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Influence of micropropagation through somatic embryogenesis on somaclonal variation in coffee (Coffea arabica): assessment of variations at the phenotypical, cytological, genetic and epigenetic level

Bobadilla Landey R.. 2013. Montpellier : UM2, 186 p.. Thèse de doctorat -- Biologie intégrative des plantes.

Somaclonal variation (SV) is a major concern in all micropropagation systems. It is described as the phenotypic variation displayed in in vitro-derived regenerants and it is believed to be originated from a large array of genetic and epigenetic mechanisms. Highly productive Coffea arabica hybrids are clonally disseminated in Meso-American region through somatic embryogenesis (SE). The objective of the present work in coffee is to evaluate the trueness-to-type of SE, to understand better the mechanisms involved in SV and further optimize SE conditions. We assessed the variations in the propagated plants at the phenotypic, cytogenetic (chromosome counting), genetic (mutations/AFLP, activation of transposable elements/S-SAP) and epigenetic (methylation/MSAP) level by using two complementary approaches. First, with 2 hybrids we studied industrial culture conditions expected to be weakly mutagenic thanks to the combined use of short term proliferation period (6 months) and low auxin supply (0-1.4 µM 2,4-D). Two proliferation systems i.e. secondary embryogenesis and embryogenic suspensions were compared, the latter being more productive and economic. AFLP and MSAP molecular analyses on 145 somatic seedlings showed that genetic and epigenetic polymorphisms between mother plants and emblings were extremely low, i.e. ranges of 0¿0.003% and 0.07¿0.18% respectively, with no significant difference between the proliferation systems. No plant was found to cumulate more than 3 methylation polymorphisms. For the two hybrids tested, massive phenotypic observations in nursery and field plots showed very low levels of SV (0.9% from 800,000 plants). Cytological analysis showed abnormal chromosome numbers (41-43, 45) in most of coffee somaclonal variants and normal numbers (44) in phenotypically normal plants. Stressful experimental conditions were also applied by using extended proliferation periods (4, 12 and 27 months) for three independent embryogenic lines established for the Caturra var. in presence of high growth regulator concentrations (4.5 µM 2,4-D, 17.8 µM 6-BA) to understand the mechanisms of culture ageing on SV. The proliferation time strongly affected the SV frequency among the 180 regenerated plants and in a highly similar way with the three embryogenic lines. No variant was found after 4 months proliferation although 30% and 94% phenotypic variants were observed in plants derived from 12 and 27 month-old cultures, respectively. Regardless the culture age and the embryogenic line, no polymorphisms were found in the 124 plants analyzed neither with AFLP nor with S-SAP using 13 different transposable elements from several families, and very limited polymorphisms were found in the methylation patterns using MSAP markers (0.049-0.087%). However, similarly to plants derived from under industrial conditions, phenotypic variants systematically showed abnormal chromosome numbers and normal plants systematically showed normal numbers. This work showed that SE based on embryogenic suspensions is reliable for true-to-type propagation of selected C. arabica varieties. It also demonstrated the importance of culture age on SV and hence the non random nature of this phenomenon. The genetic and epigenetic alterations are particularly limited during SE. The main change in most of phenotypic variants was aneuploidy showing that mitotic aberrations play a major role in SV in coffee. (Résumé d'auteur)...

Mots-clés : coffea arabica; micropropagation; embryon somatique; variation phénotypique; marqueur génétique; epigenetics [en]; multiplication végétative; régénération in vitro; recherche interdisciplinaire; cytologie; agronomie; biologie moléculaire; amélioration des plantes; Évaluation; variation somaclonale; clone; amérique centrale; nicaragua; brésil; colombie; guatemala; mexique; Épigénétique

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