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Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures.

Ribas AF, Dechamp E, Champion A, Bertrand B, Combes MC, Verdeil JL, Lapeyre F, Lashermes P, Etienne H - BMC Plant Biol. (2011)

Bottom Line: When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars.All the selected plants were proved to be transformed by PCR and Southern blot hybridization.This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre de Coopération Internationale en Recherche Agronomique pour le Développement-Département des Systèmes Biologiques (CIRAD-BIOS), UMR-RPB (CIRAD, IRD, Université Montpellier II), 911 Avenue Agropolis, BP 64501, 34394 Montpellier, France.

ABSTRACT

Background: Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica.

Results: We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization.

Conclusion: Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes.

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PCR detection of the HPTII hygromycin resistance gene in transgenic coffee plants. The plants were produced from 7-month-old embryogenic callus cultures co-cultivated with A. tumefaciens strain LBA1119 carrying out the pMDC32 binary vector. M - Molecular weight DNA markers (1 kb), C - untransformed coffee plant (control), 1-19 transgenic coffee plants derived from independent transformation events, B - blank, PCR mix without DNA, P - plasmid. The arrow indicates the fragment corresponding to the HPTII gene (506 bp).
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Figure 8: PCR detection of the HPTII hygromycin resistance gene in transgenic coffee plants. The plants were produced from 7-month-old embryogenic callus cultures co-cultivated with A. tumefaciens strain LBA1119 carrying out the pMDC32 binary vector. M - Molecular weight DNA markers (1 kb), C - untransformed coffee plant (control), 1-19 transgenic coffee plants derived from independent transformation events, B - blank, PCR mix without DNA, P - plasmid. The arrow indicates the fragment corresponding to the HPTII gene (506 bp).

Mentions: PCR analysis was performed to detect the HPTII hygromycin resistance gene in the DNA of 60 putatively transformed plants derived from independent transformation events (1 plant/transformation event). Bands indicating the presence of the HPTII gene were systematically detected in the analyzed plants (Figure 8) indicating the high efficacy of hygromycin selection. No band was observed in untransformed plants or in the blank negative control. Southern blot analysis was performed to determine the number of insertion sites of the HPTII gene into the coffee genome (Figure 9). Eleven plants from independent transformation events were analyzed. Five of them presented one transgene insertion site (event n° 2, 3, 9, 10, 11). The others presented multiple copy insertion sites: two (n°5 and 7), between four and seven (n°4, 6, 8, 12).


Agrobacterium-mediated genetic transformation of Coffea arabica (L.) is greatly enhanced by using established embryogenic callus cultures.

Ribas AF, Dechamp E, Champion A, Bertrand B, Combes MC, Verdeil JL, Lapeyre F, Lashermes P, Etienne H - BMC Plant Biol. (2011)

PCR detection of the HPTII hygromycin resistance gene in transgenic coffee plants. The plants were produced from 7-month-old embryogenic callus cultures co-cultivated with A. tumefaciens strain LBA1119 carrying out the pMDC32 binary vector. M - Molecular weight DNA markers (1 kb), C - untransformed coffee plant (control), 1-19 transgenic coffee plants derived from independent transformation events, B - blank, PCR mix without DNA, P - plasmid. The arrow indicates the fragment corresponding to the HPTII gene (506 bp).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3111370&req=5

Figure 8: PCR detection of the HPTII hygromycin resistance gene in transgenic coffee plants. The plants were produced from 7-month-old embryogenic callus cultures co-cultivated with A. tumefaciens strain LBA1119 carrying out the pMDC32 binary vector. M - Molecular weight DNA markers (1 kb), C - untransformed coffee plant (control), 1-19 transgenic coffee plants derived from independent transformation events, B - blank, PCR mix without DNA, P - plasmid. The arrow indicates the fragment corresponding to the HPTII gene (506 bp).
Mentions: PCR analysis was performed to detect the HPTII hygromycin resistance gene in the DNA of 60 putatively transformed plants derived from independent transformation events (1 plant/transformation event). Bands indicating the presence of the HPTII gene were systematically detected in the analyzed plants (Figure 8) indicating the high efficacy of hygromycin selection. No band was observed in untransformed plants or in the blank negative control. Southern blot analysis was performed to determine the number of insertion sites of the HPTII gene into the coffee genome (Figure 9). Eleven plants from independent transformation events were analyzed. Five of them presented one transgene insertion site (event n° 2, 3, 9, 10, 11). The others presented multiple copy insertion sites: two (n°5 and 7), between four and seven (n°4, 6, 8, 12).

Bottom Line: When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars.All the selected plants were proved to be transformed by PCR and Southern blot hybridization.This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centre de Coopération Internationale en Recherche Agronomique pour le Développement-Département des Systèmes Biologiques (CIRAD-BIOS), UMR-RPB (CIRAD, IRD, Université Montpellier II), 911 Avenue Agropolis, BP 64501, 34394 Montpellier, France.

ABSTRACT

Background: Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica.

Results: We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization.

Conclusion: Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes.

Show MeSH
Related in: MedlinePlus