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. 2016 Apr 6;36(5):548–561. doi: 10.1093/treephys/tpw016

Norway spruce embryogenesis: changes in carbohydrate profile, structural development and response to polyethylene glycol

Lukáš Hudec 1, Hana Konrádová 1, Anna Hašková 1, Helena Lipavská 1,2
Editor: Janice Cooke
PMCID: PMC4886291  PMID: 27052433

Abstract

Two unrelated, geographically distinct, highly embryogenic lines of Norway spruce (Picea abies (L.) Karst.) were analysed to identify metabolic traits characteristic for lines with good yields of high-quality embryos. The results were compared with corresponding characteristics of a poorly productive line (low embryo yield, scarce high-quality embryos). The following carbohydrate profiles and spectra during maturation, desiccation and germination were identified as promising characteristics for line evaluation: a gradual decrease in total soluble carbohydrates with an increasing sucrose : hexose ratio during maturation; accumulation of raffinose family oligosaccharides resulting from desiccation and their rapid degradation at the start of germination; and a decrease in sucrose, increase in hexoses and the appearance of pinitol with proceeding germination. We propose that any deviation from this profile in an embryonic line is a symptom of inferior somatic embryo development. We further propose that a fatty acid spectrum dominated by linoleic acid (18 : 2) was a common feature of healthy spruce somatic embryos, although it was quite different from zygotic embryos mainly containing oleic acid (18 : 1). The responses of the lines to osmotic stress were evaluated based on comparison of control (without osmoticum) and polyethylene glycol (PEG)-exposed (PEG 4000) variants. Although genetically distinct, both highly embryogenic lines responded in a very similar manner, with the only difference being sensitivity to high concentrations of PEG. At an optimum PEG concentration (3.75 and 5%), which was line specific, negative effects of PEG on embryo germination were compensated for by a higher maturation efficiency so that the application of PEG at an appropriate concentration improved the yield of healthy germinants per gram of initial embryonal mass and accelerated the process. Polyethylene glycol application, however, resulted in no improvement of the poorly productive line.

Keywords: anatomy, desiccation, fatty acid composition, lipids, maturation, osmotic stress, Picea abies, pinitol, raffinose family oligosaccharides, saccharides, somatic embryogenesis

Introduction

In conifers, somatic embryogenesis (SE) has become a valuable supplement to conventional plant propagation and breeding (Gupta and Grob 1995, Park 2002, Lelu-Walter et al. 2013). Factors currently limiting commercialization of SE include low initiation efficiency in recalcitrant genotypes, low culture survival, culture decline causing low or no embryo production, or maturation irregularities resulting in low germination and slow somatic seedling growth (Pullman and Buchanan 2008, Bonga et al. 2010). Therefore, widespread use of the technique depends on our knowledge of conditions controlling the process and on the ability to distinguish lines that will produce large numbers of somatic trees.

In spruce, considerable progress has been made in the development of protocols for initiation and maintenance of embryogenic cultures, embryo maturation, germination and conversion into plantlets (Bonga et al. 2010, Elhiti and Stasolla 2012). Maturation is the critical phase for early somatic embryos, which must achieve the physiological state necessary for successful entry into subsequent phases. This includes appropriate phytohormone levels, accumulation of storage compounds and development of desiccation tolerance, underlined by respective changes in gene expression (Stasolla et al. 2002, 2003). Although somatic embryo development is more variable than zygotic development, both embryo types exhibit substantial anatomical and physiological resemblances (Yeung 1995, Pullman and Buchanan 2008). The important differences have been reported in detail for Picea glauca (Fowke et al. 1994): zygotic embryos nourished by the surrounding gametophyte tissue were smaller compared with somatic embryos, and additional differences were found in size and arrangement of cotyledons and shape of the apical meristem. In several conifers, differences in biochemical characteristics were also described in the level of storage compounds (Klimaszewska et al. 2004, Grigová et al. 2007). These traits might have a significant impact on embryo behaviour during germination. It is worth considering the distinct characteristics of somatic embryos as natural adaptations to entirely specific conditions in the immediate vicinity of the embryo, and therefore not necessarily as signs of inferior somatic embryo development.

