Molecular characterization by RAPDS and micropropagation of Italian hazelnut cultivars

The production of certified plant materials requires the application of fast and reliable techniques to identify and propagate selected genotypes. The use of both molecular methods and phenotypic observations could accelerate the identification process. The availability of micro-propagation techniques allows the development of healthy and homogeneous plant materials. In this work a molecular analysis of hazelnut genotypes (Corylus avellana) was undertaken in order to obtain a fingerprinting for the Italian typical cultivars. A collection of 13 genotypes including the main cultivars from the Italian regions of cultivation (Piemonte, Lazio, Campania, and Sicilia) as well as two cultivars from Turkey and Great Britain was evaluated. DNA was extracted by the method of Doyle and Doyle (1990) and the analysis conducted by the method of Random Amplified Polymorphic DNAs (RAPDs) using six decamer oligonucleotides. The results were used to produce a similarity matrix with the coefficient of Nei and Li (1979) and a phylogenetic analysis. The primers produced a total of 37 polymorphic bands and a distinct electrophoretic profile was obtained for each of the Italian cultivars 'Tonda delle Langhe' (Piemonte), 'Tonda Gentile Romana' (Lazio), 'Tonda Rossa', 'Tonda Bianca', 'Tonda di Giffoni' (Campania). The phylogenetic analysis with the UPGMA method showed that cultivars from the same location did not always cluster together. In vitro shoot cultures were initiated using single buds and nodal shoot segments excised from greenhouse-grown suckers of the widespread Italian varieties. The explants were collected at different phenological plant stages. The medium MS (1/2 strength) supplemented with IBA (0.05 mg/l), GA(3) (0.4 mg/l) and increased concentrations of BAP or zeatin was used. During the initiation phase, both the proliferation and the elongation rate were significantly increased using explants collected during the spring. Cold storage of branches collected during winter time greatly improved the morphogenetic capacity and reduced the contamination and oxidation of tissues. A genotypic effect on micro-propagation was also observed among varieties. 'Tonda Romana', 'Tonda di Giffoni' and 'Ghirara' developed shoots with good growth habit using explants collected during both winter and spring. Generally zeatin (1 mg/l) seemed more suitable than BAP (0.5 mg/l) in the initial phase of in vitro establishment. 'Montebello' and 'Avellana Speciale' improved their response to micro-propagation when actively growing explants were used.