Embryogenic tissue (ET) is important for genetic modification and plant re-generation. The proliferation ability and vigor of ET are crucial for plant propagation via somatic embryogenesis. In this study, ET was induced from mature zygotic embryos in blue spruce (Picea pungens Engelm.). There were significant differences in ET induction between two provenances, i.e. 78.8 ± 12.5% and 62.50 ± 12.8% respectively. Effects of 2,4-Dichlorophenoxy acetic acid (2,4-D), 6-Benzyl amino-purine (6-BA) and/or sucrose on ET proliferation and somatic embryo (SE) maturation were further investigated with four cell lines. The highest ET proliferation rate reached 1473.7 ± 556.0% biweekly. Concentrations of 2,4-D or 6-BA applied at tissue proliferation stage impacted SE maturation among the cell lines, whereas sucrose showed less effects. The highest rate, 408 ± 230 mature SEs/g FW, was achieved in SE maturation cultures. This research demonstrated that the culture conditions, i.e. the specific concentrations of 2,4-D and BA, at ET proliferation stage affected not only ET growth, but also the quality of ET for SE maturation. This study revealed the necessity and benefit in developing both the general and the genotype-specific protocols for efficient production of mature SEs, or somatic plants in blue spruce.

Picea pungens (Engelm.), known for its blue-green needles, has become a likable ornamental species in northeast China since 2000. Nonetheless, a lack of propagation methods that can maintain genetic fidelity and develop seedlings at a large scale prevents the further expansion of the species. Somatic embryogenesis (SE), paired with cryopreservation technologies, may provide a valid alternative. Picea pungens SE is not new, but its practical application has been limited due to low efficiencies in SE initiation and maturation as well as a lack of effective cryopreservation technology. In this study, experiments were carried out to overcome the limitations by modifying culture media. For initiation, the efficiency was enhanced by adjusting concentrations of 2.4-dichlorophenoxy acetic acid (2,4-D), 6-benzyl amino–purine (6-BA) or sucrose supplemented to the induction medium. The concentrations of 4.0 mg/L 2,4-D, 2 mg/L 6-BA, and 5 to 10 g/L sucrose were found optimal in maximizing initiation efficiency. For maturation, the efficiency, expressed as the number of mature somatic embryos per gram of fresh mass cultured (E/gFM), varied greatly with the choices of the basal medium and concentration of abscisic acid (ABA) of the maturation medium. Based on our results, the judicial choices were using the DCR medium as the basal medium and 10 mg/L ABA. The maturation efficiency could also be improved by adjusting the maturation medium’s osmotic pressure by manipulating the concentrations of carbohydrate and Gelrite and culture density. While the maturation medium, using sucrose as carbohydrate source or supplemented with a low (<8 g/L) Gelrite concentration, facilitated maturation, optimal selections were truly genotype-dependent. Our results also suggest that, while the optimal culture density varied with genotype, in general it is needless to culture more than 100 mg embryogenesis tissues per dish (size: 10 × 1.5 cm). Based on this study, the optimum pretreatment for embryogenesis tissue cryopreservation was culturing the tissues on the proliferation medium with 0.4 mol/L sorbitol for 24 h, followed by treatment with 5% Dimethyl sulfoxide. This study significantly improved the initiation (achieved a frequency of 0.56) and embryo maturation efficiencies (achieved 1030 E/gFM) and established an effective preculturing protocol for cryopreservation (recovered 1354 E/gFM) for the species. The protocols developed here, paired with the available ones for other SE steps in the literature, form a well-refined SE technology intended for commercial application to Picea pungens.