Nurse tissue for embryo rescue: testing new conifer somatic embryogenesis methods in a F1 hybrid pine

Key message This is the first report of (1) somatic embryogenesis in the hybrids of Pinus attenuata with Pinus radiata var. cedrosensis and (2) use of Pinus radiata somatic embryogenic tissue as nurse tissue to facilitate initiation from immature hybrid pine zygotic embryos. Embryogenic cell lines were successfully initiated from first generation reciprocal hybrids of Pinus radiata var. cedrosensis and Pinus attenuata, using P. radiata protocols. This is also the first report of P. radiata somatic embryogenic tissue used as a nurse to facilitate initiation from excised zygotic embryos. Two media formulations were tested, Glitz and Glitz2. Both media are based on a modified Litvay medium. The medium Glitz differs from Glitz2 in absence of casein, an increase in l-glutamine concentration and addition of asparagine. Overall analyses of treatment effects showed the excised zygotic embryo treatments, with or without the nurse on Glitz medium, were significantly better for cell line establishment (47 and 45 %, respectively) than either of the megametophyte treatments or the Glitz2 excised embryo treatment (p <= 0.05). Total cell line establishment irrespective of hybrid cross combination, collection time and cultural treatment was 32 %. Establishment percentages within each of the reciprocal hybrids, five crosses of each, across all four collections and cultural treatments, were not significantly different despite significant differences in zygotic embryo development (p <= 0.05). From response to the initiation treatments tested in this research, P. attenuata appears to be a much better mother than P. radiata var. cedrosensis with 39 % of cell lines established compared to only 23 % from P. radiata var. cedrosensis. However, analyses of variance results did not indicate that this difference between the two hybrid types was significant, probably due to the large amount of variation among the individual crosses within each of the hybrid types.