Improving initiation, genotype capture, and family representation in somatic embryogenesis of Pinus radiata by a combination of zygotic embryo maturity, media, and explant preparation

Abstract

The principal aim of this investigation was to improve somatic embryogenesis initiation and to enhance representation of families and genotypes within those families of Pinus radiata D. Don. A total of 19 open-pollinated seed families, many with unrelated and weakly related parents, were tested. Optimum stage of cone maturity for initiation success was tested by five collections made at 1 week intervals, spanning the developmental period from pro-embryo to cotyledonary embryos. Two media were compared; embryo-development media (EDM6) and a modified Litvay medium (Glitz). Two zygotic embryo explant-preparation techniques were tested; embryos with retained megagametophytes and excised embryos. Proliferating embryogenic tissues were obtained from all four treatments (2850 explants per treatment, 570 per collection time) for the 19 families. The best initiation rates were achieved with a combination of Glitz medium with excised zygotic embryos, with 55% of explants from all collections and all families combined giving rise to proliferating embryogenic tissue. At the optimal collection time for each of the families, this treatment gave a range of 47%–97% initiation success with an average of 70% per family.

The effect of culture medium formulation on Pinus nigra somatic embryogenesis

“…The initiation frequencies obtained for Pinus nigra (maximum 10.3 and 10.4% ) in the present experiments are lower in comparison with initiation frequencies for Pinus radiata with 55% initiation frequency on average (Hargreaves et al 2009) or 20-53% for Pinus strobus (Klimaszewska et al 2001). The initiation of somatic embryogenesis in conifers is under strong genetic control as has been mentioned earlier in the text (Niskanen et al 2004).…”

Simple and efficient protocols for the initiation and proliferation of embryogenic tissue of Douglas-fir

“…We have successfully adapted recently developed protocols for the initiation of embryogenic tissue of P.radiata (Hargreaves et al 2009(Hargreaves et al , 2011 to Douglas-fir immature zygotic embryos, using simple techniques and media available in the public domain.…”

“…In most cases, methodologies and media from somatic embryogenesis initiation to somatic embryo (SE) germination were patented (Pullman and Gupta 1994;Gupta 1996;Gupta and Pullman 1996;Gupta et al 2004;Timmis et al 2011) protecting the use of these for commercial application and available only for research purposes. Recent progress with improving somatic embryogenesis initiation and early cell line proliferation in P. radiata that resulted in average initiation rates of 70% of the 19 open and 20 control-pollinated families tested were very encouraging (Hargreaves et al 2009(Hargreaves et al , 2011. Because of the success of these new protocols, we wanted to test their success in other commercially important species such as Douglas-fir.…”

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

“…2a). Using megagametophytes with immature embryos as explants is advantageous owing to the Immature embryos enclosed in megagametophytes have been used as explants in several conifer species as Abies alba (Gajdošová et al 1995;Krajňáková et al 2013a), Abies nordmanniana (Nörgaard and Krogstrup 1991), Juniperus communis (Helmersson and von Arnold 2009), Taxus brevifolia and T. cuspidata (Ewald et al 1995) but are typical for the species of the genus Pinus, as Pinus sylvestris (Aronen et al 2009), Pinus radiata (Montalbán et al 2012;Hargreaves et al 2009), Pinus pinaster (Humanez et al 2012), Pinus nigra (Salajova and Salaj 1992), Pinus palustris (Jones and van Staden 1995), Pinus halepensis (Montalbán et al 2013) Pinus oocarpa (Lara-Chavez et al 2011). Immature zygotic embryos excised from megagametophytes have been used in Picea glauca and Picea mariana (Hakman and Fowke 1987), Picea abies (Nagmani et al 1987), Picea sitchensis (Krogstrup et al1988).…”