Time saving method for protoplast isolation, transformation and transient gene expression assay in barley

This study was conducted to establish a rapid method for barley (Hordeum vulgare L.) protoplast isolation to provide an easy-to-use procedure for the transformation and primary investigation of new gene constructs by transient gene expression assays. Protoplasts were successfully isolated from the chopped embryo and scutellum parts of mature barley seeds by digesting three hours with an enzyme mixture. Isolated protoplasts were washed in W5 washing solution, sieved through plastic meshes and then cleaned on sucrose gradient. The suitability of these directly from embryo-scutellum complexes derived protoplasts for transient gene expression studies was determined by transforming the protoplasts using the PEG (polyethylene glycol) method. Plasmid pAct1-F containing the rice Act1 promoter linked with the gus coding sequences and the nos polyadenylation signal was used in the transformation. After the PEG treatment protoplasts were cultured on KPR culture medium and the transient gus expression was assayed 24-36 hours after transformation. Up to 6% of the transformed protoplasts showed gus expression after treating the protoplasts with X-gluc. The results of this study show that the protoplasts isolated directly from dissected mature barley scutellum-embryo complexes could be used to investigate transient gene expressions in barley. This procedure requires negligible time prior the transformation experiment and so can be done in a very short time compared to the protoplast system based on a suspension culture

Nopea menetelmä ohran protoplastien eristämiseksi ja sen soveltaminen geeninsiirtoon

This study was conducted to establish a rapid method for barley (Hordeum vulgare L.) protoplast isolation to provide an easy-to-use procedure for the transformation and primary investigation of new gene constructs by transient gene expression assays. Protoplasts were successfully isolated from the chopped embryo and scutellum parts of mature barley seeds by digesting three hours with an enzyme mixture. Isolated protoplasts were washed in W5 washing solution, sieved through plastic meshes and then cleaned on sucrose gradient. The suitability of these directly from embryo-scutellum complexes derived protoplasts for transient gene expression studies was determined by transforming the protoplasts using the PEG (polyethylene glycol) method. Plasmid pAct1-F containing the rice Act1 promoter linked with the gus coding sequences and the nos polyadenylation signal was used in the transformation. After the PEG treatment protoplasts were cultured on KPR culture medium and the transient gus expression was assayed 24-36 hours after transformation. Up to 6% of the transformed protoplasts showed gus expression after treating the protoplasts with X-gluc. The results of this study show that the protoplasts isolated directly from dissected mature barley scutellum-embryo complexes could be used to investigate transient gene expressions in barley. This procedure requires negligible time prior the transformation experiment and so can be done in a very short time compared to the protoplast system based on a suspension culture.Tutkimuksessa kehitettiin menetelmä, jossa protoplasteja eristetään suoraan ohran jyvien alkion ja sirkkakilven solukosta ilman edeltävää solukkoviljelyvaihetta. Jyvät pintasteriloitiin ja niitä liuotettiin steriloidussa vedessä vuorokausi. Tämän jälkeen alkion ja sirkkakilven solukot eristettiin jyvistä ja pilkottiin pieniksi paloiksi. Protoplastien eristämiseksi kokeiltiin aluksi kolmea erilaista entsyymiliuosta, joista paras valittiin PEG (polyetyleeniglykoli)- menetelmällä tehtyihin geeninsiirtokokeisiin. Siirtokokeissa käytetty rengasmainen plasmidi-DNA pAct1-F sisälsi riisin Act1-säätelyjakson, Gus-geenin (koodaa β-glukuronidaasi- entsyymiä) ja Nos-lopetusjakson. PEG-käsittelyn jälkeen protoplasteja kasvatettiin KPR-kasvatusalustalla. Transienttinen (väliaikainen) Gus-geenin ilmentyminen määritettiin vuorokauden kuluttua käsittelemällä transformoituja protoplasteja X-gluc-entsyymisubstraatilla. Enimmillään noin kuudessa prosentissa transformoiduista protoplasteista havaittiin Gus-geenin ilmentymistä. Tulosten mukaan tämä nopea protoplastien eristystekniikka soveltuu erityisesti väliaikaisen geeni-ilmentymisen tutkimukseen, sillä ajansäästö protoplastien normaaliin suspensioviljelmään verrattuna on huomattava

Induction of gametic embryogenesis and production of doubled haploids in barley microspore culture

vokKJ

Development and characterization of a chimaeric tissue-specific promoter in wheat and rice endosperm

The recently achieved significant improvement of cereal transformation protocols provides facilities to alter the protein composition of the endosperm, for example, to increase or decrease the quantity of one of its protein components or to express foreign molecules. To achieve this goal, strong endosperm-specific promoters have to be available. The aim of our work was to develop a more efficient tissue-specific promoter which is currently used. A chimaeric promoter was assembled using the 5' UTR (1,900 bp) of the gene coding for the 1Bx17 HMW glutenin subunit protein, responsible for tissue-specific expression and the first intron of the rice actin gene (act1). The sequence around of the translation initial codon was optimized. The effect of the intron and promoter regulatory sequences, using different lengths of 1Bx17 HMW-GS promoter, were studied on the expression of uidA gene. The function of promoter elements, promoter length, and the first intron of the rice actin gene were tested by a transient expression assay in immature wheat endosperm and in stable transgenic rice plants. Results showed that insertion of the rice act1 first intron increased GUS expression by four times in transient assay. The shortest 1Bx17 HMW-GS promoter fragment (173 bp) linked to the intron and GUS reporter gene provided almost the same expression level than the intronless long 1Bx17 HMW-GS promoter. Analysis of the stable transformant plants revealed that 173 nucleotides were sufficient for endosperm-specific expression of the uidA gene, despite 13 nucleotides missing from the HMW enhancer sequence, a relevant regulatory element in the promoter region

Expression of Cholera Toxin B Subunit in Transgenic Rice Endosperm

The synthetic cholera toxin B subunit (CTB) gene, modified according to the optimized codon usage of plant genes, was introduced into a plant expression vector and expressed under the control of the Bx17 HMW (high molecular weight) wheat endosperm-specific promoter containing an intron of the rice act1. The recombinant vector was transformed into rice plants using a biolistic-mediated transformation method. Stable integration of the synthetic CTB gene into the chromosomal DNA was confirmed by PCR amplification analysis. A high level of CTB (2.1% of total soluble protein) was expressed in the endosperm tissue of the transgenic rice plants. The synthetic CTB produced only in the rice endosperm demonstrated strong affinity for G(M1)-ganglioside, thereby suggesting that the CTB subunits formed an active pentamer. The successful expression of CTB genes in transgenic plants makes it a powerful tool for the development of a plant-derived edible vaccine