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These studies were conducted to determine the most effective methods for increasing shoot elongation during the initial proliferation stage of micropropagation in two dwarfing apple, Malus 3domestica (Borkh.), rootstock cultivars. Several experiments were conducted to compare explant collection date, exposure to chilling (5 ± 1 8C) temperatures, and varying concentrations of plant growth regulators in Murashige and Skoog (MS) media. Microshoot growth of ‘Geneva 41’ (‘G.41’) was very low and unaffected by chilling duration from 0 to 8 weeks or by gibberellic acid (GA3) concentration from 0 to 1.0 mg·L, but was improved by an additional subculture which increased shoot length from 1 to 15 mm. In ‘Geneva 30’ (‘G.30’), shoot elongation was most affected by date, chilling explants, and by optimizing cytokinin concentration and type. Explant collection date in April increased shoot growth compared with August or November. Microshoot growth of ‘G.30’ was increased by chilling nodal explants for 4 and 6 weeks when explants were collected in August and November, but not in April. Eight weeks chilling was detrimental for explants collected in April, and generally had little or no effect with August and November. The cytokinin 6-benzylaminopurine (BA) increased shoot number to a greater extent than thidiazuron (TDZ) or zeatin (ZT), and was also more effective for increasing shoot elongation with concentrations of 0 to 2.0 mg·L. In ‘G.30’, GA3 increased shoot growth at the optimum concentration of BA, but not with lower concentrations. ‘G.30’ microshoots were fewer and shorter with 24-epibrassinolide (EBR) at concentrations of 0.1 and 1.0 mg·L. Chemical names: N-phenyl-N’(1,2,3-thiadiazol-5-yl)urea (TDZ), 6-(4-hydroxy-3-methylbut-2-enylamino)purine (ZT). 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, singleand 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 andMcDougald, 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 exposingmicropropagated 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 (DeKlerk, 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 to promote both an increase in shoot number and shoot length of apple (James and Thurbon, 1981; Kere sa 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 Received for publication 20 Jan. 2016. Accepted for publication 21 Apr. 2016. This project was funded in part by a grant from the Maine Agricultural Center and by the USDA National Institute of Food and Agriculture Hatch project NC140. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product, and does not imply its approval to the exclusion of other products that also may be suitable. Maine Ag. and Forest Exp. Stn. no. 3479. Corresponding author. E-mail: rmoran@maine.edu. HORTSCIENCE VOL. 51(7) JULY 2016 899 PROPAGATION AND TISSUE CULTURE

Increasing In Vitro Shoot Elongation and Proliferation of ‘G.30’ and ‘G.41’ Apple by Chilling Explants and Plant Growth Regulators