Bottlenecks in Pinus radiata somatic embryogenesis: improving maturation and germination

Abstract

Commercial deployment of clonal trees via somatic embryogenesis (SE) could increase forest productivity over conventional tree breeding techniques. However, some technical advances need to be made to use SE in clonal forestry with Pinus radiata. For example, the conversion of embryonal mass (EM) into plants is at present a major bottleneck. For this reason, maturation experiments were carried out to determine the effect of the initial amount of EM, activated charcoal (AC) and the best combination of abscisic acid (ABA), sucrose and amino acid concentration in the maturation medium. Germination was evaluated on different media formulations with and without AC. When 100 mg of EM were suspended in liquid medium without AC, cotyledonary somatic embryos were obtained in all the maturation media tested. Maturation medium supplemented with 60 lM ABA, 6% sucrose, and embryo development medium amino acid mixture produced the highest number of cotyledonary somatic embryos, between 10 and 1,550 embryos per gram of EM fresh weight. Approximately half of the tested 25 lines produced more than 600 embryos per gFW. Embryo development was the best when somatic embryos were germinated in half strength modified Quoirin and Lepoivre medium supplemented with 2 g L -1 AC. This protocol simplified and improved SE maturation and germination due to the elimination of subcultures, the large number of somatic embryos obtained from a very low amount of EM, and the elimination of pre-germination treatments, resulting in a significant saving of cost and labor.

Enhancing initiation and proliferation in radiata pine (Pinus radiata D. Don) somatic embryogenesis through seed family screening, zygotic embryo staging and media adjustments

“…Furthermore, a certain degree of morphological organization in the proliferating ET must be present prior to maturation treatment, as maturation is not induced if the embryogenic heads of the early filamentous embryos are too small (Jalonen and Von Arnold 1991). In our experiments, the ECL proliferating on EDM medium matured into somatic embryos satisfactorily (Montalbán et al 2010).…”

The effect of culture medium formulation on Pinus nigra somatic embryogenesis

“…Out of six cell lines tested in two of them (E267, E306) the somatic embryo development was restricted to the precotyledonary stage. The cell line-dependent somatic embryo maturation is a well known phenomenon in Pinus nigra (Salajova and Salaj 2005) and other conifer species (Jones and van Staden 2001, El Meskaoui and Tremblay 2001, Montalbán et al 2010) and could be the determining factor for somatic seedlings regeneration (Ramarosandratana et al 2001). Recently, Robinson et al (2009) proposed a model predicting the regenerative capability of conifer embryogenic cultures by metabolomics.…”

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

“…The somatic embryo maturation is a complex developmental process characterised by internal histodifferentiation as well as morphological changes, the most apparent of them is appearance of cotyledons. The maturation is regulated by several factors; one of the most important is the genotype and therefore the maturation process is cell line dependent (Krajnaková et al 2009;Alvarez et al 2013;Fischerová et al 2008;Montalbán et al 2010;Vondráková et al 2014). Another important factor is the medium composition used for maturation.…”