Technische Universität München Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung and Umwelt Department für Pflanzenwissenschaften Lehrstuhl für Zierpflanzenbau Effect of different cultural conditions on micropropagation of rose (Rosa sp. L.) and globe artichoke (Cynara scolymus L.) Fernanda Schneider Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung and Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Agrarwissenschaften genehmigten Dissertation. Vorsitzender: Univ.-Prof. Dr.rer.hort. Dr.rer.hort.habil. Joachim Meyer Prüfer der Dissertation: 1. Univ.-Prof. Dr.rer.nat. Dr.rer.nat.habil. Gert Forkmann 2. Univ.-Prof. Dr.agr. Dr.agr.habil. Dieter R. Treutter Die Dissertation wurde am 11.04.2005 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung and Umwelt am 06.06.2005 angenommen. To my father (in memoriam) Zusammenfassung Zur Optimierung der Mikrovermehrung von Rosen und Artischocken wurde der Einfluss ausgewählter Kulturfaktoren während der in-vitro Phasen untersucht, sowie die Nachwirkung dieser Faktoren im weiteren Kulturverlauf geprüft, vor allem bei der Etablierung ex vitro. Die Entwicklung von Rosen in vitro wurde durch die Nährbodenverfestigung stark beeinflusst, die besten Ergebnisse wurden mit 6,0 g/l Agar erzielt, während die Verwendung von Gelrite bzw. eine Kultur mit Flüssigmedien sich als weniger geeignet erwiesen. Die Entwicklung während der Bewurzelungsphase wurde vor allem durch Auxingaben, Saccharosekonzentration und Gefäßverschluss beeinflusst. Die Kultur von Sprossen auf 0,1 mg L-1 NAA im Vergleich mit 0,5 mg L-1 NAA führte zu besserer Sprossentwicklung und einer höheren Etablierungsrate ex vitro. Eine Reduktion der Saccharosekonzentration in der Bewurzelungsphase ist vorteilhaft. Die Verwendung von Glaskappen, Steristopfen oder Aluminiumfolie als Verschluss ergab bessere Resultate als ein Verschluss mit Plastikfolie. Während der Bewurzelungsphase war ein deutlicher Effekt der Temperatur festzustellen: Die Wurzelbildung war bei 16°C besser als bei 20 bzw. 24°C, während das Sprosswachstum durch die höheren Temperaturen gefördert wurde. Die Dauer der Bewurzelungsphase soll nach den vorliegenden Ergebnissen etwa fünf Wochen betragen. Bei der Übertragung des Pflanzenmaterials in unsterile Bedingungen konnte die Erfolgsrate durch die Verwendung von Substraten mit erhöhter Luftkapazität gesteigert werden, z.B durch die Verwendung einer Substratmischung 1 Teil Torf / 3 Teile Perlite. Insgesamt war bei Rosen in allen Phasen ein ausgeprägter Einfluss des Genotyps auf die Entwicklung festzustellen. Bei der Mikrovermehrung von Artischocken führt eine Auxin- / Cytokininkombination 2,0 mg L-1 NAA + 2,0 mg L-1 BA zwar zu einer hohen Vermehrungsrate, jedoch in der anschließenden Bewurzelungsphase wurden bessere Ergebnisse erzielt, wenn in der vorausgegangenen Vermehrungsphase mit 0,2 mg L-1 NAA + 0,2 mg L-1 BA geringere Konzentrationen oder mit 2,0 mg L-1 NAA + 2,0 mg L-1 KIN ein schwächer wirksames Cytokinin benutzt wurde. Als Auxingabe zur Bewurzelung war 0,5 mg L-1 NAA geeignet, eine Zusatz von Gibberellinsäure erwies sich als nachteilig. Untersuchungen zum Einfluss von Saccharose und Lichtintensität wurden durch Chlorophyllfluoreszenzmessungen begleitet und zeigten bei geringerer Sacharosekonzetration und niedrigerer Lichtintensität bessere Pflanzenentwicklung in der Bewurzelungsphase. Geringere F /F -Werte bei 210 µmol m-2 s-1 v m im Vergleich zu 110 µmol m-2 s-1 weisen auf eine Photoinhibition hin. Abstract The objective of the present study was to investigate the influence of different cultural conditions in the multiplication, rooting and acclimatization phases on the growth of rose and globe artichoke and their effects on the subsequent micropropagation phases. The growth of rose plants in vitro was affected by the type of gelling agent added to the culture medium. The best results concerning plant growth were found by using agar as gelling agent at the concentration of 6 g L-1. On the other hand, the use of gelrite as gelling agent in the culture medium cannot be recommended for the micropropagation of rose cvs. ‘Frisco’ and ‘Lambada’, as well as the liquid medium in rose cv. ‘Kardinal’. Moreover, explants grown at 0.1 mg L-1 of NAA (auxin) in the rooting phase presented better growth and higher survival rate in the acclimatization phase when compared to explants grown at 0.5 mg L-1, as well as at 10 g L-1 sucrose in comparison to 20 or 40 L-1. The type of closure used also played an important role in the rooting phase. The use of glass, steristop