Factors affecting white spruce somatic embryogenesis and embryo conversion

Bibliography: p. 192-212

Growth and Physiological Responses of Norway Spruce (Picea abies (L.) H. Karst) Supplemented with Monochromatic Red, Blue and Far-Red Light

Monochromatic red light (R) supplementation is more efficient than blue light (B) in promoting Norway spruce (Picea abies (L.) H. Karst) growth. Transcriptome analysis has revealed that R and B may regulate stem growth by regulating phytohormones and secondary metabolites; however, the effects of light qualities on physiological responses and related gene expression in Norway spruce require further study. In the present study, three-year-old Norway spruce seedlings received sunlight during the daytime were exposed to monochromatic B (460 mm), monochromatic R (660 nm), monochromatic far-red light (FR, 730 nm), and a combination of three monochromatic lights (control, R:FR:B = 7:1:1) using light-emitting diode (LED) lamps for 12 h after sunset for 90 day. Growth traits, physiological responses, and related gene expression were determined. The results showed that light quality significantly affected Norway spruce growth. The stem height, root collar diameter, and current-year shoot length of seedlings treated with R were 2%, 10% and 12% higher, respectively, than those of the control, whereas seedlings treated with B and FR showed significantly lower values of these parameters compared with that of the control. The net photosynthetic rate (Pn) of seedlings under R treatment was 10% higher than that of the control, whereas the Pn values of seedlings treated with FR and B were 22% and 33%, respectively, lower than that of the control. The ratio of phosphoenolpyruvate carboxylase to ribulose-1,5-bisphosphate carboxylase/oxygenase (PEPC/Rubisco) of seedlings after the R treatment (0.581) was the highest and 3.98 times higher than that of the seedlings treated with B. Light quality significantly affected the gibberellic acid (GAs) levels, which was 13% higher in seedlings treated with R (6.4 g/100 ng) than that of the control, whereas, the GAs level of seedlings treated with B and FR was 17% and 19% lower, respectively, than that of the control. In addition, seedlings treated with R achieved the lowest ratio of leaf chlorophyll content to fresh weight (8.7). Compared to the R and control treatments, seedlings received FR treatment had consistently lower values of the quantum yield of electron transport beyond Q(A)(-) (primary quinone, phi Eo) and efficiency, with which a trapped exciton moves an electron into the electron transport chain beyond Q(A)(-) (psi o), while higher values of the relatively variable fluorescence at the J step and normalized relatively variable fluorescence at the K step (W-k). The values of phi Eo, psi O, V-J and W-k in seedlings treated with B were similar to those in the control group. The expression of genes associated with light signal transduction, such as PHYTOCHROME C (PHYC), ELONGATED HYPOCOTYL5 (HY5), CONSTITUTIVE PHOTOMORPHOGENIC 1-2 (COP1-2), and PHYTOCHROME INTERACTING FACTOR 3 (PIF3), was significantly higher in seedlings under B treatment than those under other light treatments. Nevertheless, significant differences were not observed in the expression of COP1-2, HY5, and PIF3 between the R treatment and the control. The expression value of COP1-2 was significantly lower in R than FR light treatments. In conclusion, compared with the control, R promotes, whereas B and FR inhibit Norway spruce growth, which was accompanied by physiological changes and genes expression regulation that may be relate to a changing phytochrome photostationary state (PSS) with the supplemental R in seedlings

Comparison of endogenous cytokinins, ABA and metabolites during female cone bud differentiation in low and high cone-producing genotypes of lodgepole pine

In lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm.), cone bud initiation within long-shoot buds varies according to genotype. We chose to study hormone profiles of two genotypes that differed significantly in cone yield. Phytohormone profiles were established by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in multiple reaction monitoring (MRM) mode with samples collected from genotypes 299 and 233, the typically high and low cone producers. Generally, concentrations of trans-zeatin-O-glucoside were higher in genotype 299, whereas dihydrozeatin concentrations were higher in genotype 233. Both isopentenyl adenine and isopentenyl adenosine were present at higher concentrations in genotype 233. The ratio of total quantifiable zeatin (Z)-type cytokinins to isopentenyl (iP)-type cytokinins was approximate three-fold higher in genotype 299 during female cone bud differentiation. In genotype 299, ABA concentration was significantly lower than in genotype 233 on the first sampling date, while the phaseic acid concentration was lower consistently throughout the period investigated. Dihydrophaseic acid was present in low concentrations in most samples of genotype 233, but was not quantifiable in genotype 299. Our study reveals that long-shoot buds of the high cone-producing genotype had higher ratios of Z-type cytokinins to iP-type cytokinins than were found in the low cone-producing genotype. High cone-producing buds also contained less ABA, phaseic acid and dihydrophaseic acid during female cone bud differentiation.Peer reviewed: YesNRC publication: Ye

