RESUMEN
Holm oaks (Quercus ilex L.) can suffer severe infection by the oomycete Phytophthora cinnamomi Rands; the production of more tolerant plants is, therefore, required. Embryo formation is a key period in the establishment of epigenetic memory. Somatic embryos from three holm oak genotypes were elicited, either over 3 days or 60 days, with methyl-jasmonate, salicylic acid (SA), ß-aminobutyric acid (BABA), or benzothiadiazole (all at 50 µM and 100 µM), or 10% and 30% of a filtered oomycete extract (FILT10 and FILT30) to activate plant immune responses. The number of embryos produced and conversion rate under all conditions were recorded. Some elicited embryos were then exposed to P. cinnamomi in dual culture, and differential mycelial growth and the progression of necrosis were measured. The same was performed with the roots of germinated embryos. Within genotypes, significant differences were seen among the elicitation treatments in terms of both variables. Embryos and roots of 60-day BABA, SA, or FILT10 treatments inhibited mycelium growth. The 3-day BABA (either concentration) and 60-day FILT10 induced the greatest inhibition of necrosis. Mycelium and necrosis inhibition were compared with those of tolerant trees. Both inhibitions might be a defense response maintained after primed embryo germination, thus increasing the likelihood of tolerance to infection.
RESUMEN
The genetic and epigenetic stability (analysis of DNA methylation using MSAP markers) of mint (Mentha x piperita L.) apices was studied after each step of a cryopreservation protocol, by encapsulation-dehydration. The effect of the addition of an antioxidant (ascorbic acid) during one of the protocol steps was also evaluated. Eight-week old in vitro recovered shoots from apices after each step of the protocol were genetically stable when compared to control in vitro shoots, using RAPD and AFLP markers. The addition of ascorbic acid in the medium with the highest sucrose concentration did not improve recovery and did not have any effect on stability. Apices sampled immediately after each step showed increased epigenetic differences as the protocol advanced, compared to in vitro control apices, in particular related to de novo methylation events. However, after one-day in vitro recovery, methylation status was similar to control apices. To improve the quality of methylation data interpretation, a simple and fast method for MSAP markers analysis, based on R programming, has been developed which allows the statistical comparison of treatments to control samples and its graphical representation.
