1. INDUCTION OF AGROBACTERIUM 2-3h ADDITION OF AS 200 M final AGROBACTERIUM GROWTH in MG/L (Tingay et al., 1997) CENTRIFUGATION RESUSPENSION IN INOCULATION MEDIUM 1 / 10 MS2 ( 1 / 10 MS, 30 g/l sucrose, 2 mg/l 2,4-D, pH5.2) at OD 600 <1 INOCULATION 2-3h EXPLANTS RINSE AND BLOT DRY CO-CULTIVATION 3d on 1 / 10 MS2 + 4d on MS2 GUS ASSAY T-DNA transfer in oat occurs after long co-cultivations Long co-cultivations are required for observation of GUS expression, which suggests that T-DNA transfer in oat is slower than T-DNA transfer in dicots. Long co-cultivations were also found to improve T-DNA transfer in barley 2,7, wheat 7 and rice 8. EFFECT OF CO-CULTIVATION TIME on transformation efficiency. Tissue: embryo, Co-cult. Medium: MS2, Strain: LBA4404/pAL166/pAL156, Co- cult. Time: 2-9 d. 1 / 10 MS2 0d 0.5d 2d 3d MS2 7d 6.5d 5d 4d B A Co-cultivation on low salt medium improved transformation efficiency, however reduced embryogenesis and growth of tissues were observed during the weeks that followed co-cultivation on 1 / 10 MS2. Reduction of co-cultivation on 1 / 10 MS2 to 3d, followed by 4d on MS2, was found to be optimal for efficient T-DNA transfer without loss of embryogenesis of oat tissues. EFFECT OF LOW SALT CONCENTRATION on transformation efficiency. A-Tissue: emb. callus and embryo axis, Strain: AGL1/pAL155/pAL156, Co-cult.: 7d on 1 / 10 MS2. B-Tissue: embryo axis, Strain: AGL1/pAL155/pAL156, Co-cult.: 0 to 3d on 1 / 10 MS2 followed by 4 to 7d on MS2. AS in inoculation medium: M EFFECT OF ACETOSYRINGONE (AS) on transformation efficiency. Tissue: embryo axis, Co-cult. Medium: 1 / 10 MS2, Strain: AGL1/pAL155/pAL156, Co- cult. Time: 7d. The highest level of GUS expression was obtained when Agrobacterium was pre-induced before centrifugation, and inoculation and co-cultivation were done in the absence of AS. These results were confirmed in two other experiments. Moreover, the growth of oat tissue was negatively affected by prolonged co- cultivations in the presence of AS. EFFECT OF WOUNDING AND VACUUM APPLICATION on transformation efficiency. Treatment: tissue was bombarded with gold particles prior to inoculation, or vacuum was applied on intact embryo axes during inoculation, Tissue: embryo axis, Co-cult. Medium: MS2, Strain: AGL1/pAL155/pAL156, Co-cult. Time: 9d. Transformation efficiency can be improved by either wounding the explants or by vacuum application during inoculation. FACTORS INFLUENCING T-DNA TRANSFER IN OATS Sophie J. Perret and Phillip Morris Cell Biology Department, Institute of Grassland and Environmental Research,Plas Gogerddan, Aberystwyth SY23 3EB, UK sophie.perret@bbsrc.ac.uk & phillip.morris@bbsrc.ac.uk INTRODUCTION: Plant transformation via Agrobacterium tumefaciens can result in simpler integration patterns and lower copy number of transgenes than direct gene transfer and in the independent segregation of co-transferred genes. While the number of reports of Agrobacterium-mediated transformation of other cereals 1-4 is increasing, there has been so far no report of transformation of oat by this method. We describe here the observation of GUS expression in oat tissue transformed with the gus gene via A. tumefaciens and propose a transformation protocol based on the study of variables affecting transformation efficiency. MATERIAL & METHODS: Tissues: embryogenic callus, embryo, embryo axis, leaf base of genotypes Melys and Bullion. Agrobacterium strains: LBA4404/pAL166/pAL156 contains virG WT, AGL1/pAL155/pAL156 contains virG 542 and AGL1/pSoup/pAL156 contains no additional virG (based on pSoup/pGreen binary vectors 5 ). Both strains carry the bar and gus genes on their T-DNA. Acetosyringone (AS): 200 mM. Transformation media: MS2 (MS salts, 30 g/l sucrose, 2 mg/l 2,4-D, pH5.2) and 1 / 10 MS2 ( 1 / 10 MS salts, 30 g/l sucrose, 2 mg/l 2,4-D, pH5.2). Callus induction media:MS2.TA (MS salts, 20 g/l sucrose, 2 mg/l 2,4-D, 150 mg/l asparagine, 0.5 mg/l thiamine, pH5.8) or