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Dorota Blatsios Dorota Blatsios Zróżnicowanie kolekcji różaneczników (Rhododendron L.) pod względem wybranych cech dekoracyjnych, użytkowych, polimorfizmu DNA oraz efektywności mikrorozmnażania. Praca doktorska wykonana w Katedrze Genetyki, Hodowli Roślin i Nasiennictwa Uniwersytetu Przyrodniczego we Wrocławiu pod kierunkiem dr hab. inż. Renaty Galek, prof. UPWr. Wrocław 2019 1 Podziękowania Na powstanie i ostateczny kształt mojej pracy doktorskiej miało wpływ wiele osób, którym chciałbym w tym miejscu podziękować. Przede wszystkim pragnę złożyć podziękowania kierownikowi Arboretum Leśnego im. Prof. Stefana Białoboka przy Nadleśnictwie Syców Panu Stanisławowi Sęktasowi za umożliwienie przeprowadzenia badań oraz za ogromną pomoc merytoryczną. Również dziękuję mojej Pani Promotor prof. Renacie Galek za opiekę merytoryczną, za cenne uwagi i sugestie, za zaangażowanie, dzięki któremu możliwe było napisanie tej pracy. Chcę także podziękować dr Bartoszowi Kozakowi za cenne wskazówki podczas prac i analiz molekularnych. Serdeczne podziękowania składam mgr inż. Annie Maciejewskiej-Hoza za wsparcie w trakcie prowadzenia badań, za krytyczne uwagi i codzienną nieocenioną pomoc. Dziękuję również pracownikom Katedry Genetyki, Hodowli Roślin i Nasiennictwa za pomoc w wykonaniu pomiarów laboratoryjnych oraz za wsparcie merytoryczne. Osobne, równie ważne podziękowania, składam mojemu Partnerowi i Synowi, którzy wspierali mnie, pomagali przy wykonywaniu badań polowych i mobilizowali do pracy. Również dziękuję Rodzicom, a szczególnie Mamie za pomoc w pianiu pracy. 2 SPIS TREŚCI 1. Wstęp i cel pracy .................................................................................................................... 6 Spis skrótów użytych w pracy .................................................................................................... 8 2. PRZEGLĄD LITERATURY ................................................................................................. 9 2.1 Przynależność systematyczna rodzaju Rhododendron L. ........................................ 9 2.2 Zasięg i występowanie ............................................................................................. 9 2.3 Charakterystyka rodzaju Rhododendron L. ........................................................... 10 2.4 Charakterystyka cytogenetyczna rodzaju Rhododendron L. ................................. 13 2.5 Hodowla różaneczników ........................................................................................ 13 2.6 Kierunki hodowli ................................................................................................... 15 2.7 Zastosowanie .......................................................................................................... 15 2.8 Metody hodowli ..................................................................................................... 19 2.8.1 Metody tradycyjne ....................................................................................... 19 2.8.2 Metody biotechnologiczne .......................................................................... 21 2.8.2.1 Metody in vitro ...................................................................................... 21 2.8.2.2 Poliploidyzacja ...................................................................................... 23 2.8.2.3 Transformacje ....................................................................................... 24 2.8.2.4 Markery molekularne ............................................................................ 25 2.9 Zmienność morfologiczna różaneczników w kontekście zróżnicowania genetycznego ..................................................................................................................... 29 3. MATERIAŁ ......................................................................................................................... 30 3.1 Materiał badawczy ................................................................................................. 30 3.2 Lokalizacja miejsca badań ..................................................................................... 30 4. METODY ............................................................................................................................. 31 4.1 Analiza zróżnicowania cech morfologicznych kolekcji różaneczników .............. 31 4.1.1 Pomiary i obserwacje polowe ............................................................................ 31 4.1.2 Metody statystyczne .......................................................................................... 32 4.1.3 Rozmnażanie wegetatywne różaneczników in vivo .......................................... 33 4.2 Doświadczenia in vitro .......................................................................................... 