Messa a punto di tecniche molecolari per l'identificazione precoce di virosi e risanamento di materiale propagato di nuovo ottenimento in Lilium
Author(s)
Trinchello, Debora
Date Issued
February 27, 2009
Type
Doctoral Thesis
Abstract
Lilies have become the most important floral crop in Italy in the last 20 years and this has created a need to produce high-quality, healthy bulbs free from virus. In recent year, a reverse transcription-polymerase chain reaction (RT-PCR) technique has been used for detection of specific virus strains in some plant species. This method showed the potential to have a greater sensitivity than ELISA. In order to obtain a fast and efficient protocol to detection of Lily symptomless virus (LSV) and Cucumber Mosaic Virus (CMV) in Asiatic hybrid lily lines, a simple and original method was established by RT-PCR. LSV is a mayor virus in Lilium (Marani e Bertaccini ,1984) that decrease plant growth vigour and the quality of the cut flower, yet infected plants show no distinct symptoms. When LSV combines with CMV (Cucumber Mosaic Virus) the symptoms are more heavy: the plants become more susceptible to pathogenous like Botritys e Fusarium. Stock bulbs ( with new agronomiques and commercials characteristics like pollenless, new shapes and colours of flower,) of Asiatic hybrid lily lines (‘409’, ‘599’, ‘20’, ‘00158’, ‘0045’, ‘0097’, ‘0035’, ‘00136’, ‘216’, ‘505’) were used as explants. From the scales were achieved new bulblets by ‘in vitro’ propagation. The new bulblets were analysed by RT-PCR. RT-PCR were performed using ‘Enhanced Avians HS RT-PCR’ Sigma Kit. Degenerates primers were used and these primers were designed according to amino acid sequences of the coat protein in 4 LSV and 17 CMV and used to amplify 483, 408 bp specific fragments from LSV, CMV respectively, by PCR. From bulblets virus infected were excised shoot meristems and micropropagated. RT-PCR was carried out and subsequently on the bulblets, also virus infected, were made thermotherapy (42 days at 35 °C) and a second meristem cut was made from heat treated material. The line ‘599’ and ‘409’ were analyzed either by ELISA ( achieved by University of Bologna, Division of Sciences and Agroenvironmental Tecnologies) or by RT-PCR to compare the efficiency of two different tests in Asiatic hybrid lily. Subsequently RT-PCR was applied to verify the sanitary status of ‘in vitro’ meristem derived plants. The other lines were analyzed only by RT-PCR. The virus-free lily bulblets grew vigorously ‘in vitro’ and acclimatized promptly. After the first year of culture, the plants virus-free were vigorous and produced many leaves. On virus-free lily were carried out, periodically, other surveys that have always given negative results, namely the absence of virus infection.
Additional information
Dottorato di ricerca in Ortoflorofrutticoltura
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