Douglas fir embryogenic tissue initiation

Abstract

Somatic embryogenesis (SE) is expected to play an important role in the future of US forests by providing increased productivity, sustainability, and uniformity. For broad scale implementation to occur, SE technology must work with a variety of genetically diverse trees. Douglas fir (Pseudotsuga menziesii (Mirb) Franco) is the dominant tree in the Pacific Northwest and has great economic and recreational value. We have developed a highly effective medium for initiation of embryogenic tissue of Douglas fir that contains ABA, biotin, brassinolide, folic acid, MES, pyruvic acid and can be used as a gelled medium or in a gelled-liquid medium overlay system. When tested with many high-value crosses over 2 years, initiation tests averaged initiation in the range of 40-57%. Additionally, a time-and labor-saving tetrazolium chloride embryo staining technique was developed to evaluate seed health and screen out seed sources likely to perform poorly in the initiation process.

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

“…Similar results (17-23% initiation rates) were achieved by Hong et al (1992). More successful work (40-57% initiation rates) followed, incorporating complex media supplements, dissection methods and ZE health evaluation (Pullman et al 2005(Pullman et al , 2009a. 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.…”

“…A modified Litvay medium was also recently used to initiate embryogenic lines in Douglas-fir (Kong and von Aderkas 2011), although this paper focussed on cryopreservation of immature SEs and did not report on the success of this medium for embryogenic tissue initiation. BM medium formulation has been successfully used by other researchers for Douglas-fir initiation in North America, initially named WTC (Pullman and Gupta 1994;Gupta 1996) and later BM 1 (Gupta et al 2004), and with slight alterations to the formulation, 121 (Pullman et al 2005(Pullman et al , 2009a. Somewhat surprisingly, BM 1 was unsuccessful with New Zealand plant material in our laboratory conditions, irrespective of ZE developmental stage.…”

“…This corresponds to ZEs at the precotyledonary to early cotyledonary stages. Traditionally, the optimal time reported for embryo selection in Douglas-fir is 4-6 weeks after fertilization, when the apical dome of a ZE begins to form, but before the first appearance of cotyledonary primordia (precotyledonary stage; Durzan and Gupta 1987; Gupta Pullman et al 2005Pullman et al , 2009a. Of course, it is possible that using ZEs that were less developed may have resulted in even higher initiation rates, but in 2012-2013 only one collection was made.…”

“…It should, however, be noted that the ZE dissection method employed in the present work was different than that of these authors, who used BM 1 medium with ZE suspensors still attached to megagametophytes. Interestingly, recent work by Pullman et al (2009a) indicated improved results on BM 1 medium if a range of supplements were added. These include abscisic acid, pyruvic acid and brassinolide.…”

“…This explant (ZE) and medium (Glitz) combination was found to be far superior for initiation of P. radiata embryogenic tissue, with an overall average initiation rate of 55% compared to the previous standard initiation protocol of 16% (Hargreaves et al 2009). The majority of published papers and patents reported the use of excised ZEs still attached to the megagametophytes as the best explant for initiation of embryogenic tissue of Douglas-fir Gupta et al 1994Gupta et al , 2004Pullman et al 1994Pullman et al , 2005Pullman et al , 2009a. Although none of these authors provided an explanation for why this is done, as opposed to removing the ZE completely, we assume that the belief is that the ZEs would receive growth regulators and/or nutrition from fluids accumulating in the corrosion cavity (Carman et al 2005) that will assist in the initiation of embryogenic tissue.…”