Biological characterization of young and aged embryogenic cultures of Pinus pinaster (Ait.)

Abstract

Pinus pinaster (Ait.) somatic embryogenesis (SE) has been developed during the last decade, and its application in tree improvement programs is underway. Nevertheless, a few more or less important problems still exist, which have an impact on the efficiency of specific SE stages. One phenomenon, which had been observed in embryogenic tissue (embryonal mass, EM) initiated from immature seed, has been the loss of the ability to produce mature somatic embryos after the tissue had been cultured for several months. In an attempt to get insight into the differences between young cultures of EM (3-mo-old since the first subculture) of P. pinaster that produced mature somatic embryos and the same lines of significantly increased age (18-mo-old, aged EM) that stopped producing mature somatic embryos, we analyzed in both types of materials the levels of endogenous hormones, polyamines, the global DNA methylation, and associated methylation patterns. In addition, we included in the analysis secondary EM induced from mature somatic embryos. The analysis showed that the two tested genotypes displayed inconsistent hormonal and polyamine profiles in EM cultures of a similar phenotype and that it might be difficult to attribute one specific profile to a specific culture phenotype among genotypes. Experiments were also undertaken to determine if the global DNA methylation and/or the resulting methylation pattern could be manipulated by treatment of the cultures with a hypomethylating drug 5-azacytidine (5-azaC). An aged EM was exposed to different concentrations and durations of 5-azaC, and its response in culture was established by fresh mass increases and somatic embryo maturation potential. All of the analyses are new in maritime pine, and thus, they provide the first data on the biochemistry of EM in this species related to embryogenic potential.

Analysis of genetic stability at SSR loci during somatic embryogenesis in maritime pine (Pinus pinaster)

“…In pine species, it is widely accepted that the maturation ability tends to decrease with ECL age. The same observation is true for P. pinaster (Klimaszewska et al 2009). It has been hypothesized that the reduced ability to regenerate from such embryogenic tissues is related to genetic instability.…”

Conifer somatic embryogenesis – an efficient plant regeneration system for theoretical studies and mass propagation

“…Some examples of mature somatic embryo yield (number) calculated per 1 g of fresh weight: 64 for Abies fraseri (Kim et al 2009), 36 for Abies cephalonica (Krajňáková et al 2009), 187 in Pinus pinaster (Alvarez et al 2013), 321 in Pinus sylvestris (Aronen et al 2009), 269 for Picea glauca (Kong and Yeung 1995), 65 for Picea mariana (Tremblay and Tremblay 1995), 300-960 for hybrid of Larix (Lelu et al 1994). An undesirable phenomenon -loss of ability to produce mature somatic embryos -has been observed in embryogenic tissues of many species (Klimaszewska et al 2009). This phenomenon can be avoid -at least partially -by cryopreservation and subsequent thawing of tissues.…”

Somatic Embryogenesis for More Effective Breeding and Deployment of Improved Varieties in Pinus spp.: Bottlenecks and Recent Advances

“…the cumulative proliferation time from (i) initiation to cryopreservation (4.2 months) and (ii) line regrowth from the cryopreserved stock to the maturation experiment. mDCR modified DCR medium from Gupta and Durzan (1985); mLV modified LV medium from Litvay et al (1985) 2006; Klimaszewska et al 2009;Humánez et al 2012). Biological parameters (dry weight, water content) and biochemical analyses (total protein and carbohydrate contents) showed that cotyledonary se harvested after 10 or 14 weeks did not differ markedly and thus confirmed that harvesting cotyledonary se after 12 weeks is appropriate (Morel et al 2014b).…”

“…As it is a potent cytokinin with a putative role in ageing and vigour in pine (Valdés et al 2003;Klimaszewska et al 2009), CPPU supplementation during the Fig. 19.2).…”