Regenerative potential, metabolic profile, and genetic stability of Brachypodium distachyon embryogenic calli as affected by successive subcultures
- 427 Downloads
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).
Compliance with ethical standards
Conflicts of interest
The authors declare that they have no conflicts of interest.
- Borisjuk L, Neuberger T, Schwender J, Heinzel N, Sunderhaus S, Fuchs J, Hay JO, Tschiersch H, Braun HP, Denolf P, Lambert B, Jakob PM, Rolletschek H (2013) Seed architecture shapes embryo metabolism in oilseed rape. Plant Cell 25:1625–1640. https://doi.org/10.1105/tpc.113.111740 CrossRefPubMedPubMedCentralGoogle Scholar
- Bragg JN, Anderton A, Nieu R, Vogel JP (2015) Brachypodium distachyon. In: Wang K (ed) Agrobacterium protocols, 3rd edn. Springer, New York, pp 17–33Google Scholar
- Capron A, Chatfield S, Provart N, Berleth T (2009) Embryogenesis: pattern formation from a single cell (ed) Arabidopsis Book. American Society of Plant Biologists, Rockville, pp 1–28Google Scholar
- Cuadros-Inostroza A, Caldana C, Redestig H, Kusano M, Lisec J, Penã-Cortés H, Willmitzer L, Hannah MA (2009) Target search a bioconductor package for the efficient preprocessing of GC-MS metabolite profiling data. BMC Bioinforma 10:1–12. https://doi.org/10.1186/1471-2105-10-428 CrossRefGoogle Scholar
- De Verno LL (1995) An evaluation of somaclonal variation during somatic embryogenesis. In: Jain SM, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants, 1st edn. Kluwer Academic Publishers, Dordrecht, pp 361–377Google Scholar
- Domžalska L, Kędracka-Krok S, Jankowska U, Grzyb M, Sobczak M, Rybczyński JJ, Mikuła A (2017) Proteomic analysis of Cyathea delgadii Sternb. Stipe explants reveals differentially expressed proteins involved in fern somatic embryogenesis. Plant Sci 258:61–76. https://doi.org/10.1016/j.plantsci.2017.01.017. CrossRefPubMedGoogle Scholar
- Fras A, Maluszynska J (2004) The correlation between the chromosome variation in callus and genotype of explants of Arabidopsis thaliana. Genetica 121:145–154. https://doi.org/10.1023/B:GENE.0000040375.18684.82 CrossRefPubMedGoogle Scholar
- Ge X, Zhang C, Wang Q, Yang Z, Wang Y, Zhang X, WuZ HY, Wu J, Li F (2015) iTRAQ Protein profile differential analysis between somatic globular and cotyledonary embryos reveals stress, hormone, and respiration involved in increasing plantlet regeneration of Gossypium hirsutum L. J Proteome Res 14:268–278. https://doi.org/10.1021/pr500688g CrossRefPubMedGoogle Scholar
- Hashimoto T (2015) Microtubules in plants. Arabidopsis Book 13:e0179. https//doi.org/10.1199/tab.0179
- Igamberdiev AU, Bykova NV, Lea PJ, Gardeström P (2001) The role of photorespiration in redox and energy balance of photosynthetic plant cells: a study with a barley mutant deficient in glycine decarboxylase. Physiol Plant 111:427–438. https://doi.org/10.1034/j.1399-3054.2001.1110402.x CrossRefPubMedGoogle Scholar
- Jaikumar NS, Snapp SS, Sharkey TD (2016) Older Thinopyrum intermedium (Poaceae) plants exhibit superior photosynthetic tolerance to cold stress and greater increases in two photosynthetic enzymes under freezing stress compared with young plants. J Exp Bot 67:4743–4753. https://doi.org/10.1093/jxb/erw253 CrossRefPubMedPubMedCentralGoogle Scholar
- Karp A (1994) Origins, causes and uses of variation in plant tissue cultures. In: Vasil IK, Thorpe TA (eds) Plant cell tissue culture. Kluwer, Dordrecht, pp 139–151Google Scholar
- Kellogg EA (2015) Description of the family, vegetative morphology and anatomy. In: Kellogg EA (ed) The families and genera of vascular plants: flowering plants. Monocots, Poaceae. Springer, New York, pp 3–23Google Scholar
- Lee MB, Jeon WB, Kim DY, Bold O, Hong MJ, Lee YJ, Park JH, Seo YW (2011) Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21 with two binary vectors containing hygromycin resistance and GUS reporter genes. Crop Sci Biotechnol 14:233–238. https://doi.org/10.1007/s12892-011-0080-9 CrossRefGoogle Scholar
