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INDUCTION OF AGROBACTERIUM2-3h
ADDITION OF AS200 M final
AGROBACTERIUM GROWTHin MG/L (Tingay et al., 1997)
CENTRIFUGATION
RESUSPENSION ININOCULATION MEDIUM 1/10MS2(1/10MS, 30 g/l sucrose, 2 mg/l 2,4-D, pH5.2)
at OD600<1
INOCULATION2-3h
EXPLANTS
RINSE AND BLOT DRY
CO-CULTIVATION3d on 1/10MS2 + 4d on MS2
GUS ASSAY T-DNA transfer in oat occurs after long co-cultivationsLong 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 barley2,7, wheat7 and rice8.
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2 3 6 9Co-cultivation time (d)
OD 0.1 OD 0.6 OD 1.7
EFFECT OF CO-CULTIVATION TIME on transformation efficiency.
Tissue: embryo, Co-cult. Medium: MS2, Strain: LBA4404/pAL166/pAL156, Co-cult. Time: 2-9 d.
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MS2 7d 6.5d 5d 4d
BA
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 1/10MS2.
Reduction of co-cultivation on 1/10MS2 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 1/10MS2. B-Tissue: embryo axis, Strain: AGL1/pAL155/pAL156, Co-cult.: 0 to 3d on 1/10MS2 followed by 4 to 7d on MS2.
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medium (M)
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EFFECT OF ACETOSYRINGONE (AS) on transformation efficiency.
Tissue: embryo axis, Co-cult. Medium: 1/10MS2, 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.
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No treat. wound vacuum
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 OATSSophie J. Perret and Phillip Morris
Cell Biology Department, Institute of Grassland and Environmental Research,Plas Gogerddan, Aberystwyth SY23 3EB, UK [email protected] & [email protected]
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 cereals1-4 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 virGWT,
AGL1/pAL155/pAL156 contains virG542 and AGL1/pSoup/pAL156 contains
no additional virG (based on pSoup/pGreen binary vectors5). 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 1/10MS2 (1/10MS 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 L36.• 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 + Timentin4 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
REGENERATION
Leaf b
ases
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.
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POTENTIAL OF DIFFERENT OAT EXPLANTS for transformation.
Tissue: various, Strain: AGL1/pSoup/pAL156, Co-cult.: 3d on 1/10MS2 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 basesComment: 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.
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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): 311-3242. Wu, H. X. et al. (1998) Plant Cell Tissue and Organ Culture, 54 (3): 161-1713. Cheng, M. et al. (1997) Plant Physiology, 115 (3): 971-9804. Ishida, Y. et al. (1996) Nature Biotechnology, 14 (6): 745-7505. Hellens, R. P. et al. (2000) Plant Molecular Biology, 42 (6): 819-8326. Gless, C. et al. (1998) Plant Cell Reports, 17(6-7): 441-4457. Guo, G. Q. et al. (1998) Cereal Research Communications, 26 (1): 15-228. Zhang, J. et al. (1997) Molecular Biotechnology, 8 (3): 223-231
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