Predicting the regenerative capacity of conifer somatic embryogenic cultures by metabolomics Andrew R. Robinson 1, †, Rebecca Dauwe 1, †, Nicholas K. Ukrainetz 1 , Ian F. Cullis 1 , Rick White 2 and Shawn D. Mansfield 1, * 1 Department of Wood Science, University of British Columbia, Vancouver, BC, Canada 2 Department of Statistics, University of British Columbia, Vancouver, BC, Canada Received 23 June 2009; revised 28 August 2009; accepted 29 August 2009. *Correspondence (fax 604 822 9104; e-mail shawn.mansfield@ubc.ca) †These authors contributed equally to this work. Keywords: metabolite profiling, Loblolly Pine (Pinus taeda), somatic embryogenesis, gas chromatography– mass spectrometry, stepwise variable selection, Bayesian information criterion. Summary Somatic embryogenesis in gymnosperms is an effective approach to clonally propa- gating germplasm. However, embryogenic cultures frequently lose regenerative capacity. The interactions between metabolic composition, physiological state, geno- type and embryogenic capacity in Pinus taeda (loblolly pine) somatic embryogenic cultures were explored using metabolomics. A stepwise modelling procedure, using the Bayesian information criterion, generated a 47 metabolite predictive model that could explain culture productivity. The model performed extremely well in cross- validation, achieving a correlation coefficient of 0.98 between actual and predicted mature embryo production. The metabolic composition and structure of the model implied that variation in culture regenerative capacity was closely linked to the physiological transition of cultures from the proliferation phase to the maturation phase of development. The propensity of cultures to advance into this transition appears to relate to nutrient uptake and allocation in vivo, and to be associated with the tolerance and response of cultures to stress, during the proliferation phase. Introduction Coniferous tree species are of significant ecological and commercial importance, and have been the subjects of intensive breeding and reforestation efforts worldwide. However, as a consequence of inherently long life cycles, slow sexual maturation, and frequently unavoidable dilu- tion of desirable traits because of genetic segregation and gene flow, sexually based improvement systems for tree species are inefficient, costly and time-consuming. Somatic embryogenesis (SE) is a vegetative propagation technique based on in vitro culture of plant tissue that permits the clonal replication of unlimited copies of subject tree geno- types (Merkle and Dean, 2000; Stasolla et al., 2002). This technology has the added advantage of facilitating enhanced clonal testing and silviculture applications in spe- cies that have traditionally been recalcitrant to vegetative propagation (e.g. Pinus sylvestris). Recalcitrance against SE culture initiation and consis- tently poor or progressively diminishing regenerative capacity in established cultures are long-recognized phenomena in conifer species – particularly in pines (Becwar et al., 1990; Tautorus et al., 1991; Liao and Amerson, 1995a; Li and Huang, 1996; Li et al., 1997). A primary example of such recalcitrance in pine species is the inability or reduced ability of established, proliferating SE cultures supporting early-stage embryos to respond to standard maturation conditions, and develop viable cotyle- donary embryos (Liao and Amerson, 1995b; Li et al., 1998). Although recently there has been a considerable refinement of technology for the initiation and prolifera- tion of SE tissue and subsequent generation of mature somatic embryos in pines and other conifers (Li et al., 1998; Klimaszewska et al., 2001; Pullman et al., 2003, 2009; Stasolla and Yeung, 2003; Lipavska and Konradova, 2004; Lelu-Walter et al., 2006, 2008; MacKay et al., 2006; Park et al., 2006; Vales et al., 2007), SE cultures from many species continue to exhibit a high degree of variability that reduces the reliability and efficacy of viable embryo generation. The physiological basis of the com- mon developmental block in pine somatic embryo matura- tion remains poorly understood, and as such genotype-, ª 2009 The Authors 952 Journal compilation ª 2009 Blackwell Publishing Ltd Plant Biotechnology Journal (2009) 7, pp. 952–963 doi: 10.1111/j.1467-7652.2009.00456.x