Transgenic barley by particle bombardment: Dissertation

In this study, particle bombardment was adapted for the production of stably transformed cell lines of barley and transgenic barley plants (Hordeum vulgare L.). Suspension culture of barley (cv. Pokko, VTT-G-93001) initiated from immature embryos was used as preliminary material for the optimization of particle bombardment parameters. Genes coding for b-glucuronidase (uidA) and neomycin phosphotransferase (nptII) were used as markers in separate plasmids. The helium modification of the BiolisticR PDS-1000 particle delivery system yielded at least three times higher transient expression values than the gunpowder modification. When bombarded with the uidA marker gene, an average of 7000 blue spots per gram of suspension culture were produced. Stably transformed lines were obtained by bombarding with the unlinked marker genes and by growing the bombarded samples on GeneticinR selection medium. Neomycin phosphotransferase (NPTII) activity was detected from 96 % of the GeneticinR-resistant lines and 40 % of them also expressed the unlinked uidA marker gene. Stable transformations were confirmed by Southern blot hybridization. The transformation method developed for suspension culture of barley was utilized for activation of the anthocyanin pathway. Maize anthocyanin biosynthesis is controlled by two specific regulatory genes. Representatives of these maize regulators, Lc and C1, were also able to activate the normal anthocyanin pathway in suspension culture of barley. Stably transformed pigmented lines were obtained and further cloning by selecting aggregates of 100 to 500 cells during 34 subculturings resulted in highly productive cell lines. The best cell line produced 1.9 mg total anthocyanins per gram fresh weight calculated as cyanidin-3-arabinoside. The spectrum of anthocyanins in transgenic lines was shown to be the same as in pigmented parts of leaves of barley. Delphinidin-arabinoside and peonidin-galactoside could be identified on the basis of HPLC-analysis. Stable transformation was confirmed by Southern blot hybridization. Particle bombardment parameters of immature embryos of barley (cv. Kymppi) were optimized by studying the transient expression after bombardment of the uidA marker gene. Further optimization of the amount of DNA in particle coating and of the load of DNA-coated particles per bombardment increased the transformation frequency, and an average of 50 blue spots per embryo was obtained. Transgenic fertile barley was obtained by bombarding the embryonic axis side of immature embryos with the nptII marker gene. Bombarded embryos were grown to plants without additional selection pressure and NPTII activity was screened in small plantlets. One plant out of 227 expressed the transferred gene. Altogether this plant (T0) produced 146 fertile spikes, of which ten yielded transgenic offspring (T1). Four sets of siblings were further studied. The transferred nptII gene was detected by the PCR technique, NPTII activity was observed and the integration was confirmed by Southern blot hybridization. In the order of appearance the transgenic tillers of the chimeric T0 plant studied in detail were the 13th, 30th, 36th and 50th. All these tillers and their progeny carried the same integration pattern, indicating that they originated from a single transformation event. Therefore it is evident that these transgenic shoots originated from one of the side shoot meristems. To prove this, immature and mature embryos of barley were bombarded with the uidA marker gene to the embryonic axis side. After histochemical staining, transformation events were localized in longitudinal sections of embryos. The results showed that successful transformation occurred in the nodal region of embryos containing the secondary shoot meristems capable of producing non-chimeric transgenic shoots. The inheritance and stability of the transferred nptII gene was followed through six generations. NPTII activity was found in all the studied generations and Southern blot hybridization confirmed the inheritance without rearrangements. The agronomic evaluation of the transgenic plants ensured that their overall appearance was normal. When transgenic plants were compared to non-transformed Kymppi plants, the growth, flowering and ripening rhythms were observed to be identical. Particle bombardment is a novel gene transfer method for the production of transgenic barley. The experiments described showed that it is possible to obtain transgenic plants by bombardment of the nodal region of an embryo. Furthermore, it was shown that the transgenic plants were normal and that the transferred gene was inherited through six subsequent generations.