Ploidy instability in long-term in vitro cultures of Coffea arabica L. monitored by flow cytometry

Abstract

Somatic embryogenesis of Coffea arabica L. has been mainly carried out in liquid medium for clonal and mass propagation of elite lines. This in vitro system involves suspension cultures of embryogenic aggregates, with high multiplication rate and unorganized growth. These characteristics are linked to the occurrence of somaclonal variation (SV), especially considering that cell aggregates are usually maintained for long periods in media supplemented with the synthetic auxin 2,4-dichlorophenoxyacetic acid. Because SV detection has been considered essential in in vitro tissue cultures, flow cytometry (FCM) was applied to verify ploidy instability in embryogenic cell aggregates of C. arabica, throughout successive subcultures. FCM allowed us to detect the occurrence of non-true-to-type aggregates in all samples collected after approximately 4 months in liquid medium. These aggregates showed octaploid and/or aneuploid cells, with DNA ploidy level being corroborated by chromosome counting. Considering this result, we recommend a limit of <4 months for true-to-type mass propagation of C. arabica cell aggregate suspensions. Besides, FCM was an important tool to detect SV at an early stage of tissue culture in this species, proving to be very useful for quality control in clonal propagation and in the introduction of somaclones to breeding programs.