Molecular cloning of Ve promoters from Gossypium barbadense and G. hirsutum and functional analysis in Verticillium wilt resistance |
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Authors: | Lixia Liu Weiqiao Wang Jun Yang Yan Zhang Guiyin Zhang Zhiying Ma Xingfen Wang |
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Affiliation: | 1.Department for Innovation in Biological, Agro-food and Forest systems,University of Tuscia,Viterbo,Italy;2.Department of Ecological and Biological Science,University of Tuscia,Viterbo,Italy;3.Hawkesbury Institute for the Environment,University of Western Sydney,Penrith,Australia;4.Consiglio per la Ricerca in Agricoltura e l’Analisi dell’Economia Agraria – Forestry Research Centre,Arezzo,Italy |
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Abstract: | Global warming is affecting Mediterranean ecosystems, in which Arbutus unedo represents an important species. Mesophyll protoplasts are convenient material to study plant DNA integrity, nevertheless their release from mature plants along the year has not been reported in sclerophyll species. In this respect, in the present study the chance to isolate protoplasts from leaf tissues of A. unedo in different seasons was investigated. The digestion was obtained using Macerozyme R10 (1%) and Cellulase Onozuka RS (2%), with 0.6 M of mannitol, incubated for 4 h at 25 °C in the dark. In spring, soft leaves of different ages were studied to identify the most suitable material and protoplast yields were significantly influenced, with the still expanding 4th leaf characterized by the highest amount. Protoplast release decreased during the growing season when leaves become partially hardened, while their obtainment was quite impossible in summer and winter, due to leaf hardening. In November, an increase of incubation time (16 h) and of Cellulase RS (4%) or mannitol concentration (0.8 M) was needed with chilled leaves from the field; conversely, a good source for protoplasts were the green soft leaves coming from detached and pruned branches placed in controlled environment at spring temperatures. The employment of polyvinylpyrrolidone-40 during isolation procedure is discussed. The isolated protoplasts were employed to assess the DNA integrity, by using single cell gel electrophoresis (SCGE). The X-irradiated nucleoids were significantly damaged confirming that the present approach could be used in the selection of elite material of A. unedo devoted to fruit plantation. |
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