Several treatments have been reported to improve the quality of mature somatic embryos and increase mature embryo yields. Exposure to osmotic stress has been shown to affect the process (Attree and Fowke 1993, Stasolla et al. 2002), and osmotic stress in vitro mimics the natural decrease in water availability within conifer seeds at the end of embryo maturation (Attree et al. 1995). The technology of SE often includes desiccation as a step promoting switch from maturation to germination (Attree and Fowke 1993, Gupta and Grob 1995, Stasolla and Yeung 2003). Alternatives to desiccation have been proposed, for example, low temperature treatment (Konrádová et al. 2003, Pond 2005) or reduced glutathione (Stasolla et al. 2004). Osmotic stress during maturation has been shown to be beneficial for further embryo development (von Arnold et al. 2002, Maruyama and Hosoi 2012). A considerable osmotic effect was provided by saccharides, primarily as medium supplements for carbon and energy (Tremblay and Tremblay 1995, Tereso et al. 2007). In Picea mariana and Picea rubens, embryo yields increased with increasing sucrose concentrations. The replacement of a proportion of sucrose with polyols did not reduce embryo number, implying that sucrose acted as an osmoticum. However, high sucrose concentrations (9–12%) had negative effects on embryo maturation (Tremblay and Tremblay 1991). In P. glauca × engelmannii, 6% mannitol doubled the yield of precotyledonary embryos, but inhibited embryo transition to the cotyledonary stage. An increase in mannitol content in the medium (13–20%) was necessary to prevent precocious germination (Roberts 1991). Despite some supportive effects on embryo maturation, low molecular weight osmotica were not optimal as they gradually penetrated into the cells and subsequently lowered the cell's intrinsic osmotic potential, resulting in a lessening of osmotic stress. Therefore, the stress imposed was only temporary and interference with cellular metabolism cannot be avoided (Attree and Fowke 1993).

High-molecular-weight compounds, such as polyethylene glycol (PEG) 4000 or dextrans, do not enter protoplasts and, thus, sustain constant low water availability for the culture, closely mimicking conditions experienced by seed-resident embryos (Attree and Fowke 1993, von Arnold et al. 2002). Attree et al. (1995) showed for P. glauca that only embryos maturing in the presence of PEG 4000 (optimum concentration 7.5%) or dextrans possessed enough storage compounds, survived desiccation and could be stored at –20 °C for 1 year. In the presence of 7.5% PEG, a ninefold increase in storage lipids was recorded in P. glauca somatic embryos (Attree et al. 1992), and the complete spectrum of storage proteins was achieved only as a result of PEG treatment at an optimum concentration of 7.5% (Misra et al. 1993).

Discussion still continues about the overall benefits of PEG. There is a general consensus that PEG is able to improve maturation and increase the number of mature embryos (Attree et al. 1995, Kong and Yeung 1995, Find 1997, Belmonte et al. 2005, Maruyama and Hosoi 2012). However, the impact on embryo quality and consequently on post-maturation development remains obscure. Evidence exists that PEG can have negative effects on root protrusion and post-germinative growth of conifer somatic embryos. Mainly structural alterations in the subapical region, the hypocotyl or the root cap and the collapse of the root apical meristem quiescent centre have occurred (Kong and Yeung 1995, Find 1997, Tereso et al. 2007). In P. glauca, Stasolla et al. (2003) documented PEG-induced positive changes in gene expression connected with formation of the embryo body plan (ZWILLE or SCARECROW homologues), and the transcription pattern of genes involved in carbohydrate and nitrogen metabolism denoted the character of the mature embryo as a storage sink, especially in the presence of PEG. A microarray study of Norway spruce revealed that several stress response genes were up-regulated during the transition from embryo differentiation to late embryogenesis (Vestman et al. 2011). The effect of osmotic stress caused by PEG is still far from clear and further investigations are needed.

Previously, using the Picea abies (L.) Karst. embryogenic line AFO541, we found that certain features of maturing embryos were either positively or negatively influenced by PEG, depending on the concentration. For example at 7.5% PEG, ruptures and rows of dead cells appeared within hypocotyls, whereas no damage was observed at 3.75% PEG (Svobodová et al. 1999). Similarly, carbohydrate metabolism was significantly unbalanced at 7.5% PEG, but not at half the concentration (Lipavská et al. 2000). At 3.75 and 5% PEG, the characteristics analysed most closely resembled those found in zygotic embryos (Gösslová et al. 2001), from which we concluded that 3.75% PEG was beneficial for this particular line.