34 4.2.1 Dezynfekcja materiału roślinnego – wyprowadzenie kultury ........................... 34 4.2.2 Doświadczenia w kulturach in vitro .................................................................. 35 4.2.3 Metody statystyczne – doświadczenia in vitro .................................................. 35 4.3 Ocena Polimorfizmu DNA .................................................................................... 35 4.3.1 Izolacja genomowego DNA .............................................................................. 35 3 4.3.2 Ocena jakości wyizolowanego DNA – elektoforeza żelowa ............................. 36 4.3.3 Ocena zróżnicowania genetycznego przy użyciu markerów ISSR–PCR .......... 36 4.3.4 Metody statystyczne .......................................................................................... 37 4.4 Badanie opinii ........................................................................................................ 37 5. WYNIKI ............................................................................................................................... 39 5.1 Charakterystyka mieszańców Rhododendron L. pod względem wybranych cech morfologicznych ................................................................................................................ 39 5.1.1 Ocena mieszańców Rhododendron L. pod względem badanych cech .............. 39 5.1.1.1 Ocena kolekcji mieszańców Rhododendron L. pod względem pokroju krzewów ............................................................................................................ 39 5.1.1.2 Ocena kolekcji mieszańców Rhododendron L. pod względem cech liścia .................................................................................................................. 40 5.1.1.3 Ocena kolekcji mieszańców Rhododendron L. pod względem cech kwiatu ................................................................................................................ 44 5.1.1.4 Ocena kolekcji mieszańców Rhododendron L. pod względem zabarwienia kwiatów ......................................................................................... 51 5.1.1.5 Ocena kolekcji mieszańców Rhododendron L. pod względem wielkości krzewów, liści i kwiatów ................................................................................... 53 5.1.2 Analiza zależności badanych cech w kolekcji mieszańców Rhododendron L. 56 5.1.3 Ocena udziału poszczególnych cech kształtujących zmienność w kolekcji mieszańców Rhododendron L. ................................................................................... 58 5.1.4 Pogrupowanie analizowanych obiektów na podstawie 36 cech z wykorzystaniem analizy skupień ............................................................................. 60 5.2 Analiza rozmnażania wegetatywnego różaneczników in vivo .............................. 63 5.3 Analiza procesu mikrorozmnażania Rhododendron L. ......................................... 63 5.3.1 Analiza wpływu rodzaju eksplantatu wyjściowego – segmentów nodalnych i fragmentów pędu z merystemem apikalnym na regenerację mieszańca nr 59 ........... 63 5.3.2 Analiza wpływu segmentów nodalnych na regenerację mieszańca 59 ............. 64 5.3.3 Analiza wpływu segmentów nodalnych na regenerację mieszańca nr 96 ......... 65 5.3.4 Analiza wpływu rodzaju eksplantatu wyjściowego - segmentów nodalnych i fragmentów pędu z merystemem apikalnym na regenerację mieszańca nr 96 ........... 65 5.3.5 Synteza analizy wzrostu eksplantatów pędowych dwóch mieszańców Rhododendron L. ........................................................................................................ 67 5.3.6 Ocena wpływu genotypu, materiału wyjściowego wykorzystanego do inicjacji kultur oraz pożywki na wysokość roślin dwóch mieszańców różaneczników - nr 59 i nr 96 .......................................................................................................................... 68 5.3.7 Ocena wpływu genotypu, materiału wyjściowego wykorzystanego do inicjacji kultur oraz pożywki na liczbę liści u dwóch mieszańców różaneczników - nr 59 i nr 96 ................................................................................................................................ 70 5.4 Analiza zróżnicowania genetycznego z wykorzystaniem markerów ISSR ........... 71 5.5 Analiza badania opinii publicznej .......................................................................... 72 4 5.5.1 Pytanie nr 1: Czym kierujesz się przy wyborze odmiany różaneczników? ...... 72 5.5.2 Pytanie nr 2: Który kolor kwiatów jest dla Ciebie najbardziej atrakcyjny? ...... 72 5.5.3 Pytanie nr 3: Czy uważasz, że kolorowy rysunek na płatkach kwiatów (jak na zamieszczonym zdjęciu) jest dla Ciebie ważny przy wyborze odmiany? .................
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