Plant Regeneration via Somatic Embryogenesis in Larix principis-rupprechtii Mayr

Prince Rupprecht’s larch (Larix principis-rupprechtii Mayr) is a native conifer in North China with great economic and ecological values. Somatic embryogenesis (SE) is a powerful tool for the mass clonal propagation in plants. In this study, we described a high-efficiency SE system via indirect pathways and investigated the effect of genotype, culture conditions and phytohormones on SE. Immature zygotic embryos (IZEs) of L. principis-rupprechtii Mayr were used as explant materials. In the induction stage, embryogenic tissues (ETs) were induced on mLV medium supplemented with 2.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 1.0 mg L−1 6-benzylaminopurine (6-BA). The initiation frequencies showed significant differences (p < 0.05) among 20 genotypes of open-pollinated mother trees with the highest induction frequency reaching 30%. For tissue proliferation, proliferation in liquid medium was more efficient compared with proliferation in semi-solid medium, providing a multiplication rate of 3.12 in an 8-day subculture period. As a necessary exogenous plant growth regulator (PGR) for somatic embryo maturation in conifers, abscisic acid (ABA) was optimized at 16 mg L−1 in this system. Next, an orthogonal test on osmotic pressure factors showed 50 g L−1 sucrose, 7 g L−1 phytagel and 75 g L−1 polyethylene glycol (PEG) was the optimal combination for somatic embryo maturation in L. principis-rupprechtii Mayr. Moreover, the dispersion culture method provided a more efficient somatic embryo maturation, up to 545 per gram of fresh weight (FW). Finally, 2 g L−1 of active charcoal (AC) was found to increase the somatic embryo germination rate to 63.46%. The improved protocol of SE will serve as a foundation for establishing mass propagation and genetic transformation of L. principis-rupprechtii Mayr

Analysis of Phytohormone profiles during male and female cone initiation and early differentiation in long-shoot buds of Lodgepole pine

In lodgepole pine (Pinus contorta Dougl. ex Loud. var. latifolia Engelm), cone initiation and gender differentiation are site-specific in long-shoot buds with female cones in the distal portion and male cones in the proximal portion. By using high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in multiple reaction monitoring (MRM) mode, cytokinins, indole-3-acetic acid (IAA), gibberellins (GAs), abscisic acid (ABA) and their selected metabolites, were investigated in developing long-shoot buds from multiple genotypes. Spatially, higher concentrations of trans-zeatin riboside (t-ZR) and dihydrozeatin (dhZR) existed in the distal parts of long-shoot buds, whereas concentrations of isopentenyl adenosine (iPA), IAA, GA24, ABA, ABA glucose ester (ABA-GE) and phaseic acid (PA) were higher in the proximal parts in all investigated genotypes. In long-shoot buds of genotypes with a history of high female cone yield, concentrations of t-ZR and the ratio of zeatin-type to isopentenyl-type cytokinins were higher in the entire buds, whereas dhZR or IAA was higher either in the distal or in the proximal part respectively. In low female cone yielding genotypes, concentrations of c-ZR, iPA, ABA-GE and PA were higher in both of the parts, whereas ABA was higher mainly in the distal part with higher GA24 in the proximal part. Temporally, concentrations of several hormone-related compounds showed obvious changes in late June and late July, prior to male and female cone bud differentiation. This study reveals that the local hormonal status in a long-shoot bud at specific developmental stages may play an important role in gender determination and cone yield.Peer reviewed: YesNRC publication: Ye

Cryo-Treatment Enhances the Embryogenicity of Mature Somatic Embryos via the lncRNA–miRNA–mRNA Network in White Spruce