Compilation of a list of traits that are closely linked to regular somatic embryo development is important for elaboration of knowledge on the process in the basic research. This can only be achieved if structural, physiological, biochemical and molecular markers of regular SE are identified. Analyses performed with the Norway spruce embryogenic line AFO541 provided basic information regarding selected anatomical and biochemical features of embryo maturation under PEG-induced osmotic stress (Svobodová et al. 1999, Lipavská et al. 2000, Konrádová et al. 2002, Grigová et al. 2007). However, it remains unclear whether the results are of general value or were specific only for that genotype.

In this study, we examined and compared two unrelated embryogenic lines sharing essential macroscopic traits, with the ultimate aim of searching for common features (structural characteristics and patterns of carbohydrate and fatty acids (FAs)) that can serve as potential markers of high-yield embryogenesis in spruce. The results were compared with selected characteristics of one poorly productive line (low embryo yield, scarce high-quality embryos). In addition, we assessed the response of the lines to different levels of PEG-induced osmotic stress during maturation, and correlated the results with post-maturation development in order to estimate the overall impact of the treatment on somatic plant production.

Materials and methods

Plant material

Three embryogenic lines of Norway spruce (P. abies) were studied: C110 and C107 were initiated from immature zygotic embryos (IEB Czech Academy of Sciences; Dr Martin Vágner from cones collected from the arboretum of FGMRI in Kostelec nad Černými Lesy (Czech Republic, 50°0'N, 14°51'E) in 1998) and AFO541 (obtained from Dr Bercêtche, AFOCEL, Nangis, France; the line was initiated from zygotic seedlings (Ruaud et al. 1992), the seeds were collected in the region of Gerardmer, Vosges, France, 48°05'N, 6°50'E, season 1990–91).

Somatic embryogenesis initiation

For all three lines, Gupta and Durzan (GD) medium (Gupta and Durzan 1986) containing 3% (w/v) sucrose (Lachema, Brno, Czech Republic), 0.75% (w/v) agar (grade ‘plant cell culture tested’ cat. no. A7921), 5 µM 2,4-dichlorophenoxyacetic acid, 2 µM kinetin and 2 µM benzyladenine (all Sigma, St. Louis, MO, USA) were used. Cultivation was performed in Magenta vessels (type GA-7-3, Magenta™ Corp., Chicago, IL, USA). Mature zygotic embryos for lipid analysis were isolated from cones collected in north-eastern Moravia (Beskydy Mountains, 49°30'N, 18°32'E), in January 2002, and stored at −80 ± 1 °C until extraction.

Culture maintenance

Embryonal-suspensor mass (ESM) was maintained by weekly sub-cultures on medium of the same composition as during initiation. The cultures were maintained in the dark, at 25 ± 0.5 °C.

Maturation

Nine pieces (∼200 mg each) of ESM (1 week after transfer to fresh medium) were transferred to Magenta vessels with maturation GD medium supplemented with 3% (w/v) sucrose, 0.75% (w/v) agar and 20 µM abscisic acid (ABA) (Sigma). Abscisic acid was filter-sterilized and added after autoclaving (121 °C, 0.144 MPa, 20 min). The cultures were sub-cultured weekly and maintained in the dark at 25 ± 0.5 °C. The effects of 3.75, 5 or 7.5% (w/v) PEG 4000 (Sigma) were examined. Polyethylene glycol was added to complete maturation medium composition and was present during the whole maturation period.

Desiccation

Cotyledonary embryos (after 6 and 5 weeks of maturation for control and PEG-treated cultures, respectively) were exposed to high relative humidity for 3 weeks. The embryos (∼40 embryos) were placed into small open Petri dishes (diameter 5 cm) with dry filter paper, and these were then placed in a larger closed Petri dish (diameter 12 cm) containing three layers of filter paper moistened with 2 ml of sterile distilled water. The larger Petri dishes were sealed with two wraps of parafilm. Desiccation was at 20 ± 0.5 °C and 16 h photoperiod (irradiance 400 µmol m−2 s−1).

