Articles
COMPARISON OF TWO TRANSFORMATION METHOD EFFICIENCY USING PELARGONIUM × HORTORUM ‘PANACHÉ SUD’ PROTOPLASTS AND LEAF DISCS AS EXPLANTS
Article number
743_17
Pages
127 – 132
Language
English
Abstract
Because water resources must be preserved by both plant producers and consumers, the use of ornamental plants that maintain aesthetic qualities under drought stress is of great interest.
We have tested P. × hortorum cultivars and found that resistance was related to sugar accumulation.
Since drought resistance is a complex phenomenon, we choose to face the problem via genetic transformation.
Plant regeneration systems from leaf protoplasts and leaf discs of P. × hortorum Panaché Sud was set up (Hassanein and Dorion, 2005, 2006) as well as genetic transformation of leaf discs via Agrobacterium tumefaciens (Hassanein et al., 2005). Direct protoplast transformation via electroporation was studied.
The best conditions for direct gene transfer were as follows: 20-25 µg/ml pFAJ3000, three electric pulses of 33µF capacitance and 250 V cm-1 on a protoplast and plasmid suspension of 1800 µS.cm-1 conductivity.
The transformation efficiency was 1.5%. Comparing both transformation methods, we have stated that indirect transformation is more rapid (17 weeks against 22 weeks) and easier to handle while direct transformation could be more efficient.
Indeed, using the same material (one leaf), it is possible to obtain 54 transformed plants via Agrobacterium transfer versus theoretically 47.000 via direct transfer. nptII gene was observed after PCR analysis in all the clones transformed via Agrobacterium (119). uidA gene was present in 78% of the clone (PCR analysis) while only 66% expressed it (histochemical GUS assay). PCR analyses were easy and robust with plants arising from indirect transformation.
In contrast many difficulties were encountered analyzing not only clones (10 out of 29) arising from direct gene transfer but also control plants.
Nevertheless, npt II gene could be present and uidA gene absent in the main part of the tested clones.
We have tested P. × hortorum cultivars and found that resistance was related to sugar accumulation.
Since drought resistance is a complex phenomenon, we choose to face the problem via genetic transformation.
Plant regeneration systems from leaf protoplasts and leaf discs of P. × hortorum Panaché Sud was set up (Hassanein and Dorion, 2005, 2006) as well as genetic transformation of leaf discs via Agrobacterium tumefaciens (Hassanein et al., 2005). Direct protoplast transformation via electroporation was studied.
The best conditions for direct gene transfer were as follows: 20-25 µg/ml pFAJ3000, three electric pulses of 33µF capacitance and 250 V cm-1 on a protoplast and plasmid suspension of 1800 µS.cm-1 conductivity.
The transformation efficiency was 1.5%. Comparing both transformation methods, we have stated that indirect transformation is more rapid (17 weeks against 22 weeks) and easier to handle while direct transformation could be more efficient.
Indeed, using the same material (one leaf), it is possible to obtain 54 transformed plants via Agrobacterium transfer versus theoretically 47.000 via direct transfer. nptII gene was observed after PCR analysis in all the clones transformed via Agrobacterium (119). uidA gene was present in 78% of the clone (PCR analysis) while only 66% expressed it (histochemical GUS assay). PCR analyses were easy and robust with plants arising from indirect transformation.
In contrast many difficulties were encountered analyzing not only clones (10 out of 29) arising from direct gene transfer but also control plants.
Nevertheless, npt II gene could be present and uidA gene absent in the main part of the tested clones.
Publication
Authors
A. Hassanein, K. Loridon, N. Dorion
Keywords
direct gene transfer, electroporation, geranium, transgenic plant, Agrobacterium tumefaciens, PCR analysis
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