- Lema-Rumińska J, Śliwińska E (2015) Evaluation of the genetic stability of plants obtained via somatic embryogenesis in Chrysanthemum × grandiflorum. Acta Sci Pol. Hortorum 14:131–139Google Scholar
- Magnaval C, Noirot M, Verdeil JL, Blattes A, Huet C, Grosdemange F, Buffard-Mo J (1995) Free amino acid composition of coconut (Cocos nucifera L.) calli under somatic embryogenesis induction conditions. J Plant Physiol 146:155–161. https://doi.org/10.1016/S0176-1617(11)81982-6 CrossRefGoogle Scholar
- Mishiba K, Okamoto T, Mii M (2011) Increasing ploidy level in cell suspension cultures of Doritaenopsis by exogenous application of 2,4-dichlorophenoxyacetic acid. Physiol Plant 112:142–148. https://doi.org/10.1034/j.1399-3054.2001.1120119.x CrossRefGoogle Scholar
- Nadwodnik J, Lohaus G (2008) Subcellular concentrations of sugar alcohols and sugars in relation to phloem translocation in Plantago major, Plantago maritima, Prunus persica, and Apium graveolens. Planta 227:1079–1089. https://doi.org/10.1007/s00425-007-0682-0 CrossRefPubMedPubMedCentralGoogle Scholar
- Nascimento-Gavioli MCA, Cangahuala-Inocente GC, Steinmacher D, Ree JF, Steiner N, Guerra MP (2017) Physiological and biochemical features of embryogenic and non-embryogenic peach palm (Bactris gasipaes Kunth) cultures. In Vitro Cell Dev Biol Plant (1):1–8. https://doi.org/10.1007/s11627-017-9805-x
- Oliveira EJ, Koehler AD, Rocha DI, Vieira LM, Pinheiro MVM, Matos EM, Cruz ACF, Silva TCR, Tanaka FAO, Nogueira FTS, Otoni WC (2017) Morpho-histological, histochemical, and molecular evidences related to cellular reprogramming during somatic embryogenesis of the model grass Brachypodium distachyon.Protoplasma. 254:2017–2034. https://doi.org/10.1007/s00709-017-1089-9
- Otto FJ (1990) DAPI staining of fixed cells for highresolution flow cytometry of nuclear DNA. In: Darzynkiewiez Z, Crissman HA, Robinson JP (eds) Methods in cell biology. Academic, San Diego, pp 105–110Google Scholar
- Sharifi G, Ebrahimzadeh H, Ghareyazie B, Gharechahi J, Vatankhah E (2012) Identification of differentially accumulated proteins associated with embryogenic and non-embryogenic calli in saffron (Crocus sativus L.) Proteome Sci 10:3. https://doi.org/10.1186/1477-5956-10-3 CrossRefPubMedPubMedCentralGoogle Scholar
- Singh D (1981) The relative importance of characters affecting genetic divergence. Indian J Genet Plant Breed 41:237–245Google Scholar
- Trafford K, Haleux P, Henderson M, Parker M, Shirley NJ, Tucker MR, Fincher GB, Burton RA (2013) Grain development in Brachypodium and other grasses: possible interactions between cell expansion, starch deposition, and cell-wall synthesis. J Exp Bot 64:5033–5047. https://doi.org/10.1093/jxb/ert292 CrossRefPubMedGoogle Scholar
- Wójcikowska B, Jaskóła K, Gąsiorek P, Meus M, Nowak K, Gaj MD (2013) LEAFY COTYLEDON2 (LEC2) promotes embryogenic induction in somatic tissues of Arabidopsis, via YUCCA-mediated auxin biosynthesis. Planta 238(3):425–440. https://doi.org/10.1007/s00425-013-1892-2 CrossRefPubMedPubMedCentralGoogle Scholar
- Xu Z, Zhang C, Zhang X, Liu C, Wu Z, Yang Z, Zhou K, Yang X, Li F (2013) Transcriptome profiling reveals auxin and cytokinin regulating somatic embryogenesis in different sister lines of cotton cultivar CCRI24. J Int Plant Biol 55:631–642. https://doi.org/10.1111/jipb.12073
- Yordem BK, Conte SS, Ma JF, Yokosho K, Vasques KA, Gopalsamy SN, Walker EL (2011) Brachypodium distachyon as a new model system for understanding iron homeostasis in grasses: phylogenetic and expression analysis of Yellow Stripe-Like (YSL) transporters. Ann Bot 108:821–833. https://doi.org/10.1093/aob/mcr200 CrossRefPubMedPubMedCentralGoogle Scholar
- Zombori Z, Szécsényi M, Györgyey J (2011) Different approaches for Agrobacterium-mediated genetic transformation of Brachypodium distachyon, a new model plant for temperate grasses. Acta Biol Szeged 55:193–195Google Scholar