Germination

Embryos were germinated on GD medium supplemented with 1% (w/v) sucrose, 0.8% (w/v) agar and 0.4% (w/v) charcoal (grade ‘suitable for plant cell culture’, Sigma, cat. no. C9157), without plant growth regulators. Germination occurred at 25 °C and 16 h photoperiod. According to their morphologies, germinated embryos were sorted into two categories (Figure 1): Class 1 were well-developed green germinants with elongated hypocotyls and cotyledons, apical buds and growing roots, or germinants with slightly delayed development of either the root or shoot, and Class 2 were embryos with severely abnormal germination or without sign of germination.

Figure 1.

Figure 1.

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Categories of P. abies germinants used for determining germinating somatic embryo quality after 3 weeks of germination. Class 1: well-developed green germinants with elongated hypocotyls and cotyledons, terminal buds and roots having protruded or embryos with slightly delayed development of either roots or shoot poles. Class 2: embryos with severely irregular germination or complete abortion of germination.

Saccharide content and spectrum determination

The procedure of Lipavská et al. (2000) was used for soluble saccharide extraction and determination. Samples of ESM (∼200 mg fresh weight), or isolated cotyledonary embryos (∼70 mg fresh weight), were freeze-dried and dry weight was determined. The material was boiled with 80% (v/v) methanol (0.5 ml) at 75 °C for 15 min, the solvent was vacuum-evaporated and the residue was resuspended in an appropriate amount of Milli-Q ultrapure water (Millipore). The samples were analysed using high-performance liquid chromatography with refractometric detection and an IEX Pb2+ form column (Watrex, Praha, Czech Republic), mobile phase: Milli-Q water and flow rate: 0.5 ml min−1.

Anatomical examination

Embryonal-suspensor mass squashes were stained with trypan blue (O'Brien and McCully 1981). The double-staining procedure for median longitudinal paraffin sections of embryos (12 µm) with alcian blue and nuclear fast red (Poláčková and Beneš 1975) was used. This allowed identification of meristematic areas by their typical meristematic cell morphology, and also by the intensive red colouring of chromatin in active nuclei. Alcian blue counterstains acidic mucopolysaccharides and glycoproteins within cell walls. Anatomical specimens were evaluated using a light microscope.

Lipid analysis

Samples (minimum dry mass of 30 mg) were mixed with 0.5 ml 1 : 1 (v/v) methanol : chloroform, vortexed and filtered. The extraction was repeated three times and the extracts were combined. Fatty acid methyl esters in the lipid extracts were separated on an AHP 5995 gas chromatograph system (Agilent, Santa Clara, CA, USA). The methyl esters were prepared according to Metcalfe and Wang (1981). The samples were injected onto a 25 m × 0.25 mm × 0.1 m Ultra-1 capillary column (Supelco, Bellefonte, PA, USA) (injector and detector temperatures of 300 °C) under a temperature programme of 5 min at 50 °C, increasing at 10 °C min–1 to 320 °C, and 15 min at 320 °C. Helium carrier gas was at a flow of 0.52 ml min–1. The methyl esters were identified by comparison of retention times with those of standards (kit RM-7, Supelco).

Experimental design and statistics

Samples were collected at the end of 1-week sub-cultivation interval, and 5–15 samples of whole ESM (proliferation phase and earlier weeks of maturation when embryo excision was not possible) and 5–6 samples of excised embryos (10–30 embryos per sample, depending on the embryo developmental stage) were examined. Using NCSS 9 statistical software (NCSS, LCC Kaysville, UT, USA), the data obtained were checked for normal distribution, and analysed by one-way analysis of variance (ANOVA) and by Fisher's least significant difference multiple comparison test (for normally distributed data) or the Kruskal–Wallis multiple comparison z-value test (if data were not distributed normally). Differences were examined at α ≤ 0.05 level.

Results

Two P. abies embryogenic lines (C110 and AFO541) of different ages and origins (primary explant, provenance) were tested for responses to PEG (0, 3.75, 5 and 7.5%) applied during maturation. Structural development, FA spectra and carbohydrate level during maturation, and carbohydrate content and spectra during desiccation and germination were followed, together with characteristics of process efficiency. The lines were responsive to ABA and provided high yields of viable, morphologically similar embryos. The data were compared with corresponding characteristics of a poorly productive embryogenic line (C107).

