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Vgrosso Tesid.Pdf 1.0 PARTE GENERALE ........................................................................................................................ 6 1.1 Cenni storici ................................................................................................................................... 6 1.2 Lo stato del commercio e dell’industria delle Orchidee................................................................ 8 1.3 Il miglioramento genetico nelle Orchidee: dall’incrocio classico alle biotecnologie .................... 9 1.3.1 Micropropagazione .................................................................................................................. 11 1.3.2 Embriogenesi somatica ............................................................................................................ 13 1.3.3 Variazione Somaclonale ........................................................................................................... 15 1.3.4 Manipolazioni cellulari: Protoplasti ed Ibridazione somatica .................................................. 17 1.3.4.1 I Protoplasti ........................................................................................................................... 17 1.3.4.2 L’ibridazione somatica ........................................................................................................... 18 1.3.4.3 Manipolazioni cellulari nelle Orchidee .................................................................................. 21 1.4 La citogenetica delle orchidee ..................................................................................................... 22 1.4.1 Il Genoma delle Orchidaceae ................................................................................................... 22 1.4.2 Analisi citogenetiche ................................................................................................................ 24 1.4.3 Ibridazione in situ fluorescente - FISH ...................................................................................... 26 1.5 La poliploidia ed il fenomeno della endopoliploidia nelle Orchidee ........................................... 27 1.5.1 Poliploidizzazione in vitro ......................................................................................................... 30 1.6 Nuovi metodi per l’analisi di cellule vegetali: la Citofluorimetria a flusso .................................. 32 1.6.1 Come funziona un citometrio a flusso ..................................................................................... 33 1.6.2 La separazione cellulare (flow Sorting) .................................................................................... 36 1.7 La citometria a flusso nelle piante .............................................................................................. 37 1.7.1 Fluorocromi specifici per il DNA ............................................................................................... 37 1.7.2 Analisi del contenuto di DNA in nuclei in sospensione ............................................................ 38 1.7.3 Analisi e sorting dei protoplasti ................................................................................................ 43 1.8 Caratterizzazione dei materiali vegetali tramite marcatori molecolari ...................................... 44 1.8.1 Marcatori RAPD ........................................................................................................................ 45 1.8.2 Marcatori SSR ........................................................................................................................... 46 2 1.9 Obiettivi del Dottorato ................................................................................................................ 47 2.0 MATERIALI E METODI ...................................................................................................................... 51 2.1 Materiale vegetale ...................................................................................................................... 51 2.1.1 Sterilizzazione degli espianti .................................................................................................... 52 2.2 Coltura in vitro ............................................................................................................................. 52 2.2.1 Coltura espianti ........................................................................................................................ 52 2.2.2 Induzione embriogenesi somatica ........................................................................................... 52 2.2.3 Coltura in sospensione di PLBs ................................................................................................. 53 2.3 Isolamento dei protoplasti .......................................................................................................... 53 2.3.1 Soluzioni enzimatiche ............................................................................................................... 54 2.3.2 Isolamento protoplasti da foglia .............................................................................................. 54 2.3.3 Isolamento protoplasti da PLBs ................................................................................................ 55 2.3.4 Purificazione dei protoplasti su gradiente ............................................................................... 55 2.3.5 Analisi della vitalità ................................................................................................................... 57 2.4 Ibridazione somatica ................................................................................................................... 57 2.4.1 Fusione tramite PEG ................................................................................................................. 57 2.4.2 Elettrofusione dei protoplasti .................................................................................................. 59 2.5 Coltura dei protoplasti ................................................................................................................ 59 2.6 Poliploidizzazione ........................................................................................................................ 61 2.7 Citometria a flusso....................................................................................................................... 61 2.7.1 Isolamento di nuclei in sospensione ........................................................................................ 62 2.7.2 Analisi del contenuto di DNA ................................................................................................... 62 2.7.3 Analisi della ploidia ................................................................................................................... 63 2.7.4 Analisi e separazione citofluorimetrica degli ibridi .................................................................. 63 2.8 Analisi marcatori molecolari ........................................................................................................ 64 2.8.1 Estrazione del DNA genomico .................................................................................................. 64 2.8.2 Analisi RAPD ............................................................................................................................. 65 3 2.8.3 Analisi SSR................................................................................................................................. 66 2.8.4 Analisi dei profili elettroforetici ............................................................................................... 67 2.9 Analisi citologiche ........................................................................................................................ 67 2.9.1 Colorazione Feulgen ................................................................................................................. 67 2.9.2 FISH su nuclei in sospensione ................................................................................................... 68 2.9.3 Analisi microscopiche ............................................................................................................... 69 2.10 Analisi morfologiche .................................................................................................................. 69 2.10.1 Analisi delle dimensioni e della densità stomatica ................................................................ 69 2.10.2 Analisi morfometriche ............................................................................................................ 70 2.11 Analisi statistiche ....................................................................................................................... 70 3.0 RISULTATI ........................................................................................................................................ 72 3.1 Micropropagazione degli espianti ............................................................................................... 72 3.2 Induzione di protocormi .............................................................................................................. 75 3.3 Coltura in Sospensione dei PLBs .................................................................................................. 77 3.4 Isolamento protoplasti ...............................................................................................................
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