Stimulation of somatic embryo growth and development in Picea spp. by polyethylene glycol

Abstract

Abstract:The objective of the presented study was to compare the effect of osmotic stress, induced by various concentrations of polyethylene glycol (PEG) MW 4000, on the development, maturation, and conversion of somatic embryos of Picea abies (model species) and P. omorika (endemic species) into somatic seedlings. Embryogenic tissue (ET) of P. abies (line A and B) and P. omorika were incubated on a maturation medium with 20 µM abscisic acid (ABA), 1 µM indole-3-butyric acid (IBA) and PEG 4000 at 0, 5, and 7.5%. After 5 weeks, the number of somatic embryos produced, and the number of cotyledonary somatic embryos per 1 gram of ET, was assessed. Cotyledonary somatic embryos were selected and transferred to a Margara germination medium for two months. Their growth, as measured by hypocotyl and radicle length, was evaluated after one month, and the somatic embryos were transferred to fresh medium. After the second month of incubation, the conversion frequency of germinated embryos into the seedlings was assessed. In general, ETs of both of the tested spruce species cultured on a maturation medium amended with 5% PEG 4000 exhibited a tendency to produce a higher total number of embryos, as well as a higher number of cotyledonary embryos. The B line of P. abies produced a significantly greater number of cotyledonary embryos, relative to the control (no PEG). PEG only stimulated hypocotyl growth in embryos of both lines (A and B) of P. abies during the germination stage. Somatic embryos of Picea omorika were characterized by poor growth of both hypocotyls and radicles, irrespective of PEG treatment. The addition of PEG 4000 to the maturation medium did not improve the conversion frequency of germinated somatic embryos into somatic seedlings in either spruce species after a month of incubation on Margara medium. The effect of PEG 4000 on somatic embryogenesis efficiency was more evident in the ET of the two P. abies lines, relative to the P. omorika ET line, during both the maturation and the germination stage. Results indicated that the P. omorika ET line was almost completely insensitive to all of the PEG concentrations tested.

Different Roles of Auxins in Somatic Embryogenesis Efficiency in Two Picea Species

“…High exogenous auxin levels in the maintenance medium interfere with the polar auxin gradient that is established during embryogenesis and prevent correct apical-basal embryo patterning [76][77][78]. Therefore, to enable further somatic embryo development in conifers, the PGRs initially added to the induction and proliferation media are replaced with mild auxin (indole-3-butyric acid (IBA)), abscisic acid (ABA), and polyethylene glycol (PEG) [74,[79][80][81]. The elimination of auxins and cytokinins is commonly used to improve the quality of somatic embryos in conifers [82].…”

“…In turn, Hu et al [85] demonstrated the possibility of obtaining mature somatic embryos of Cunninghamia lanceolata after using a low NAA concentration (1.08 µM) that was eight-times lower than that of the cytokinins used. Our previous studies revealed the possibility of generating mature somatic embryos of P. abies and P. omorika after the maintenance of ETs on a medium supplemented with picloram (9 µM) and BA (4.5 µM) [80]. Comparing the effects of 2,4-D, NAA and picloram used at the proliferation stage on the maturation stage in this study, we did not observe a statistically significant impact on the number of produced SESs or embryos at the cotyledonary stage in either spruce species.…”

Somatic Embryogenesis of Norway Spruce and Scots Pine: Possibility of Application in Modern Forestry

“…It was also proven that high-molecular-weight compounds such as polyethylene glycol (PEG) 4000 had a positive effect on somatic embryo quality and increased the number of mature spruce embryos [80,82,83]. However, their impact on post-maturation development is still unclear, and further investigations are needed [80,84]. An alternative to osmotic stress treatment during the maturation stage may be reduced glutathione (GSH) application to the medium [85] or low-temperature treatment [86,87].…”