Sequence of embryo structural development

A detailed study of anatomical characteristics of AFO541 somatic embryos without PEG treatment and under PEG exposure was reported previously (Svobodová et al. 1999). The developmental pattern proved to be very stable over time and in subsequent experiments (Lipavská et al. 2000, Konrádová et al. 2002, Kubeš et al. 2014). Comparison with C110 embryos revealed similar characteristics regarding typical anatomical traits at particular stages (Figure 2a–c): early somatic embryo (meristematic dome, suspensor), cylindrical embryo (protoderm formed), precotyledonary embryo (apical meristem, root cap and procambium initiated) and cotyledonary embryo (all embryonal organs established, histo-differentiation completed). However, timing (the onset of a particular developmental stage) differed slightly between the highly embryogenic lines, with C110 somatic embryos developing faster. In C110, the final cotyledonary stage appeared after 4 weeks of maturation (Table 1), while in AFO541, it was 1 week later. In C107, the developmental sequence was very similar to C110, but the yields were at least one order of magnitude lower (∼3.7 ± 3.3 cotyledonary embryos per 1 g of initial fresh weight of proliferating ESM) with a large proportion of embryos exhibiting structural abnormalities at the microscopic level (Figure 2h and i). For comparison, the highly responsive lines AFO541 and C110 provided per 1 g of initial fresh weight of ESM in average 116 ± 36 and 75 ± 30 embryos, respectively.

Figure 2.

Figure 2.

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Anatomical structure of maturing P. abies somatic embryos, line C110 (a–g) and line C107 (h and i). Line C110: (a–c) 0% PEG. (a) Early somatic embryo (Week 0). (b) Precotyledonary somatic embryo (Week 3). (c) Cotyledonary somatic embryo (Week 6). (d–g) Root cap structure under different PEG concentrations, 1 week after the onset of cotyledonary stage. (d) 0% PEG. (e) 3.75% PEG. (f) 5% PEG. (g) 7.5% PEG. Arrows—disintegration of the root cap tissue. Line C107 (0% PEG): (h) precotyledonary somatic embryo (Week 3). Arrow—irregularity of protodermis formation. (i) Cotyledonary somatic embryo (Week 6). Bar represents 500 µm (a, b, c and i) or 250 µm (d–h). Staining—trypan blue (a), alcian blue and nuclear fast red (b–i).

Table 1.

Effect of PEG exposure on the onset of developmental stages during P. abies somatic embryo maturation, lines C110 and AFO541. Semi-quantitative microscopic evaluation of prevailing (75% and more) developmental stages in a given week. ESE, early somatic embryo; Cy, cylindrical stage; Pc, precotyledonary stage; Co, cotyledonary stage—highlighted by shading.

Week PEG 0%
 PEG 3.75%
 PEG 5%
 PEG 7.5%
C110 AFO541 C110 AFO541 C110 AFO541 C110 AFO541
1 ESE Cy ESE ESE ESE ESE ESE ESE
2 Cy Cy Pc Cy Pc Cy Pc Cy
3 Pc Cy Co Co Co Co Co Pc
4 Co Pc Co Co Co Co Co Pc/Co
5 Co Co Co Co Co Co Co Co
6 Co Co Co Co Co Co Co Co
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Marked changes in embryonic inner structures were revealed after the application of PEG. In C110, the root cap was most influenced. In the absence of PEG, the root cap was short and tissue disintegration began as early as 1 week after the onset of the cotyledonary stage (Figure 2d). In the presence of 3.75% PEG, this disorder was not recorded during the same developmental period (Figure 2e). Higher PEG concentrations (5 and 7.5%), however, resulted in the formation of ruptures and intercellular spaces within the pericolumnar tissues (Figure 2f and g). Exposure to PEG osmotic stress led to a speeding up of the process in both highly embryogenic lines by ∼1 week (Table 1). No improvements were recorded in C107 exposed to PEG.

Carbohydrate content and spectrum

The results of morphological analyses allowed the connection of structural developmental stages of embryos with biochemical status. Proliferating ESM was characterized by a high sugar content, with hexoses (glucose and fructose) strongly dominating over sucrose (Figure 3a). After transfer to maturation medium, the total carbohydrate content of cultures increased temporarily, but from Week 2, a continued decrease until the end of cultivation was recorded. This decrease was mainly a result of decreasing content of hexoses; sucrose displayed the opposite trend. In the poorly productive C107, the pattern observed for a highly embryogenic line was largely disturbed (Figure 4a).

Figure 3.

Figure 3.

