Nuclear rDNA instability inin vitro-generated plants is amplified after sexual reproduction with conspecific wild individuals

Abstract

Using micropropagation through tissue culture has become the most used approach worldwide for mass production for the conservation of endangered species. However, the screening of somaclonal variations generated using in vitro culture is usually restricted to the first generation of micropropagated plants, when they have not yet been released in the field. Accordingly, the fate of genetically modified regenerants after sexual reproduction is usually not assessed and changes in the genetic structures of species are unknown. In this work, we assess the cytogenetic stability of two rDNA gene families in the offspring of experimental crosses between accessions generated after in vitro culture and wild individuals of Cistus heterophyllus (Cistaceae). The cytogenetic rDNA profiles (45S rDNA, 5S rDNA) of 118 accessions including wild and in vitro micropropagated individuals and bi-directional artificial crosses between wild and in vitro-generated plants were assessed by fluorescence in situ hybridization (FISH) and Ag-NOR staining. Plants regenerated by micropropagation showed a lower size of the FISH signals in a 45S rDNA site, but this condition was not present in the wild accessions. Three new cytogenetic and cytological variants were present in 36% of the experimental progeny, involving the amplification of one additional 45S rDNA site and the presence of heteromorphic nucleoli. rDNA-based genomic instability was present after sexual reproduction between wild and in vitro-generated plants. The results of this study discourage the use of micropropagated materials for plant conservation unless comprehensive surveys of the genetic integrity and stability of the regenerants are performed after crossing between wild and micropropagated plants.

Dissimilar molecular and morphological patterns in an introgressed peripheral population of a sand dune species (Armeria pungens, Plumbaginaceae)

“…To explore if intragenomic polymorphisms for the ribosomal sequences (ITS and IGS) could be slowed down by the number and different genomic location of ribosomal loci, we determined the number of ribosomal loci, both 45S and 5S rDNA, through FISH analysis following the procedures described in Rosato et al (). We analysed seven samples of A. macrophylla from Camarinal, seven samples of A. pungens from Cape Trafalgar, as representative of the non‐introgressed populations, and one from distantly related and widespread A. maritima Willd.…”

Precise evaluation of tissue culture-induced variation during optimisation of in vitro regeneration regime in barley

“…Reduced DNA methylation was observed in triticale regenerants, and it continued decreasing for the next few generative cycles before slightly increasing (Machczyńska et al 2014a). Thus, tissue culture-induced variation at the molecular level can be transmitted to progeny (Rosato et al 2016), and several generative cycles may or may not stabilise it (Machczyńska et al 2014a;Orłowska et al 2016). Further studies are needed to determine whether regenerant variation can be controlled.…”

Obtención de material vegetal de reproducción de Cistus heterophyllus carthaginensis (Cistaceae), especie catalogada En Peligro de Extinción en la Comunidad Valenciana (España)

“…No obstante, recientemente, en colaboración con el equipo de Biología Molecular del Jardín Botánico de la Universitat València, se planteó comprobar si : 24-37 el material de C. heterophyllus producido in vitro podía exhibir mutaciones del ADN ribosomal nuclear (Rosato et al, 2016), como las ya detectadas en otras plantas -por ejemplo Limonium perplexum (véase Ibáñez, 2013)-que habían sido producidas mediante esta misma técnica.…”

“…carthaginensis indican que se han producido mutaciones (Rosato et al, 2016), siguiendo además un esquema muy parecido al observado en Limonium perplexum. Estas mutaciones o variaciones somaclonales, fueron trasmitidas a la descendencia mediante los cruzamientos realizados con plantas mutantes y también a la descendencia resultante de los cruzamientos con plantas no mutadas (Rosato et al, 2016). Por lo tanto, el empleo de ejemplares mutantes en acciones de translocación puede generar cambios en las estructuras genéticas de las poblaciones y las especies.…”