L3 6. Selection medium: MS2, 3 mg/l PPT, pH5.8. Regeneration medium: MSK (MS salts, 30 g/l sucrose, 0.2 mg/l kinetin, 3 mg/l PPT, 0.8% agar, pH 5.6). Unless otherwise stated, media were solidified with 0.3% Phytagel. After co-cultivation, all media were supplemented with 150 mg/l Timentin. CULTURE ON CALLUS INDUCTION MEDIUM + Timentin 4 weeks DETERMINATION OF TRANSFORMATION EFFICIENCY: As no transient expression was observed immediately after transformation, GUS expression was analysed 4-5 weeks after transformation on 10 explants and transformation efficiency was expressed as the mean number of GUS expressing foci per explant. When embryo and embryo axes were transformed, the GUS assay was performed on the non-embryogenic tissue that had developped, while the embryogenic tissue was transferred to selection medium. SELECTIONREGENERATION Leaf bases Other tissues Five weeks after the start of selection, GUS expression in embryogenic callus was limited to small spots, no large blue cluster was observed. No GUS expression was detected 10 weeks after the start of selection. No transformed plant regenerated. The disappearance of GUS expressing tissue after selection and the absence of plant regeneration suggest that the selectable marker gene was not expressed in these tissues. Non-expression of a complete bar gene sequences could be due to silencing or to malfunction of the construct itself. Alternatively, the gene sequence integrated in the genome could be incomplete. This problem will be further investigated ACKNOWLEDGEMENTS: We thank MAFF for providing funding under the Crop Molecular Genetics Programme and Dave Lonsdale (IPSR Norwich) for providing the Agrobacterium strains. IGER is grant aided by BBSRC. POTENTIAL OF DIFFERENT OAT EXPLANTS for transformation. Tissue: various, Strain: AGL1/pSoup/pAL156, Co-cult.: 3d on 1 / 10 MS2 and 3d on MS2. Transformation efficiency was similar in non-embryogenic tissue of embryo and embryo axis. Transformation efficiency was similar in embryogenic callus and leaf bases Comment: GUS assay on non-embryogenic tissue resulted in single cell count, while cell clusters were counted on embryogenic tissue. CONCLUSIONS: Factors affecting T-DNA transfer to oat tissue have been identified and an improved protocol for Agrobacterium transformation has been established. The use of embryo or embryo axis as explant provides a good system to study additional factors that could further increase transformation rates, while leaf bases and embryogenic callus could be used for stable transformation and production of transgenic plants. GUS EXPRESSION IN OAT TISSUES transformed with Agrobacterium. A, GUS expressing cells in embryogenic calli 7 weeks after co-cultivation. B, GUS expressing cells in embryo axis-derived non embryogenic tissue 4 weeks after co- cultivation. C, GUS expressing, transformed (t) leaf base-derived embryo 4 weeks after co-cultivation. nt: non-transformed embryos. ABC t nt Determination of transformation efficiency was based on small, independent cell clusters in embryogenic callus (A) and leaf bases, and on loose, individual cells on non-embryogenic tissue that had developped from embryos and embryo axes (B). Observation of a GUS expressing somatic embryo on leaf base tissue (C) indicates that production of transgenic oat plants via Agrobacterium is achievable. REFERENCES : 1. Khanna, H. K. and Raina, S. K. (1999) Australian Journal of Plant Physiology, 26 (4): 311-324 2. Wu, H. X. et al. (1998) Plant Cell Tissue and Organ Culture, 54 (3): 161-171 3. Cheng, M. et al. (1997) Plant Physiology, 115 (3): 971-980 4. Ishida, Y. et al. (1996) Nature Biotechnology, 14 (6): 745-750 5. Hellens, R. P. et al. (2000) Plant Molecular Biology, 42 (6): 819-832 6. Gless, C. et al. (1998) Plant Cell Reports, 17(6-7): 441-445 7. Guo, G. Q. et al. (1998) Cereal Research Communications, 26 (1): 15-22 8. Zhang, J. et al. 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