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Changes in content and spectrum of endogenous non-structural carbohydrates during P. abies somatic embryo maturation, line C110. (a) 0% PEG. (b) 3.75% PEG. (c) 5% PEG. (d) 7.5% PEG. (e) Sucrose : hexose ratio in mature P. abies somatic embryos, 2 weeks after the onset of cotyledonary stage (6-week maturation for 0% PEG; 5-week maturation for 3.75, 5 and 7.5% PEG). Week 0—ESM just before the transfer to maturation medium; E—isolated embryos; 4E, 5E, 6E, 7E and 8E—maturation of isolated embryos from 4th, 5th, 6th, 7th and 8th weeks, respectively. Different letters above columns in (e) show statistically significant differences. One-way ANOVA and multiple comparison Tukey–Kramer test or Kruskal–Wallis z-value test for normally or not normally distributed data, respectively, were used for statistical analysis, α = 0.05. Bars above columns—standard deviations for total soluble saccharides, n = 5–13.

Figure 4.

Figure 4.

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Changes in the content and spectrum of endogenous non-structural carbohydrates during P. abies somatic embryo maturation, line C107. (a) 0% PEG. (b) 5% PEG. E—isolated embryos; 4E, 5E, 6E, 7E and 8E—maturation of isolated embryos from 4th, 5th, 6th, 7th and 8th weeks, respectively. Bars above columns—standard deviations for total soluble saccharides, n = 5–10.

In PEG-treated C110 embryos, the decrease in total carbohydrate content began immediately after transfer of the culture to maturation medium, without an initial increase typical for the control. When treated with PEG, the embryos contained less total carbohydrates than the control (Figure 3b–d). A shift in individual saccharides occurred—the sucrose : hexose ratio was significantly higher after the treatment with 5% PEG (Figure 3e; 2 weeks after cotyledons appearance). In C107, exposure to 5% PEG also led to a lower level of total carbohydrates, but the pattern was dissimilar to C110 (Figure 4b).

Fatty acid composition of lipid reserves in highly embryogenic lines

In Norway spruce somatic embryos, the following FAs were detected: palmitic (16 : 0), palmitoleic (16 : 1), hexadecadienic (16 : 2), stearic (18 : 0), oleic (18 : 1), 7-octadecenic (7–18 : 1), linoleic (9.12–18 : 2), 5.9-octadecadienic (5.9–18 : 2), linolenic (18 : 3), arachidonic (20 : 0) and eicosenic (20 : 1) acids. In somatic embryos, the most abundant FAs were linoleic (18 : 2), oleic (18 : 1), palmitic (16 : 0) and 5.9-octadecadienic (5.9–18 : 2) acids. The spectra were very similar in both lines and differed from the mature zygotic embryo FA spectrum in the same way, with linoleic acid (18 : 2) being the major FA in somatic embryos and oleic acid (18 : 1) in zygotic embryos. The exposure to PEG led to slight shifts in ratios of particular FAs, mainly an increase in 7-octadecenic (7–18 : 1), linolenic (18 : 3) and palmitic (16 : 0) acids, and a decrease in palmitoleic (16 : 1) acid. The changes induced by PEG were similar in both lines (Figure 5).

Figure 5.

Figure 5.

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Fatty acid composition of lipids in mature P. abies somatic (lines C110 and AFO541) and zygotic embryos (ZE). Somatic embryos: 2 weeks after cotyledon appearance (6-week maturation for 0% PEG; 5-week maturation for 5% PEG). Zygotic embryos: mature zygotic embryos isolated from cones sampled in north-eastern Moravia (Beskydy Mountains) in January 2002. Fatty acids: palmitic (16 : 0), palmitoleic (16 : 1), hexadecadienic (16 : 2), stearic (18 : 0), oleic (18 : 1), 7-octadecenic (7–18 : 1), linoleic (9.12–18 : 2), 5.9-octadecadienic (5.9–18 : 2), linolenic (18 : 3), arachidonic (20 : 0) and eicosenic (20 : 1) acids. n = 5–7.

