Regeneration of fertile barley plants from protoplasts and production of transgenic barley by electroporation:Dissertation

In this study tissue culture methods for barley (Hordeum vulgare L. cv. Kymppi, an elite cultivar) were developed in order to produce a regenerable protoplast system for the stable transformation of barley by electroporation. Embryogenic suspension cultures were initiated from immature embryo- and anther-derived calli. Both materials gave rise to homogenous suspension cultures. The effects of inorganic nitrogen source and phosphate on growth and regeneration of the suspensions were studied using the Box-Wilson statistical experimental design. Maximum growth was obtained with an ammonium concentration of 5.0 mM, nitrate concentration of 25.0 mM and phosphate concentration of 1.4 mM. No effects of inorganic nitrogen and phosphate on the regeneration of plants were observed. Albino plantlets were regenerated from protoplasts isolated from suspension cultures. Culture conditions for the regeneration of plants from isolated microspores were optimized. The subculture regime and the sugar concentration of the medium had a marked effect on the regeneration of green plantlets from mechanically isolated microspores of barley. An almost tenfold increase in the yield of green plants was obtained by shortening the suspension culture time of the developing proembryo mass from four to three weeks. A further twofold increase was obtained by increasing the maltose concentration of the microspore isolation medium from 0.175 M to 0.250 M and of the culture medium from 0.175 M to 0.325 M. In the optimal conditions a mean of 169 green plants per spike were regenerated. Cultures of isolated microspores were used for isolation of protoplasts. The protoplasts were cultured embedded in agarose and with a nurse culture. The plating efficiency varied from 0.002% to 0.015%. Plantlets regenerated from protoplasts were potted in soil within 4 - 5 months of collecting the spikes for microspore culture. Of 150 plants regenerated, 95% were fertile. Electroporation parameters and protoplast culture conditions were optimized for improved transformation frequencies using protoplasts isolated from the suspension cultures. Three plasmid constructions were used, one containing the neomycin phosphotransferase gene (nptII) under the control of the CaMV 35S transcript promoter and the other two containing the beta-glucuronidase gene (uidA) and the first intron of the maize Adh1 gene under the CaMV 35S transcript promoter or the maize Adh1 promoter. The levels of transient gene expression were evaluated after different electroporation treatments. Electroporation at 800 V/cm, 200 - 300 mF in amino acid buffer was found to be optimal for the transformation of suspension protoplasts. Pretreatment of the protoplasts with heat shock and culture with feeder cells further improved the transformation efficiency. Protoplasts isolated from cultured microspores were electroporated with plasmid DNA containing the nptII gene and cultured to produce transgenic plants. From 6.5 x 106 protoplasts treated, three green plants were regenerated. Enzyme assay performed from leaf extracts of these plants indicated that all three contained the transferred nptII gene. Southern blot hybridization performed with genomic DNA from leaves of two of the positive plants confirmed the incorporation of the transferred gene and the progeny of the plants inherited the new trait. Electroporation is a new method for the production of transgenic barley plants. The experiments described showed that it is possible to establish barley microspore cultures from which regenerable protoplasts can be isolated. Additionally it was shown that the protoplasts can be used to produce transgenic barley plants by electroporation