 1993). Chilling increases in vitro shoot growth when it occurs to stock plants of Rubus (Wu et al., 2009), explants of apricot (Koubouris and Vasilakakis, 2006), and shoot proliferation cultures of Prunus tenella (Bouza et al., 1992). Chilling ‘Jork’ apple shoot cultures during shoot proliferation increases growth of shoots that have no obvious growth inhibition (De Klerk, 1992), but has not been tested on apple cultivars with obvious signs of stunted growth.
The effect of chilling duration during apple nodal culture establishment has not been tested, but increasing chilling from 1 to 2 weeks increases subsequent shoot proliferation in established cultures (De Klerk, 1992). In the case of apricot explants (Koubouris and Vasilakakis, 2006), a short chilling duration of 4 to 12 d is sufficient to increase shoot number, but not elongation. In sour cherry, 6 to 8 weeks was optimum for promoting shoot growth, but longer than 8 weeks was detrimental (Borkowska, 1986).
Cytokinin in the media is necessary for proliferation, but can inhibit shoot elongation at concentrations that promote rapid increase in shoot number (Elliott, 1972; Hutchinson, 1984). In general, BA is preferred to other cytokinin types, and the optimum concentration is 1 mg·L−1 to promote both an increase in shoot number and shoot length of apple (James and Thurbon, 1981; Kereša et al., 2012; Lane and McDougald, 1982; Pua et al., 1983). TDZ causes greater proliferation than BA, but can also inhibit elongation to a greater extent (Huetteman and Preece, 1993; Marin et al., 1993; van Nieuwkerk et al., 1986). Compared with BA, kinetin results in fewer but longer shoots (Lundergan and Janick, 1980) and may be more effective for genotypes that display poor shoot elongation.
Adding auxin or GA3 to the media can increase shoot length (Kane, 2005; Reeves et al., 1985), but the effect is variable and dependent on a number of other factors such as the concentration of cytokinin in the media. Pua et al. (1983) reported that low concentration of BA combined with GA3 increases shoot number and length, but GA3 decreases shoot length with a high BA concentration in the media. Increasing concentration of indole-3-butyric acid (IBA) increases shoot length, but only when GA3 is also present (Yepes and Aldwinckle, 1994). A greater understanding of the GA3 effectiveness and interactions with other growth regulators may improve propagation of recalcitrant genotypes.
Brassinosteroid promotes in vitro shoot multiplication and elongation in apple when applied directly to foliage (Schaefer et al., 2002), but this method is labor intensive and increases the chance for contamination. When added to media, EBR can promote shoot elongation at a concentration below 1 mg·L−1 in Dioscorea sp. (Engelmann-Sylvestre and Engelmann, 2013) and Capsicum annuum (Franck-Duchenne et al., 1998). As an additive to media, it has not been tested for increasing in vitro shoot growth in apple cultivars that display shoot growth inhibition.
The purpose of these studies was to fill substantial gaps in our understanding of the effects of explant collection date, chilling nodal explants, and media concentration of the plant growth regulators GA3, EBR, BA, ZT, and TDZ on shoot growth of ‘G.30’ and ‘G.41’ apple during the initial proliferation stage of micropropagation. The focus of this research was to identify factors that improve initial shoot proliferation. Therefore, the effect of these factors on continued proliferation and subsequent rooting was not examined.
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