Regeneration and transformation by particle bombardment in leek (Allium ampeloprasum L.)

In this thesis the results are presented of experiments aiming at the genetic modification of leek ( Allium ampeloprasum L.). Leek is a vegetable grown for its edible (false) stem and belongs to the Alliaceae, together with onion ( Allium cepa ) and garlic ( Allium sativum ). The production of leek is mainly confined to Europe. In the last few years production has increased along with consumer demands. It is propagated through seeds and gives rise to heterogeneous progeny. Problems in cultivation of leek are rust ( Puccinea allii , P.mixtu ), yellow stripe virus and the lack of uniformity. The most suitable system able to cope with these problems seems to be hybrid breeding. However, hybrid breeding is hampered by the lack of a suitable emasculation system or male sterility system and the severe inbreeding depression. Therefore, emphasis has been put on the application of genetic modification in order to solve some of these problems. This relatively new technique opens the possibility to add or alter traits which cannot easily be achieved with conventional breeding methods. The most important prerequisites for genetic modification were investigated during this thesis research. The first prerequisite is an efficient regeneration system. Starting from seed, a cyclic somatic embryogenesis regeneration system was developed with long term regeneration potential, providing a regeneration system where it should be possible to obtain true transformants from chimeric structures (Chapter 2).A comparison was made between the first cycle, starting from zygotic embryos, and latter cycli, starting with somatic embryos. All genotypes tested were able to produce somatic embryos although genotypic differences in somatic embryo production occurred. The first cycle, using zygotic embryos, was the most effective in somatic embryo production compared to the later cycli using somatic embryos. On average the first cycle produced 23.8 somatic embryos per zygotic embryo and the later cycli ranged from 11.1 to 16.0 somatic embryos per initial somatic embryo. Shoot regeneration from somatic embryos was satisfactory, obtaining normal looking greenhouse plantlets.In Chapter 3 this cyclic somatic embryogenesis system was analyzed for its suitability in a genetic transformation system, like particle bombardment. The relatively new reporter gene luciferase was used in the transformation experiments. Leek, like most monocotyledons, seemed to be very persistent to selective media. Neither the selective agent kanamycin nor hygromycin could prevent leek cells from growing. The selective agent phosphinothricin had a better inhibitory effect on cell growth. A histological and morphological study showed that regeneration occurred from the deeper cell layers inside the somatic embryo, in the same way that leaves are produced on a mature leek plant. These cell layers are hardly reached by particles, explaining the poor results of the transformation experiments using somatic embryos.In Chapter 4 a newly developed regeneration system from flower stalk strip explants was used to determine the optimal conditions for particle bombardment with the luciferase reporter gene. After a vernalisation period leek starts to develop a flower stalk. This flower stalk, still inside the plant, is harvested and stripped. Regeneration from these strips occurs just beneath the epidermis, which is easy to reach for the particles of the particle gun. The regeneration frequency was neither influenced by the stripping nor by the bombardments. Over 300 plantlets could be obtained from 1 flower stalk. Important factors for transformation experiments like pre-culture time, pressure, distance and coating of the particles were analyzed and optimized for these highly regenerative explants. Flower stalk strips of leek as explants, cut from the basal part of the flower stalk, pre-cultured for 2 days, should be bombarded at a distance of 5 cm, with a pressure of 1800 psi to obtain a high transient expression. Gold particles should be used and coated following the procedure of Christou (1991). After bombardment the explants should be transferred to selection medium.In Chapter 5 these optimal conditions were applied to the flower stalk strip explants. A comparison of the use of the reporter genes GUS and luciferase was made. Both genes, combined in one plasmid, showed similar expression patterns. Expression of both genes was still present 7 weeks after bombardment, but local increases in gene activity were not observed. The reporter gene luciferase facilitates the investigations in the genetic modification research as the chemical reaction with this reporter gene is non-lethal to the plant tissue whereas the reaction with GUS-reporter gene is. The non-detrimental effect of the luciferin treatment made it possible to investigate gene expression in time. Still, using a novel reporter gene means also unexpected results like the long transient expression time of the luciferase gene product even after application of the substrate luciferin. Eventually, 16 chimeric plantlets were obtained. Probably, the regeneration from flower stalk strip explants is a multicellular event. Seeds harvested from potentially chimeric plants did not show any GUS or luciferase activity after germination.In the near future leek transformation research has to focus on the development of embryogenic cell suspension or protoplast cultures, to obtain true transformants of the chimeric plants and an efficient selection system to select and favor the transgenic cells. To facilitate success in transformation research, efforts could also be directed at plastid transformation, in order to come to true breeding transgenic line