or aluminium as closure showed better results than plastic film for growth of shoots for all tested cultivars (‘Frisco’, ‘Kardinal’ and ‘Lambada’). In addition, the growth of ‘Kardinal’ was not influenced by the use of Magenta B-CAP covers with or without filter in baby food culture jars. Temperature in the rooting phase clearly influenced rooting formation. The formation of roots was improved at 16°C, whereas shoot growth showed better results at higher temperatures (20°C or 24°C), being different temperatures required for the optimum growth of roots and shoots. Additionally, a minimum duration of the rooting phase of five weeks should be used in the micropropagation of rose. Substrates with higher drainage capacity in the initial phase of acclimatization (as e.g. a mixture of peat and perlite in the proportion 1peat:3perlite v/v) increased the survival rate of plants. The use of perlite mixed with peat should be recommended in order to increase the looseness, permeability and aeration of the substrate. The growth of globe artichoke cv. ‘Green globe’ plants in vitro was affected by the type and concentration of growth regulators supplemented to the culture medium. The use of 2.0 mg L-1 NAA + 2.0 mg L-1 BA in the culture medium positively influenced the growth of shoots in the multiplication phase. However, in the subsequent phases (rooting and acclimatization) this effect was not maintained. In these phases, higher shoot development, rooting percentage, and lower mortality rate of plants were found with 0.2 mg L-1 NAA + 0.2 mg L-1 BA or with 2.0 mg L-1 NAA + 2.0 mg L-1 Kinetin in the multiplication phase. Concerning the rooting phase, the addition of auxin (NAA) to the rooting medium at a concentration of 0.5 mg L-1 is suitable for the micropropagation of globe artichoke. Moreover, the supplementation of the rooting medium with gibberellic acid (GA ) is not beneficial. Light 3 intensity of 110 µmol m-2 s-1 can be used for the micropropagation of globe artichoke, since plants were not photoinhibited under this condition. Lower values of the F /F ratio may v m indicate the occurrence of photoinhibition due to damage to photosystem II reaction centers in response to light intensity of 210 µmol m-2 s-1. The results indicate that a reduction of sucrose concentration is advantageous for the micropropagation of globe artichoke. Contents 1 Introduction……………………………………………………………………….. 1 2 Literature Overview ………………………………………………………..……. 3 2.1 Micropropagation……………………………………………………………... 3 2.1.1 Principles of tissue culture………………………………………………. 3 2.1.2 Composition of nutrient media………………………………………….. 4 2.1.2.1 Mineral nutrition……………………………………………………… 4 2.1.2.2 Carbon source……………………………………………………….. 5 2.1.2.3 Growth regulators…………………………………………………… 6 2.1.2.4 Gelling agents……………………………………………………….. 8 2.1.3 Physical environment……………………………………………………. 10 2.1.3.1 Gas exchange and relative humidity inside the vessel…………. 10 2.1.3.2 Light…………………………………………………………………... 11 2.1.3.3 Temperature…………………………………………………………. 12 2.1.4 Characteristics of micropropagated plants……………………………. 13 2.1.4.1 Anatomy…………………………………………………………….... 13 2.1.4.2 Photosynthesis: heterotrophic vs. autotrophic metabolism…..… 15 2.1.5 Substrates ex vitro…………………………………………………….…. 15 2.2 Chlorophyll fluorescence…………………………………………………..…. 17 3 Material and Methods………………………………………………………..….. 20 3.1 Plant material………………………………………………………………..… 20 3.1.1 Rose (Rosa sp. L.)………………………………………………………. 20 3.1.2 Globe artichoke (Cynara scolymus)……………………………….…… 21 3.2 Culture medium and vessel………………………………………………….. 21 3.3 Ambient conditions in vitro…………………………………………………... 23 3.4 Acclimatization ex vitro……………………………………………………..… 24 3.5 Description of experiments and treatments………………………..………. 25 3.5.1 Rose (Rosa sp. L.)…………………………………………………….…. 25 3.5.1.1 Gelling agent………………………………………………………… 25 3.5.1.2 Type of closure of vessel and sucrose concentration…………... 26 3.5.1.3 Room temperature……………………………………………….…. 27 3.5.1.4 Duration of the rooting phase……………………………………… 27 3.5.1.5 Consistency of the medium, auxin concentration and container type……………………………………………………….................. 28 3.5.1.6 Substrate in the acclimatization phase………….………………… 29 3.5.2 Globe artichoke (Cynara scolymus L.)………………….…………….. 30 3.5.2.1 Growth regulators: auxin and gibberellin…….…………………… 30 3.5.2.2 Growth regulators (auxin and cytokinins) and duration of the 31 rooting phase………………………………………………………... 3.5.2.3 Growth regulators: auxin…………………………………………… 31 3.5.2.4 Sucrose concentration, type of vessel closure and light 31 intensity………..