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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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Histological appearance of coffee embryogenic callus cultures as a function of the age of the culture. Primary embryogenic callus produced on leaf explants; note the heterogeneous structure where degenerating (arrow) and active areas co-exist; the cells are characterized by a small non-central nucleus and low starch and cytoplasm soluble protein contents, scale bar = 36.5 μM (A). Seven-month-old embryogenic callus culture; note the highly homogeneous structure mainly comprising proembryogenic masses (PEMs) whose cells exhibit a high nucleus cytoplasm ratio and dense cytoplasm rich in soluble and reserve proteins, scale bar = 73 μM (B). Twenty-six month-old embryogenic callus culture; note the heterogeneous appearance of the mix of isolated cells, degenerating areas (arrow), masses oriented towards somatic embryogenesis (ma), along with areas of cells with protein rich cytoplasm (PEMs) similar to those in younger embryogenic cultures, scale bar = 73 μM (C).
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Figure 6: Histological appearance of coffee embryogenic callus cultures as a function of the age of the culture. Primary embryogenic callus produced on leaf explants; note the heterogeneous structure where degenerating (arrow) and active areas co-exist; the cells are characterized by a small non-central nucleus and low starch and cytoplasm soluble protein contents, scale bar = 36.5 μM (A). Seven-month-old embryogenic callus culture; note the highly homogeneous structure mainly comprising proembryogenic masses (PEMs) whose cells exhibit a high nucleus cytoplasm ratio and dense cytoplasm rich in soluble and reserve proteins, scale bar = 73 μM (B). Twenty-six month-old embryogenic callus culture; note the heterogeneous appearance of the mix of isolated cells, degenerating areas (arrow), masses oriented towards somatic embryogenesis (ma), along with areas of cells with protein rich cytoplasm (PEMs) similar to those in younger embryogenic cultures, scale bar = 73 μM (C).

Mentions: Histological analysis revealed a change in the quality of embryogenic callus tissues depending on the age of the culture with no changes visible to the naked eye (Figure 6). Whereas the most homogeneous and active tissues were observed in seven and nine-month old embryogenic callus cultures that displayed the typical histological appearance of the yellow phenotype (Figure 6B), the histological appearance of the primary embryogenic callus (no proliferation period) and oldest embryogenic callus cultures was very different. Observation of primary embryogenic callus indicated a very heterogeneous structure (Figure 6A) with coexisting degenerating and active areas. The cells had a small nucleus and low starch and cytoplasm soluble protein contents. At the histological level, the old embryogenic cultures were much more heterogeneous than at seven months and appeared as a mix of isolated cells, proembryos and masses oriented towards somatic embryogenesis (Figure 6C). Other areas comprised cells with protein-rich cytoplasm similar to those in younger embryogenic cultures.


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)

Histological appearance of coffee embryogenic callus cultures as a function of the age of the culture. Primary embryogenic callus produced on leaf explants; note the heterogeneous structure where degenerating (arrow) and active areas co-exist; the cells are characterized by a small non-central nucleus and low starch and cytoplasm soluble protein contents, scale bar = 36.5 μM (A). Seven-month-old embryogenic callus culture; note the highly homogeneous structure mainly comprising proembryogenic masses (PEMs) whose cells exhibit a high nucleus cytoplasm ratio and dense cytoplasm rich in soluble and reserve proteins, scale bar = 73 μM (B). Twenty-six month-old embryogenic callus culture; note the heterogeneous appearance of the mix of isolated cells, degenerating areas (arrow), masses oriented towards somatic embryogenesis (ma), along with areas of cells with protein rich cytoplasm (PEMs) similar to those in younger embryogenic cultures, scale bar = 73 μM (C).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Histological appearance of coffee embryogenic callus cultures as a function of the age of the culture. Primary embryogenic callus produced on leaf explants; note the heterogeneous structure where degenerating (arrow) and active areas co-exist; the cells are characterized by a small non-central nucleus and low starch and cytoplasm soluble protein contents, scale bar = 36.5 μM (A). Seven-month-old embryogenic callus culture; note the highly homogeneous structure mainly comprising proembryogenic masses (PEMs) whose cells exhibit a high nucleus cytoplasm ratio and dense cytoplasm rich in soluble and reserve proteins, scale bar = 73 μM (B). Twenty-six month-old embryogenic callus culture; note the heterogeneous appearance of the mix of isolated cells, degenerating areas (arrow), masses oriented towards somatic embryogenesis (ma), along with areas of cells with protein rich cytoplasm (PEMs) similar to those in younger embryogenic cultures, scale bar = 73 μM (C).
Mentions: Histological analysis revealed a change in the quality of embryogenic callus tissues depending on the age of the culture with no changes visible to the naked eye (Figure 6). Whereas the most homogeneous and active tissues were observed in seven and nine-month old embryogenic callus cultures that displayed the typical histological appearance of the yellow phenotype (Figure 6B), the histological appearance of the primary embryogenic callus (no proliferation period) and oldest embryogenic callus cultures was very different. Observation of primary embryogenic callus indicated a very heterogeneous structure (Figure 6A) with coexisting degenerating and active areas. The cells had a small nucleus and low starch and cytoplasm soluble protein contents. At the histological level, the old embryogenic cultures were much more heterogeneous than at seven months and appeared as a mix of isolated cells, proembryos and masses oriented towards somatic embryogenesis (Figure 6C). Other areas comprised cells with protein-rich cytoplasm similar to those in younger embryogenic cultures.

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