Introduction, maintenance and utilization of male sterility in leek (Allium ampeloprasum L.) = [Inductie, handhaving en gebruik van mannelijke steriliteit in prei (Allium ampeloprasum L.)]

This thesis describes research work performed in leek in relation to efforts to obtain or exploit a male sterility system for hybrid leek breeding. Male sterile plants can be found in every leek cultivar. The male sterility trait can be explained by a nuclear monogenic inheritance or by a nucleo-cytoplasmic inheritance. The existence of a nucleo-cytoplasmic inheritance remains speculative unless the maintainer genotype will be identified. No source of CMS has been found, sofar, in leek or related forms of A. ampeloprasum L.Mutation breeding experiments during an extended period of time, using chemicals or somaclonal variation, did not result in the induction of cytoplasmic male sterile mutants in leek.The small chances for fixation of a mutation in a mitochondrial genome population and problems concerning the identification and recovery of the cytoplasmic mutation at the plant level may have been the major obstacles in obtaining a cytoplasmic male sterile mutant.An efficient callus regeneration system and the selection of leek plants with improved regeneration ability are described in this thesis. The availability of a regeneration system is an essential step in the application of somatic hybridization, somaclonal variation and transformation techniques in leek.Vegetative propagation of nuclear male sterile genotypes remains up till now the most realistic option for producing leek hybrids. A new in vitro method for mass clonal propagation of leek, based on flower stalk explants, was developed and is described in this thesis. This method offers a realistic opportunity to leek breeders for producing leek hybrids, until a less expensive genetic system for producing male sterile lines would become available

Isoelectric focusing: Sample pretreatment – separation – hyphenation

The three major goals of the research presented in this thesis are: • Investigation of electrophoretic sample pretreatment strategies. • Study and optimization of capillary isoelectric focusing for separation of proteins. • Hyphenation of isoelectric focusing to MS and iSPR. Chapter 2 gives an overview of the developments in cIEF over the past years including method improvements, new theoretical insights and applications. Chapter 3 describes the effect of several BGE additives on cIEF-UV repeatability and linearity and on protein signal intensity in MALDI-TOF MS. Chapter 4 describes the hyphenation of cIEF to MALDI-TOF MS via a spotting device. The development of the system as well as the effects of focusing and spotting times on separation efficiency are shown. A 3D plot is constructed from cIEF and MS data and the applicability is demonstrated for a degraded biopharmaceutical compound (glucagon). Chapter 5 is dedicated to the coupling of gel-IEF to iSPR via pressure blotting. The development of the system as well as the influence of the biological matrix on IEF separation and transfer efficiency is discussed. The applicability is demonstrated by the analysis of synovial fluid spiked with a known RA biomarker, i.e. citrullinated fibrinogen. Chapter 6 describes the use of the IonChip for salt removal from a protein sample prior to CZE separation (ITP-CZE mode) and the removal of interfering proteins from a peptide sample with subsequent CZE separation (CZE-CZE mode). Chapter 7 gives a general conclusion and recommendations for future research. Adjustments to the different systems and devices are proposed