The in vitro manipulation of cauliflower (Brassica oleracea L. convar. botrytis (L.) Alef. var. botrytis L.) meristematic tissues for utilisation in genetic improvement programmes
Cauliflower curd meristem activity (organogenic, plastochronic, phyllotactic) was analysed biometrically and confirmed that the curd is the product of a constant process of meristem production and branch ramification with little if any dominance between branch apices. A growth model based on curd branching pattern was developed and its mathematical expression enabled the estimation of the number of meristems carried by a curd at maturity to be over ten million which was previously widely underestimated. Analysis of the response to the in vitro culture of this meristematic tissues revealed that meristems are not predetermined to produce flower and that their shoot regeneration capacity is under several levels of control, the most important being explant physical property (size) and the culture system (nutrient supply). Optimisation of these parameters enabled the development of a low cost, semi-automated protocol for mass production of cauliflower propagules at an unprecedented scale with over 10000 propagules produced per curd. Micropropagules a few millimetres in length were produced, encapsulated in calcium alginate hydrogel, stored at 4°C for several months and used as an 'artificial seed' system of cauliflower propagation. The response to the procedure of micropropagule production is genotype-dependent with summer heading varieties being less reactive than winter heading varieties, this phenomenon was also associated with plasmalemma instability at the cellular (protoplast) level. Furthermore, this material was successfully cryopreserved in liquid nitrogen using a dehydration / vitrification method. The micropropagation protocol is of great interest when used as a regeneration system for experiments involving genetic manipulation such as genetic transformation. A preliminary study of genetic transformation by microprojectile bombardment, using the gus reporter gene, allowed transient expression in curd meristematic tissue. The fundamental and industrial implications for cauliflower breeding of the different protocols developed in this thesis are discussed.