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From chromosome doubling to DNA sequence changes: outcomes of an improved in vitro procedure developed for allotriploid “Híbrido de Timor” (Coffea arabica L. × Coffea canephora Pierre ex A. Froehner)

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Abstract

Since 1966, chromosome doubling has been performed mainly in vitro, associating anti-tubulin treatment and different plant tissues showing proliferative cells. Despite the achieved improvements, some bottlenecks have been pointed out, such as the low rate of polyploids and high rate of mixoploid plantlets. To overcome these hurdles, some approaches have indicated that indirect somatic embryogenesis (ISE) constitutes an alternative trigger for chromosome doubling, especially for homoploid and anorthoploid germplasms. In this way, a guideline has been developed for hexaploidization of the Coffea line “Híbrido de Timor” (HT) ‘CIFC 4106’ (anorthoploid, 3x = 33 chromosomes, 1C = 2.10 pg, Coffea canephora × Coffea arabica) from friable embryogenic calli (FEC) treated with colchicine. From this, a relatively high percentage (49.3%) of HT hexaploids (6x = 66 chromosomes, 2C = 4.20 pg) was obtained, without recovery of mixoploids. Besides confirmation of endomitosis induction through the obtained hexaploids, SSR markers revealed that the FEC/colchicine strategy also resulted in loss of allelic diversity in 39 regenerated HT plantlets, demonstrating its genotoxic effect. Considering these results, the present procedure resolved the main bottlenecks for chromosome doubling, which have been reported since the discovery and isolation of the anti-tubulin colchicine in 1930. Hexaploid HT plantlets have enriched Coffea germplasm banks as a new genetic resource since the resolution of their karyotype and DNA sequence. Just as the true allotetraploid C. arabica and the allotriploid HT ‘CIFC 4106’, the hexaploid HT is relevant to investigate the genomic and phenotypic changes arising from polyploidization events.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

CIM:

Callogenesis induction medium

FCM:

Flow cytometry

FEC:

Friable embryogenic callus

FEC-CT:

Friable embryogenic callus—colchicine-treated

FEC-NCT:

Friable embryogenic callus—not colchicine-treated

GA3 :

Gibberellic acid

GM:

Germination medium

HT:

Híbrido de Timor

HT-ED:

Híbrido de Timor—explant donors

ISE:

Indirect somatic embryogenesis

RM:

Regeneration medium

SE:

Somatic embryos

SSR:

Simple sequence repeat

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Acknowledgements

We would like to thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, Brasília—DF, Brazil, Grants: 443801/2014-2), the Fundação de Amparo à Pesquisa do Espírito Santo (FAPES, Vitória—ES, Brazil, grant: 65942604/2014) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, Brasília—DF, Brazil) for financial support. We also thank Eveline Teixeira Caixeta (Embrapa Café, Empresa Brasileira de Pesquisa Agropecuária, BIOAGRO, Laboratório BioCafé, Universidade Federal de Viçosa, MG, Brazil) for providing leaves in 2011 of the “Híbrido de Timor” ‘CIFC 4106’ plants.

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Authors and Affiliations

  1. Laboratório de Citogenética e Cultura de Tecidos, Departamento de Biologia, Centro de Ciências Exatas, Naturais e da Saúde, Universidade Federal do Espírito Santo, Alegre, ES, 29500-000, Brazil

    Natália Arruda Sanglard, Paulo Marcos Amaral-Silva, Stéfanie Cristina de Oliveira & Wellington Ronildo Clarindo

  2. Laboratório de Citogenética e Citometria, Departamento de Biologia Geral, Centro de Ciências Biológicas e da Saúde, Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil

    Mariana Cansian Sattler & Carlos Roberto Carvalho

  3. Departamento de Engenharia Florestal, Centro de Ciências Agrárias, Universidade Federal de Viçosa, Viçosa, MG, 36570-000, Brazil

    Andrei Caíque Pires Nunes

  4. Laboratório de Bioquímica e Biologia Molecular, Departamento de Farmácia e Nutrição, Centro de Ciências Exatas, Naturais e da Saúde, Universidade Federal do Espírito Santo, Alegre, ES, 29500-000, Brazil

    Taís Cristina Bastos Soares

Authors
  1. Natália Arruda Sanglard
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  2. Paulo Marcos Amaral-Silva
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  3. Mariana Cansian Sattler
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  4. Stéfanie Cristina de Oliveira
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  5. Andrei Caíque Pires Nunes
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  6. Taís Cristina Bastos Soares
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  7. Carlos Roberto Carvalho
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  8. Wellington Ronildo Clarindo
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Contributions

The authors SNA, SMC and CWR conducted the tissue culture experiments and in vitro chromosome doubling. CWR and OSC carried out the cytogenetic analyses. A-SPM and STCB executed the SSR molecular analyses. CWR and CCR conducted the flow cytometry analysis. NACP did the statistical analysis. All authors equally contributed for manuscript editing and revision and approved the final manuscript for submission.

Corresponding author

Correspondence to Wellington Ronildo Clarindo.

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The authors declare that they have no conflict of interest.

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Communicated by Nokwanda Pearl Makunga.

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Sanglard, N.A., Amaral-Silva, P.M., Sattler, M.C. et al. From chromosome doubling to DNA sequence changes: outcomes of an improved in vitro procedure developed for allotriploid “Híbrido de Timor” (Coffea arabica L. × Coffea canephora Pierre ex A. Froehner). Plant Cell Tiss Organ Cult 131, 223–231 (2017). https://doi.org/10.1007/s11240-017-1278-4

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