×Malosorbus florentina (Rosaceae) is a rare and endangered native species of Greece, which is considered a natural intergeneric hybrid of Malus sylvestris and Sorbus torminalis and occurs in a few localities, mostly as single trees or small groups of trees in forests with Abies and Pinus (Christensen, 1995). It is also found further north in the Balkan Peninsula, Italy, and northern Turkey (Browicz, 1983), whereas in Serbia, it is on the list of endangered to vulnerable plants (Tomović et al., 2003). Its largest populations in Greece, found in Mt. Parnitha National Park, were burned by a devastating fire in 2007 and their natural regeneration was inhibited by persistent grazing, probably by red deer.
The attractive foliage (leaves broadly ovate, toothed, deeply lobed; upper surface dark green and glabrous, lower surface grayish green and hairy; turning orange–scarlet in fall), flowers (white petals, inflorescence a five- to nine-flowered, hairy corymb or umbel; in late spring) and fruits (red or yellowish red, 8 to 14 mm in diameter; from early fall) give M. florentina particular horticultural value (Christensen, 1995) and make it suitable for use as an ornamental landscape plant or for reforestation.
In vitro techniques are appropriate for the conservation of threatened plants, because they provide rapid shoot multiplication from small quantities of initial material (Kartsonas and Papafotiou, 2007). The preferred material for use in initiation of in vitro cultures, to maximize the genetic diversity of produced plants, is normally seed, but where seeds are not available, cultures should start from explants with meristematic activity, e.g., dormant or actively growing apical or lateral buds, as a result of their genetic stability (Fay, 1994; Sarasan et al., 2006).
There is a report on establishment of in vitro cultures of ×M. florentina from seeds but the microshoots produced failed to form roots (Savić et al., 2006). However, at least in the population of ×M. florentina on Mt. Parnitha, it is difficult to find seeds because either plants do not produce fruits or fruits produced do not contain seeds (personal observations between 2004 and 2011). Apart from seeds, in vitro cultures of ×M. florentina have also been initiated from apical buds of adult plants, albeit with a very low percentage of culture establishment (7%) as a result of contamination problems and explant browning that lead to death of explants (Papafotiou and Martini, 2009a, 2009b). The multiplication of microshoots was successful (Papafotiou and Martini, 2009a, 2009b), but their rooting ability was low (Martini and Papafotiou, 2009). Thus, to develop an efficient micropropagation protocol for ×M. florentina, the problem of contamination, explant browning, and low microshoot rooting ability should be addressed.
Browning and necrosis is a common problem in cultures of woody species and have been generally attributed to the oxidation of phenolic compounds in explant tissues (Dobránszki and Teixeira da Silva, 2010; Hu and Wang, 1983). Explants from axillary shoot tips of Vitis vinifera had a higher survival rate than those from terminal positions, and a strong negative correlation was found between survival in vitro and the pre-existing phenolic content of the explants (Yu and Meredith, 1986). Shoot tip explants of ‘Koroneiki’ olive trees exhibited higher browning rates together with higher total phenol and polyphenoloxidase activity compared with single-node explants, whereas explants derived from glasshouse-growing trees presented higher survival rates and lower total phenol and polyphenoloxidase activity than corresponding explants from field-growing trees (Roussos and Pontikis, 2001a, 2001b). The season of collection has been reported to affect browning of explants (Dobránszki et al., 2000; Modgil et al., 1999; Wang et al., 1994) as well as their survival through effects on the concentration of phenolics (Thomas and Ravindra, 1997). In addition, the season of explant collection has been found to affect the survival of explants through effects on microbial contamination (Thomas and Ravindra, 1997).
The present study investigated the effect of explant origin (juvenile, adult), explant location on shoots (apical, axillary), and season of explant collection on contamination and explant browning as well as on all stages of in vitro propagation of ×M. florentina, including microshoot rooting and acclimatization. The phenolic content of explants was also determined to correlate it with browning, which has been found to affect explant survival during in vitro culture establishment (Papafotiou and Martini, 2009a, 2009b).
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