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Somatic embryogenesis from in vitro anther culture of apomictic buffel grass genotypes and analysis of regenerated plants using flow cytometry

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Abstract

Buffel grass is a fodder grass that reproduces mainly via apomixis. Using in vitro tissue culture in apomictic species provides alternatives that can be applied in a breeding program. The aims of this work were to establish a protocol for in vitro generation of apomictic buffel grass genotypes using anthers as explants and to evaluate the genetic stability of regenerated plants via flow cytometry (FCM). Three genotypes were able to induce embryogenic calli in Murashige and Skoog (MS) medium supplemented with 6 mg/l of 2,4-dichlorophenoxyacetic acid. Seedling regeneration occurred in a MS medium supplemented with 0.5 mg/l napthaleneacetic acid + 1 mg/l 6-benzylaminopurin. Seedlings were derived from somatic embryos and the morphogenic process was induced using the somatic tissue of the stamens. Induction and regeneration efficiency depended on the genotype and was affected by date of tiller collection, different pretreatments or the interaction of these variables. FCM analyses in in vitro regenerated plants showed genetic instability in their nuclear DNA content. Plants with lower nuclear DNA content may indicate DNA aneuploids (8.6 %), whereas plants that had twice the value of nuclear DNA content (4.7 %) suggest in vitro polyploidization. This variation observed in apomictic genotypes provides an opportunity to obtain new variants, which may then be included as sources of genetic variability in buffel grass breeding programs.

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Abbreviations

FCM:

Flow cytometry

2,4-D:

2,4-Dichlorophenoxyacetic acid

NAA:

Napthaleneacetic acid

BAP:

6-Benzylaminopurin

MS:

Murashige and Skoog (1962) medium

BAC:

Beginning of anther culture

PEC:

Proportion of embryogenic calli

NRS:

Number of regenerated seedlings

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Acknowledgments

We are grateful to Jaroslav Dolezel for providing the plant DNA standards used in this work. We thank P. Crespo and P. Abadie for assistance with the FCM analysis and I. Teich for assistance with statistical analyses. This research was supported by the projects INTA-AEFP-PE No. 261821 and CONICET (PIP 112 201101 0031).

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Authors and Affiliations

  1. Instituto de Fisiología y Recursos Genéticos Vegetales (IFRGV), Centro de Investigaciones Agropecuarias (CIAP), Instituto Nacional de Tecnología Agropecuaria (INTA), Avenida 11 de setiembre 4755, C.P. X5020ICA, Córdoba, Argentina

    Edgardo Carloni, Andrea Ribotta, Eliana López Colomba, Sabrina Griffa, Mariana Quiroga, Exequiel Tommasino & Karina Grunberg

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  1. Edgardo Carloni
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  2. Andrea Ribotta
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  3. Eliana López Colomba
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Correspondence to Edgardo Carloni.

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Carloni, E., Ribotta, A., López Colomba, E. et al. Somatic embryogenesis from in vitro anther culture of apomictic buffel grass genotypes and analysis of regenerated plants using flow cytometry. Plant Cell Tiss Organ Cult 117, 311–322 (2014). https://doi.org/10.1007/s11240-014-0441-4

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