Regenerative potential, metabolic profile, and genetic stability of Brachypodium distachyon embryogenic calli as affected by successive subcultures
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Brachypodium distachyon, a model species for forage grasses and cereal crops, has been used in studies seeking improved biomass production and increased crop yield for biofuel production purposes. Somatic embryogenesis (SE) is the morphogenetic pathway that supports in vitro regeneration of such species. However, there are gaps in terms of studies on the metabolic profile and genetic stability along successive subcultures. The physiological variables and the metabolic profile of embryogenic callus (EC) and embryogenic structures (ES) from successive subcultures (30, 60, 90, 120, 150, 180, 210, 240, and 360-day-old subcultures) were analyzed. Canonical discriminant analysis separated EC into three groups: 60, 90, and 120 to 240 days. EC with 60 and 90 days showed the highest regenerative potential. EC grown for 90 days and submitted to SE induction in 2 mg L−1 of kinetin-supplemented medium was the highest ES producer. The metabolite profiles of non-embryogenic callus (NEC), EC, and ES submitted to principal component analysis (PCA) separated into two groups: 30 to 240- and 360-day-old calli. The most abundant metabolites for these groups were malonic acid, tryptophan, asparagine, and erythrose. PCA of ES also separated ages into groups and ranked 60- and 90-day-old calli as the best for use due to their high levels of various metabolites. The key metabolites that distinguished the ES groups were galactinol, oxaloacetate, tryptophan, and valine. In addition, significant secondary metabolites (e.g., caffeoylquinic, cinnamic, and ferulic acids) were important in the EC phase. Ferulic, cinnamic, and phenylacetic acids marked the decreases in the regenerative capacity of ES in B. distachyon. Decreased accumulations of the amino acids aspartic acid, asparagine, tryptophan, and glycine characterized NEC, suggesting that these metabolites are indispensable for the embryogenic competence in B. distachyon. The genetic stability of the regenerated plants was evaluated by flow cytometry, showing that ploidy instability in regenerated plants from B. distachyon calli is not correlated with callus age. Taken together, our data indicated that the loss of regenerative capacity in B. distachyon EC occurs after 120 days of subcultures, demonstrating that the use of EC can be extended to 90 days.
KeywordsFlow cytometry Metabolomics Ploidy Somatic embryogenesis
The authors are grateful to the Núcleo de Análise de Biomoléculas of the Universidade Federal de Viçosa for providing the facilities for the metabolite analysis. Caio G. Otoni and Ross Thomas are also acknowledged for the English revision.
T.C.M.-R. and E.M.M. raised the in vitro plants for the experiments and performed the experiments; T.C.M.R., N.M.V., D.F., and A.N.N. performed metabolite profiling analyses. C.D.C., D.S.B., and T.C.M-R. performed statistical analysis. E.M.M and L.F.V. performed flow cytometric analysis. T.C.M.-R., D.S.B., C.D.C., F.T.S.N., L.F.V., and W.C.O. contributed to the design and interpretation of the research and to the writing of the paper.
This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Brasília, DF, Brazil) and Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) (Belo Horizonte, MG, Brazil). T.C.M.-R. was recipient of a scholarship from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Brasília, DF, Brazil).
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Conflicts of interest
The authors declare that they have no conflicts of interest.
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