Assessment of ploidy stability of the somatic embryogenesis process in Quercus suber L. using flow cytometry

Abstract

Flow cytometry analyses were used to verify the ploidy stability of Quercus suber L. somatic embryogenesis process. Leaf explants of two adult cork oak trees (QsG0 and QsG5) of the North of Portugal were inoculated on MS medium with 2,4-D and zeatin. After 3 months, calluses with embryogenic structures were isolated and transferred to fresh MS medium without growth regulators and somatic embryo evolution was followed. Morphologically normal somatic embryos (with two cotyledons) and abnormal somatic embryos (with one or three cotyledons) were used in this assay. Flow cytometry combined with propidium iodide staining was employed to estimate DNA ploidy levels and nuclear DNA content of somatic embryos and leaves from mother plants. No significant differences (P< or =0.05) were detected among embryos, and between the embryos and the mother plants. Also, after conversion of these embryos, no significant morphological differences were observed among the somatic embryo-derived plants. These results and further studies using converted plantlet leaves and embryogenic callus tissue indicate that embryo cultures and converted plantlets were stable with regard to ploidy level. As no major somaclonal variation was detected our primary goal of "true-to-type" propagation of cork oak using somatic embryogenesis was assured at this level. The estimation of the 2C nuclear DNA content for this species is similar to the previously obtained value.

Oak somatic and gametic embryos maturation is affected by charcoal and specific aminoacids mixture

“…By this approach, the cumbersome method of repeated backcrossings is achieved in one step. Problems of identification of the cellular origin and genotypic composition of anther-derived embryos were overcome through the use of molecular markers, i.e., enzymes and random amplified polymorphic DNA (RAPD; Bueno et al, 2000) and simple sequence repeats (SSR;Gomez et al, 2001), by ploidy level studies through flow cytometry (Bueno et al, 2003;Loureiro et al, 2005), and by cytological analysis of early microspore divisions (Pintos et al, 2005). All these methods corroborated the gametic origin and the genetic variability of the anther embryos from different haploid cells (microspores).…”

Somatic Embryogenesis and Cryopreservation in Forest Species: The Cork Oak Case Study

“…Loureiro et al (2005), using flow cytometry, found no ploidy or DNA content variations in cork oak embryogenic tissues or among somatic embryos ( Figure 5). …”

“…In particular, the currently available rates of success in cork oak plant regeneration by somatic embryogenesis Lopes et al, 2006;Loureiro et al, 2005;Pinto et al, 2001;2002) and in cork oak material cryopreservation (Fernandes et al, 2008;Fernandes, 2011) are highly encouraging for researchers and breeders to consider the integration of these strategies in breeding and conservation programs of this species (Figure 1). …”

“…Flow cytometry histograms of relative propidium iodide (PI) fluorescence intensity obtained after simultaneous analysis of nuclei isolated from Quercus suber and from the reference standard Glycine max (2C=2.50 pg DNA): a) leaves of the mother plant; b) somatic embryo. Histogram peak 1: nuclei at G 0 /G 1 phase of Q. suber; peak 2: nuclei at G 0 /G 1 phase of G. max, peak 3 nuclei at G 2 phase of Q. suber, peak 4 nuclei at G 2 phase of G. max (adapted from Loureiro et al, 2005).…”

“…Gross genetic variations of genomic origin affect mostly the number of chromosomes and ploidy level and can be detected by chromosome counting or flow cytometry (Loureiro et al, 2005). Chromosome mutations (e.g., inversion, translocation) and genic mutations are screened by molecular markers that detect DNA sequence modifications.…”