Articles
A SELECTION STRATEGY TO OBTAIN TRANSFORMED APRICOT PLANTS
Article number
725_106
Pages
771 – 776
Language
English
Abstract
Efficient regeneration and transformation procedures have been developed with mature tissues from apricot cultivars.
Integration of transgenes (gfp and nptII marker genes) in the plant cells have been demonstrated with transformed callus tissues by means of observation of GFP expression, exposure to high kanamycin doses, PCR and Southern blot hybridization.
However, to date, production of transformed plants had not been reported.
This work was aimed to develop a strategy based on antibiotic selection that allowed the production of the first transformed apricot plants from mature leaf tissues.
Application of two different aminoglycoside antibiotics at different concentrations and at different times during the transformation experiments have been tried.
High selection pressure with 40 µM kanamycin failed to produced transformed buds, so antibiotic concentration was reduced to 25.7 µM and applied right after co-culture, delayed for 5, 7 or 10 days or not applied.
However, only one transformed bud was obtained when kanamycin was applied immediately after co-culture and two chimeras when no selection was applied.
Given the results, different aminoglycoside antibiotics were tested at different concentrations and 40 µM paromomycine was found to better stimulate growth of transformed tissues.
Therefore, this antibiotic was used in subsequent experiments, although when applied at that concentration from the beginning of the experiments, transformed plants were not obtained.
A progressive selection strategy has been necessary to obtain transformed apricot plants.
When a low selection pressure (10 µM) was applied during the first 10 days after co-culture some transformed buds were obtained but selection efficiency was really low and many escapes were also produced.
Increasing the initial paromomycin concentration to 20 µM followed by a higher selection applied after 10 to 24 days after co-culture have greatly improved transformation and selection efficiencies.
Based on GFP expression and PCR, some of these strategies have produced over 7% of transformed buds.
Integration of transgenes (gfp and nptII marker genes) in the plant cells have been demonstrated with transformed callus tissues by means of observation of GFP expression, exposure to high kanamycin doses, PCR and Southern blot hybridization.
However, to date, production of transformed plants had not been reported.
This work was aimed to develop a strategy based on antibiotic selection that allowed the production of the first transformed apricot plants from mature leaf tissues.
Application of two different aminoglycoside antibiotics at different concentrations and at different times during the transformation experiments have been tried.
High selection pressure with 40 µM kanamycin failed to produced transformed buds, so antibiotic concentration was reduced to 25.7 µM and applied right after co-culture, delayed for 5, 7 or 10 days or not applied.
However, only one transformed bud was obtained when kanamycin was applied immediately after co-culture and two chimeras when no selection was applied.
Given the results, different aminoglycoside antibiotics were tested at different concentrations and 40 µM paromomycine was found to better stimulate growth of transformed tissues.
Therefore, this antibiotic was used in subsequent experiments, although when applied at that concentration from the beginning of the experiments, transformed plants were not obtained.
A progressive selection strategy has been necessary to obtain transformed apricot plants.
When a low selection pressure (10 µM) was applied during the first 10 days after co-culture some transformed buds were obtained but selection efficiency was really low and many escapes were also produced.
Increasing the initial paromomycin concentration to 20 µM followed by a higher selection applied after 10 to 24 days after co-culture have greatly improved transformation and selection efficiencies.
Based on GFP expression and PCR, some of these strategies have produced over 7% of transformed buds.
Authors
C. Petri, S. Lopez-Noguera, L. Burgos, N. Alburquerque
Keywords
aminoglycoside antibiotics, A. tumefaciens, gfp, nptII, P. armeniaca
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