…………………………………………………..... 3.6 Parameters evaluated………………………………………………………... 32 3.6.1 Description of the parameters………………………………………….. 32 3.6.2 Plants parameters determined in the experiments with rose (Rosa sp. L.)………. ……………………………………………………. 38 3.6.3 Plants parameters determined in the experiments with globe artichoke (Cynara scolymus L.)………………………………...……… 39 3.7 Statistical analysis……………………………….……………………………. 39 4 Results and Discussion………………………………………………………… 40 4.1 Rose (Rosa sp. L.)……………………………………………………………. 40 4.1.1 Gelling agent……………………………………………………………… 40 4.1.2 Type of closure of vessel and sucrose concentration………………... 46 4.1.3 Room temperature……………………………………………………….. 58 4.1.4 Duration of the rooting phase…………………………………………… 64 4.1.5 Consistency of the medium, auxin concentration and container type……………………………………………………………………….. 71 4.1.6 Substrate in the acclimatization phase………………………………… 79 4.1.7 Rose: conclusions and outlook…………………………………………. 84 4.2 Globe artichoke (Cynara scolymus L. cv. ‘Green globe’)…………………. 87 4.2.1 Growth regulators: auxin and gibberellin……………………………… 87 4.2.2 Growth regulators (auxin and cytokinins) and duration of the rooting phase………………………………………………………..…... 94 4.2.3 Growth regulators: auxin………………………………………………… 103 4.2.4 Sucrose concentration, type of vessel closure and light intensity……………………………………………………………......... 108 4.2.5 Globe artichoke: Conclusions and outlook…………….……………… 116 5 References………………………………………………………………………… 118 Acknowledgements……………………………………………………………… 129 Curriculum Vitae…………………………………………………………………. 130 List of Figures 1. Chlorophyll fluorescence induction kinetics (Kautsky effect) upon irradiation of a dark-adapted leaf. Initial fluorescence (F ) and maximal o fluorescence (F ) are represented……...................................................... 18 m 2. Growth chamber used for the cultivation in vitro (left) and culture of explants in the test tubes (right)………………………………………….…… 23 3. Growth chamber and tray used for the cultivation ex vitro of rose…….….. 25 4. Types of closure tested in the test tubes………………………………….…. 26 5. Baby food culture jars covered with Magenta B-CAP with filter (left) or 27 without filter (right)……………………………………………………………… 6. Schematic representation of the organization of the different treatments concerning the duration of the rooting phase. Arrows indicate the beginning of each treatment………………………………………….……….. 28 7. Plants growing in liquid medium with a hydroponic floating system based on the use of polyethylene granulated (PE)………………………….……… 29 8. Quality score degrees of shoots in relation to the growth of rose…….………………………………………………………………………… 33 9. Quality score degrees of shoots in relation to the growth of globe artichoke…………………………………………………………….…………… 33 10. Number of shoots per plant (a) and quality score of shoots (b) in the multiplication phase of rose grown under different concentrations of the gelling agents agar and gelrite in the culture medium. Means followed by the same letter are not significantly different according to Duncan’s Test (P=0.05). A description of the quality score is presented in the chapter Material and Methods (Table 3) (n=120)…………………………………….. 41 11. Development of plants of three rose cultivars (‘Kardinal’, ‘Lambada’ and ‘Frisco’) grown in test tubes with different types of closure (glass, steristop, aluminium and plastic film)………………………………………… 49 12. Number of roots per plant (a) and quality score of roots (a) of rose in the rooting phase grown in test tubes with different types of closure. Means followed by the same letter are not significantly different according to Duncan’s Test (P=0.05). A description of the quality score is presented in the chapter Material and Methods (Table 4)(n=50)………………………… 50 13. Quality score of roots (a) and number of roots per plant (b) of rose in the rooting phase grown under different sucrose concentrations in the culture medium. Means followed by the same letter are not significantly different according to Duncan’s Test (P=0.05). A description of the quality score is presented in the chapter Material and Methods (Table 4) (n=50)……….............................................................................................. 