Development of an Agrobacterium transformation system for onion (Allium cepa L.)
Degree GrantorUniversity of Canterbury
Degree NameDoctor of Philosophy
Onion (Allium cepa) bulbs of the New Zealand-bred cultivars 'Pukekohe Longkeeper' and 'Early Longkeeper' produced tumourous growths after inoculation with 25 virulent strains of Agrobactenum tumefaciens, A. rubi and A. rhizogenes. The majority of these tumours produced nopaline, indicating that tumour cells were transformed. Some excised tumours produced roots in sterile culture. Eight onion genotypes were screened in tissue culture for callus formation, regeneration of plantlets from callus and clonal multiplication by shoot proliferation. All genotypes could be clonally multiplied and four were readily regenerable from callus. A technique for plantlet multiplication, which uses longitudinally-bisected stems of in vitro-germinated onion seedlings as explants, was developed. Onion (‘Pukekohe Longkeeper', 'Southport White Globe', 'Japanese Saporo Yellow' and 'Hikeeper Fl ') protoplasts were isolated and cultured on a range of media. These protoplasts formed new cell walls and sometimes divided, but only first divisions were regularly seen. Kanamycin, geneticin (G418), hygromycin and chlorsulfuron were evaluated for their use as selective agents in onion transformation experiments. Tissues surveyed for sensitivity to these selective agents included seeds and seedlings on germination and callusing media, established callus on callusing and regeneration media, and shoot cultures on shoot proliferation medium. Hygromycin was shown to be the antibiotic most toxic to tissues of all the surveyed onion cultivars, with effects being obvious in all tissues after 4-5 weeks of culture on concentrations as low as 20 mgl-1. Kanamycin was shown to be the least toxic of the selection agents surveyed. The kanamycin analogue G418 was considerably more toxic to most onion cultures than kanamycin. However, responses of cultures to G418 were slower than those to hygromycin. The herbicide chlorsulfuron was also shown to be toxic to onion seedlings and shoot cultures. The ability of Agrobacterium tumefaciens to transfer foreign genes to A. cepa was demonstrated. A single, putatively transformed plantlet (RC1), was regenerated from an onion seedling stem via callus, following co-cultivation of stem explants with Agrobacterium strain LBA4404 harbouring the binary vector pKIWI110. In addition, 41 auxiliary or adventitious shoots which grew directly from basal plates injected in vitro with four strains of A. tumefaciens (each harbouring the binary vectors pKIWI110 or pGA643) exhibited resistance to G418 in culture. The binary vectors used carry the neomycin phosphotransferase II gene (nptII) controlled by the nopaline synthase (nos) promoter. Both RC1 and some of the shoots growing from basal plate explants produced roots when grown on culture media supplemented with G418. Southern analyses showed that fragments of DNA from RC1 and from five of the 41 G418-resistant shoots hybridized to a 1.25 kbp nptII probe. (β- glucuronidase (GUS) activity was detected in over half of the plantlets derived from basal plate tissue injected with A. tumefaciens strains LBA4404 or C58, both of which harboured pKIWI110. Molecular and phenotypic evidence suggested that the putatively transformed plants produced from injected basal plate tissues were chimeric.