A protocol for cauliflower micro-propagule production was developed and optimised for both micropropagation and artificial seed production techniques using meristematic tissues from cauliflower curd. All steps in the protocol were empirically optimised including: blending, sieving, culture methods, liquid culture media composition and plant growth regulator combinations and concentrations. The cost of the micro-propagules could be reduced by as much as 50% on the initial costings reported previously since treatments doubled the number of microshoots produced per culture unit. The research confirmed the suitability of cauliflower microshoots to be encapsulated as artificial seeds and an effective protocol for microshoot encapsulation was designed through the optimization of 1) the production of cauliflower microshoots suitable for encapsulation, 2) encapsulation procedures, 3) artificial seed artificial endosperm structure, 4) conversion materials. The possibility of culturing cauliflower artificial seeds in commercial substrates such as perlite, sand, vermiculite and compost was confirmed. The use of plant preservative mixture (PPM) for the control of contamination in cauliflower culture media and artificial seeds was optimised and the effect of this material on the development of plant material was assessed. It was confirmed that cauliflower artificial seed could be stored in a domestic refrigerator for up to 6 months which could have a great impact in cauliflower breeding programmes. The huge number of cauliflower microshoots that could be produced using this protocol and the homogeneity of the culture system, provided a tool for the molecular analysis of cauliflower microshoots (and artificial seed) abiotic stress tolerance analysis. Various treatments were conducted to improve microshoot cold tolerance and the up-regulation of the CBF/DREB1 transcription factor including low temperature acclimation, mannitol, ABA (abscisic acid) and Mo (molybdenum). Microshoots were confirmed to acclimate successfully using low temperature. Mo was shown to improve the cold tolerance of cauliflower microshoots and to up-regulate CBF/DREB1 in the absence of low temperature acclimation. Acclimation did not increase the accumulation of dehydrin proteins and it is concluded that dehydrins do not play a significant role in the cold tolerance of cauliflower microshoots. Since cauliflower breeding and seed multiplication protocols make extensive use of micropropagation, the studies reported in this research could make a significant impact by decreasing the cost of micropropagation and increasing its reliability. It also opens new perspectives for further research for cauliflower artificial seed production and the possibility of sowing these seeds directly in the field. Furthermore, this research helps to facilitate cauliflower breeding programmes by improving the understanding of abiotic stress tolerance mechanisms and the relationship between different types of abiotic stresses such as cold and drought.

Seit einigen Jahren wird die Einkapselungstechnik für die Produktion künstlicher Samen oder zur Vorbereitung der Kryokonservierung genutzt. Das Ziel dieser Untersuchungen war es, Methoden zur Einkapselung von Dendranthema- und Rosa-Sprosssegmenten zu entwickeln und dadurch "künstliche Samen" herzustellen, die für die Konversion unter In-vitro- und Ex-vitro-Bedingungen geeignet sind. Es wurde mit den Dendranthema-Sorten `PS 27´, `Snowdon Weiß´, `Topfweiß´ und `Garden mum´ und mit der Rosensorte, Rosa hybrida `Kardinal´, gearbeitet. Von 3 bis 5 Wochen alten Sprosskulturen wurden 4 bis 5 mm lange Nodiensegmente entnommen und in der Gelmatrix eingekapselt. Sowohl das Alter der Sprosskulturen (Subkulturdauer) als auch die Position der Explantate am Spross hatten einen deutlichen Einfluss auf die Konversion. Die Konversion war bei den Sprossspitzen schneller und besser als bei den Nodiensegmenten. Bei Dendranthema konnte mit 5 Wochen alten Sprosskulturen gearbeitet werden. Bei den Rosen wurden bessere Ergebnisse mit Nodiensegmenten von 3 Wochen alten Sprosskulturen erzielt. Nach Optimierung der Gelmatrixkomponenten konnte für Dendranthema eine 100%ige Konversion unter sterilen Bedingungen erreicht werden, wenn der Gelmatrix (3% Na-Alginat) MS-Salze, Vitamine, 3% Saccharose und 1 mg/l IES zugesetzt wurden. Unter unsterilen Bedingungen erreichte die Sprossbildung 73%, wenn eine Zwei-Schicht-Kapsel mit einer nährstofffreien äußeren Schicht und Nährstoffen in der inneren Schicht verwendet wurde. Rosa hybrida `Kardinal´ hatte andere Ansprüche an die Nährstoffversorgung und die Zugabe von Wachstumsregulatoren als Dendranthema. Die Konversion der eingekapselten Nodiensegmente von Rosa hybrida `Kardinal´ erreichte auf einem Agar-Wasser-Medium fast 60%, wenn der Gelmatrix MS-Salze, Vitamine, 80 g/l bzw. 90 g/l Saccharose und 2 mg/l IBS zugesetzt wurden. Es wurden auch Nodiensegmente aus dem Gewächshaus direkt zur Einkapselung verwendet. Die Konversion dieser Nodiensegmente von Dendranthema `PS 27´ erreichte auf einem Agar-Wasser-Medium 75%, wenn der Gelmatrix MS + 4 mg/l IES zugesetzt wurden. Die eingekapselten Nodiensegmente von Dendranthema `PS 27´ konnten bei 4°C in einer MS + 1,0 mg/l IES-Lösung für 3 Monate gelagert werden. / Recently, the encapsulation technique is used for the production of artificial seeds or for cryopreservation. The aim of the present studies was to develop methods for encapsulation of Dendranthema- and Rosa-shoot segments to produce artificial seeds, that are suitable for the conversion under in vitro and ex vitro conditions. Dendranthema `PS 27´, `Snowdon Weiß´, `Topfweiß´ and `Garden mum´ and Rosa hybrida `Kardinal´ were used as plant material for the encapsulation. Nodal segments of about 4-5 mm length were taken as explants for the encapsulation. The conversion was influenced by the quality of the plant material used for encapsulation. The age of shoot cultures (subculture duration) and the position of explant on the shoot affected the conversion. The conversion of the shoot tips was faster and better than that of the nodal segments. With Dendranthema, 5 weeks old shoot cultures could be used, while in Rosa nodal segments of 3 weeks old cultures responded better. After optimization of the components of the gel matrix (3% Na-Alginat), 100% conversion could be recorded under sterile conditions for Dendranthema, when the gel matrix contained MS salts, vitamins, 3% sucrose and 1 mg/l IAA. The percentage of shoot formation reached 73% under non-sterile conditions, when a two-layer-capsule was used with a nutrient free cover layer and nutrients in the inner layer. Rosa hybrida `Kardinal´ had other nutritional and hormonal requirements compared with Dendranthema. The conversion frequency of the encapsulated shoot buds of Rosa hybrida `Kardinal´ reached almost 60% on agar-water-medium, when the gel matrix obtained MS salts, vitamins, 80 g/l or 90 g/l sucrose and 2 mg/l IBS. In this work, also shoot buds from the greenhouse were encapsulated directly. The conversion frequency of these encapsulated buds of Dendranthema `PS 27´ reached 75% on agar-water-medium, when the gel matrix contained MS + 4 mg/l IES. The encapsulated shoot buds of Dendranthema `PS 27´ could be stored for 3 months at 4°C in MS liquid medium.