In conifers, somatic embryogenesis is uniquely initiated from immature embryos in a narrow time window, which is considerably hindered by the difficulty to induce embryogenic tissue (ET) from other tissues, including mature somatic embryos. In this study, the embryogenic ability of newly induced ET and DNA methylation levels was detected, and whole-transcriptome sequencing analyses were carried out. The results showed that ultra-low temperature treatment significantly enhanced ET induction from mature somatic embryos, with the induction rate from 0.4% to 15.5%, but the underlying mechanisms remain unclear. The newly induced ET showed higher capability in generating mature embryos than the original ET. DNA methylation levels fluctuated during the ET induction process. Here, WGCNA analysis revealed that OPT4, TIP1-1, Chi I, GASA5, GST, LAX3, WRKY7, MYBS3, LRR-RLK, PBL7, and WIN1 genes are involved in stress response and auxin signal transduction. Through co-expression analysis, lncRNAs MSTRG.505746.1, MSTRG.1070680.1, and MSTRG.33602.1 might bind to pre-novel_miR_339 to promote the expression of WRKY7 genes for stress response; LAX3 could be protected by lncRNAs MSTRG.1070680.1 and MSTRG.33602.1 via serving as sponges for novel_miR_495 to initiate auxin signal transduction; lncRNAs MSTRG.505746.1, MSTRG.1070680.1, and MSTRG.33602.1 might serve as sponges for novel_miR_527 to enhance the expression of Chi I for early somatic embryo development. This study provides new insight into the area of stress-enhanced early somatic embryogenesis in conifers, which is also attributable to practical applications

Effects of stem girdling on cone yield and endogenous phytohormones and metabolites in developing long shoots of Douglas-fir (Pseudotsuga menziesii)

Stem-girdling treatments were applied in early spring to stimulate cone formation in two genotypes of interior Douglas-fir (Pseudotsuga menziesii var. glauca (Beissn.) Franco). After girdling treatments, male cone yield increased significantly in the next growing season. The increase was 14-fold in genotype 9137. In genotype 9550, more than 8,700 male cones were induced from each tree whereas no male cones were found in controls. Female cone yield was zero in controls and low for girdled trees in both genotypes. Multiple phytohormone-related compounds, including gibberellins (GAs), cytokinins, indole-3-acetic acid (IAA), abscisic acid (ABA) and their selected metabolites, were analyzed in developing long shoots after girdling treatments by high performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) in multiple reaction monitoring mode. Concentrations of GA4 were slightly higher at week 2 following girdling treatment, whereas at week 8 lower GA4 concentrations were found in girdled samples. Stem girdling did not affect concentrations of IAA and major cytokinins, such as zeatin riboside and isopentenyl adenosine. Concentrations of ABA differed two-fold between the genotypes. Although girdling treatment did not cause differences in ABA concentrations, it generally resulted in higher concentrations of ABA glucose ester. Concentration increase of 7'-hydroxy ABA by girdling was only found in genotype 9550 at week 8. Girdling caused little change in concentrations of phaseic acid in both genotypes.Peer reviewed: YesNRC publication: Ye

Involvement of 5mC DNA demethylation via 5-aza-2'-deoxycytidine in regulating gene expression during early somatic embryo development in white spruce (Picea glauca)

DNA methylation plays a crucial role in the development of somatic embryos (SEs) through the regulation of gene expression. To examine the impact of DNA methylation on gene expression during early SE development in Picea glauca, the demethylation reagent 5-aza-dC (5-aza-2′-deoxycytidine) was employed to modify DNA methylation regions and levels during the pre-maturation stage of somatic embryogenesis. The application of 2.0 µM 5-aza-dC did not induce toxicity to SEs in early development. Following treatment, the global DNA methylation level decreased significantly on the 7th day of pre-maturation and the 1st week of maturation. Methylated DNA immunoprecipitation (MeDIP) sequencing revealed that differentially methylated regions, as analyzed through Gene Ontology (GO), were related to plant development and reproduction and that they were hypomethylated on the 3rd day but hypermethylated on the 7th day in 5-aza-dC-treated embryogenic tissues. These findings indicate that 5-aza-dC treatment positively impacts early SE development, which was inhibited following 7 d of treatment. The expression of MSH7, JMJ14, and CalS10 was associated with DNA methylation, epigenetic regulation, and somatic embryogenesis. Further analysis of methylated regions revealed that the expression profiles of MSH7, JMJ14, and CalS10 were correlated with altered DNA methylation, suggesting DNA methylation at 5 mC may play a role in controlling the expression of these genes and regulating the early development of SEs in P. glauca. This study offers new insights into the regulation of somatic embryogenesis in conifers