Articles
EFFICIENT TRANSFORMATION OF COMMERCIAL APRICOT CULTIVARS AND REGENERATION OF TRANSFORMED PLANTS
Article number
738_84
Pages
647 – 651
Language
English
Abstract
Efficient procedures for the regeneration of transformed plants have been developed from leaves of commercial apricot cultivars.
Integration of the nptII and gfp transgenes in the transformed plants have been demonstrated.
Based on GFP expression in the regenerated buds very high transformation efficiencies have been obtained by using specific selection strategies.
Low selection pressure during the first days after co-culture followed by progressive increase in the antibiotic concentration have allowed the production of transformed buds with efficiencies ranging from 3.8 to 5.8%. However, survival of those buds, especially strong GFP expressers, was really low yielding a final number of transformed shoots much lower (final efficiencies based on surviving buds ranged from 0.2 to 1.4%). Factors affecting the viability of putative transgenics have been evaluated.
A possible GFP toxicity was discarded by transforming with the same construction containing the uidA gene and observing similar transformation efficiencies (based on surviving shoots expressing the marker gene) as those obtained previously with gfp. An increased viability was obtained by recovering GFP positive regenerated buds the week after they were detected, limiting the time the buds remained in the regeneration medium exposed to a high thidiazuron concentration.
Also, elimination or reduction of the antibiotic concentration the first weeks after recovering the buds increased their survival by allowing a correct development of the bud.
A high selection pressure in the multiplication medium, continuously applied afterwards, reduced occurrence of escapes.
Integration of the nptII and gfp transgenes in the transformed plants have been demonstrated.
Based on GFP expression in the regenerated buds very high transformation efficiencies have been obtained by using specific selection strategies.
Low selection pressure during the first days after co-culture followed by progressive increase in the antibiotic concentration have allowed the production of transformed buds with efficiencies ranging from 3.8 to 5.8%. However, survival of those buds, especially strong GFP expressers, was really low yielding a final number of transformed shoots much lower (final efficiencies based on surviving buds ranged from 0.2 to 1.4%). Factors affecting the viability of putative transgenics have been evaluated.
A possible GFP toxicity was discarded by transforming with the same construction containing the uidA gene and observing similar transformation efficiencies (based on surviving shoots expressing the marker gene) as those obtained previously with gfp. An increased viability was obtained by recovering GFP positive regenerated buds the week after they were detected, limiting the time the buds remained in the regeneration medium exposed to a high thidiazuron concentration.
Also, elimination or reduction of the antibiotic concentration the first weeks after recovering the buds increased their survival by allowing a correct development of the bud.
A high selection pressure in the multiplication medium, continuously applied afterwards, reduced occurrence of escapes.
Authors
C. Petri, S. López-Noguera, N. Alburquerque, L. Burgos
Keywords
aminoglycoside antibiotics, A. tumefaciens, gfp, nptII, Prunus armeniaca, selection
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