Articles
SELECTABLE MARKER SYSTEMS FOR GENETIC ENGINEERING OF GRAPEVINE
Article number
603_63
Pages
485 – 490
Language
English
Abstract
The efficiency of Phosphomannose-Isomerase (PMI) and Phosphinotricin Acetyl Transferase (PAT) as selectable marker systems to regenerate genetically modified grapevines (Vitis spp) were investigated. Agrobacterium-mediated transformation of proembryogenic masses (PEMs) of Vitis vinifera L. cv Merlot and of embryo clusters of Vitis sp.
Seyval blanc and different rootstocks (V. berlandieri x V. riparia) using a GUS-intron cassette combined with the selectable marker systems were carried out.
Mannose was found to have only a minor selective effect on embryogenic tissue of grapevine.
Transformation of PEMs of Merlot with the PMI construct, yielded the highest number of GUS expressing cells and embryos on medium comprising 25% sucrose and 75% mannose (60 g/L total sugar). However, 16 weeks post transformation, 25 to 40 % of these new regenerating embryos were found to be none transgenic.
The best selection efficiency for embryo clusters of Seyval blanc was found with 60% to 75 % of mannose (20 g/L total sugar). Continued selection for 20 weeks post transformation yielded regenerating embryos, but these proved to be either chimeric or non-transgenic.
Grapevine embryogenic tissue (PEMs and embryo clusters) was highly sensitive to Phosphinotricin (PPT) treatments higher than 2.5 to 5.0 mg/L (depending on tissue and genotype). With PAT transformed PEM cells of Merlot, no GUS expressing embryos were found after a 16 week selection period (0 to 10 mg/L PPT). Using embryo clusters of Seyval blanc, new GUS expressing embryos regenerated on the original explants after about 16 weeks under 5.0 and 7.5 mg/L PPT selection.
Transformation of embryo clusters of rootstocks using the PAT as selectable marker system, yielded about 40% of putatively tansformed embryos after 8 weeks under 5 to 10 mg/L PPT selection followed by 2.5 mg/L PPT selection until week 20 post transformation.
Clonal propagation and further PCR analysis of the regenerated plants showed that most of the lines are chimeric or non-transgenic.
The results demonstrate that both selectable marker systems need further optimisation before being used as efficient selection and regeneration systems for genetically modified grapevines.
Seyval blanc and different rootstocks (V. berlandieri x V. riparia) using a GUS-intron cassette combined with the selectable marker systems were carried out.
Mannose was found to have only a minor selective effect on embryogenic tissue of grapevine.
Transformation of PEMs of Merlot with the PMI construct, yielded the highest number of GUS expressing cells and embryos on medium comprising 25% sucrose and 75% mannose (60 g/L total sugar). However, 16 weeks post transformation, 25 to 40 % of these new regenerating embryos were found to be none transgenic.
The best selection efficiency for embryo clusters of Seyval blanc was found with 60% to 75 % of mannose (20 g/L total sugar). Continued selection for 20 weeks post transformation yielded regenerating embryos, but these proved to be either chimeric or non-transgenic.
Grapevine embryogenic tissue (PEMs and embryo clusters) was highly sensitive to Phosphinotricin (PPT) treatments higher than 2.5 to 5.0 mg/L (depending on tissue and genotype). With PAT transformed PEM cells of Merlot, no GUS expressing embryos were found after a 16 week selection period (0 to 10 mg/L PPT). Using embryo clusters of Seyval blanc, new GUS expressing embryos regenerated on the original explants after about 16 weeks under 5.0 and 7.5 mg/L PPT selection.
Transformation of embryo clusters of rootstocks using the PAT as selectable marker system, yielded about 40% of putatively tansformed embryos after 8 weeks under 5 to 10 mg/L PPT selection followed by 2.5 mg/L PPT selection until week 20 post transformation.
Clonal propagation and further PCR analysis of the regenerated plants showed that most of the lines are chimeric or non-transgenic.
The results demonstrate that both selectable marker systems need further optimisation before being used as efficient selection and regeneration systems for genetically modified grapevines.
Authors
G.M. Reustle, M. Wallbraun, M. Zwiebel, R. Wolf, T. Manthey, C. Burkhardt, T. Lerm, M. Vivier, G. Krczal
Keywords
transformation, proembryogenic masses, embryo cluster, Phosphomannose Isomerase, Phosphinotricin Acteyl Transferase, rootstocks (V. berlandieri x V. riparia), Merlot
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