52 14. Quality score of shoots of rose in the acclimatization phase grown under different sucrose concentrations in the culture medium in the rooting phase. Means followed by the same letter are not significantly different according to Duncan’s Test (P=0.05). A description of the quality score is presented in the chapter Material and Methods (Table 3) (n=40)………… 55 15. Quality score of shoots (a) and number of roots per plant (b) of rose in the rooting phase grown under different room temperatures. Means followed by the same letter are not significantly different according to Duncan’s Test (P=0.05). A description of the quality score is presented in the chapter Material and Methods (Table 3)(n=55)………………………… 58 16. Development of plants of three rose cultivars (‘Kardinal’, ‘Lambada’ and ‘Frisco’) at the end of the rooting phase grown under different room temperatures……………………………………………………………………. 59 17. Transverse sections of rose leaves (semi-thin sections stained with toluidine blue). (a) Greenhouse plant, cv. ‘Lambada’. (b) Cv. ‘Lambada’ cultured in vitro at 16°C room temperature. (c) Cv. ‘Lambada’ cultured in vitro at 24°C room temperature. (d) Cv. ‘Frisco’ cultured in vitro at 24°C room temperature. Arrows indicate open stomata………………………….. 62 18. Quality score of shoots (a) and number of leaves per plant (b) of three cultivars of rose as affected by the duration of the rooting phase. Lines represent fitting regression curves. A description of the quality score is presented in the chapter Material and Methods (Table 3). Regression equations were as follows: Figure (a): ‘Frisco’ y= 6.91 -1.54x + 0.15x2, ‘Kardinal’ y= 8.37 – 2.40x + 0.35x2, ‘Lambada’ y= 8.05 – 2.38x + 0.334x2; Figure (b) ‘Frisco’ y= 4.89 – 1.34x + 0.13x2, ‘Kardinal’ y= 6.57 – 2.13x + 0.24x2, ‘Lambada’ y= 6.01 – 2.14x + 0.26x2 (n=55)………………………… 65 19. Development of plants of three rose cultivars (‘Kardinal’, ‘Lambada’ and ‘Frisco’) as affected by the duration of the rooting phase…………..… 65 20. Quality score of roots (a) and number of roots per plant (b) of three rose cultivars as affected by the duration of the rooting phase. Lines represent fitting regression curves. A description of the quality score is presented in the chapter Material and Methods (Table 4). Regression equations were as follows: Figure (a): ‘Frisco’ y=1.02 + 0.07x, ‘Kardinal’ y=0.26 + 0.72x, ‘Lambada’ y=0.98 + 0.45x; Figure (b) ‘Frisco’ y=0.01 + 0.08x, ‘Kardinal’ y=-1.69 + 1.41x - 0.13x2, ‘Lambada’ y=0.15 + 0.57x – 0.04x2 (n=55)……………………………………………………………………………. 66 21. Quality score of shoots (a) and number of leaves per plant (b) of rose as affected by the duration of the rooting phase. Means followed by the same letter are not significantly different according to Duncan’s Test (P=0.05). A description of the quality score is presented in the chapter Material and Methods (Table 3) (n=60)……………………………………… 68 22. Quality score of roots (a) and number of roots per plant (b) of rose as affected by the duration of the rooting phase. Means followed by the same letter are not significantly different according to Duncan’s Test (P=0.05). A description of the quality score is presented in the chapter Material and Methods (Table 4) (n=60)……………………………………… 69 23. Quality score of shoots (a) and roots (b) in the rooting phase of rose as affected by the concentration of NAA and the consistency of the culture medium. Means followed by the same capital letter (A,B) within the same NAA-level or by the same small letter (a,b) within the same medium consistency are not significantly different according to Duncan’s Test (P<0.05). A description of the quality scores is presented in the chapter Material and Methods (Tables 3 and 4) (n=60)……………………………... 72 24. Development of plants of rose cultivar ‘Kardinal’ in the rooting phase as affected by the concentrations of NAA and the consistency of the culture medium………………………………………………………………………….. 74 25. Variation in the volume of solid material (S), air space (A), easily available water (EAW), water buffering capacity (WBC) and less readily available water (LRAW) for the substrates tested. Per = Perlite………….. 80 26. Quality score of shoots of globe artichoke in the rooting phase grown under different concentrations of GA . Means followed by the same letter 3 are not significantly different according to Duncan’s Test (P=0.05). A description of the quality score is presented in the chapter Material and Methods (Table 3) (n=60)……………………………………………………… 88 27. Number of shoots per plant (a) and quality score of shoots (b) of globe artichoke in the multiplication phase grown under different concentrations and types of auxin and cytokinins. Means followed by the same letter are not significantly different according to Duncan’s Test (P=0.05). A description of the quality scores is presented in the chapter Material and Methods (Table 3) (n=60)……………………………………… 95