Carbohydrates in post-maturation phases in highly embryogenic lines

Carbohydrate status was determined after 3 weeks of high-humidity desiccation treatment and during the subsequent 3 weeks of germination (Figure 6). In both lines, total carbohydrate content decreased during desiccation. The end of the monitored post-maturation phase in C110 was accompanied by an increase in total carbohydrates. During desiccation, raffinose family oligosaccharides (RFO) appeared in the spectrum with substantially higher proportions in AFO541. In both lines, RFO disappeared within 1 day of germination. Pinitol appeared during the first week of germination and its content further increased as germination proceeded. The most pronounced difference between lines was found in the proportion of hexoses in the spectra. In both lines, as germination proceeded, we observed a decrease in sucrose amount accompanied by the opposite trend in hexoses. This pattern was more distinct in C110. After 3 weeks of germination, sucrose still represented the major saccharide in AFO541, while it nearly disappeared in C110. A preceding PEG treatment caused no significant changes in post-maturation carbohydrate status.

Figure 6.

Figure 6.

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Changes in the content and spectrum of endogenous non-structural carbohydrates during P. abies somatic embryo desiccation and germination, lines C110 and AFO541. 6w M: embryos after 6-week maturation; 3w D: embryos after 3-week high-humidity desiccation treatment; 1/2d G, 1d G, 3d G, 1w G and 3w G: embryos after ½ day, 1 day, 3 days, 1 week and 3 weeks germination, respectively. Bars above columns—standard deviations for total soluble saccharides, n = 5–8, isolated embryos.

Polyethylene glycol effect on somatic embryo yield and quality

A substantial increase in yield of embryos was achieved by PEG exposure (Figure 7a). Sensitivity to PEG treatment, however, differed between lines. In C110, the number of embryos formed per unit of initial ESM fresh weight increased over the whole range of concentrations tested, although there was no significant difference between 5 and 7.5% PEG. In contrast, with AFO541, 3.75 and 5% PEG yielded similar results, while 7.5% PEG resulted in a yield of embryos comparable to 3.75% PEG. In both highly embryogenic lines, the quality of the matured embryos (reflected in the proportion of high-quality germinants) decreased with an increasing concentration of PEG (Figure 7b). Response patterns differed between lines with a sharp decrease in the number of C110 Class 1 embryos in 3.75% PEG-treated cultures, in contrast to no decrease in AFO541 under the same conditions. A further increase in PEG did not yield any significant difference in the proportion of Class 1 embryos in C110, and a gradual decrease in AFO541. Figure 7c integrates the data presented in Figure 7a and b in order to estimate the beneficial or deleterious effects of PEG, on the basis of overall efficiency computed as the number of Class 1 embryos gained per unit of initial ESM. The overall efficiency of the process was higher in AFO541. This line was more PEG sensitive, as shown by the positive effect of 3.75 and 5% PEG, as well as the negative effect of 7.5% PEG. Such a dramatic reaction was not observed in C110, although a weaker beneficial effect was found for 5 and 7.5% PEG.

Figure 7.

Figure 7.

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The efficiency of P. abies SE, lines C110 and AFO541, determined as yields of embryos at the end of maturation and germination for different PEG concentrations. (a) Yield of mature cotyledonary embryos 2 weeks after the onset of cotyledonary stage (6-week maturation for 0% PEG; 5-week maturation for 5% PEG). (b) Proportion of high-quality germinants (Class 1) after 3-week germination. (c) Yield of high-quality germinants (Class 1) after 3-week germination, calculated value. Different letters above columns show statistically significant differences among variants. One-way ANOVA and multiple comparison Tukey–Kramer test or Kruskal–Wallis z-value test for normally or not normally distributed data, respectively, were used for statistical analysis, α = 0.05.

Discussion

In our previous studies, we found some attributes (anatomical features, carbohydrate content and spectrum) that were characteristic for the highly embryonic line AFO541 (AFOCEL). From a set of unrelated embryonic lines derived from plant material collected in the Czech Republic, we selected the C110 line exhibiting comparably high embryogenic potential under standard cultivation conditions, and the poorly productive C107 line. The goal of this study was to find out whether similarities found at the macroscopic level were also expressed at the structural and biochemical levels in order to identify common characteristics that were indicators of regular embryo development. A similar issue was examined in Pinus pinaster at the level of carbohydrate and proteomic analyses (Morel et al. 2014). We also addressed the still controversial question of the effects of a non-penetrating osmoticum by exposing the lines to various concentrations of PEG 4000 in the maturation medium. Polyethylene glycol is often regarded as beneficial for the maturation of conifer somatic embryos; it affects the process by changing the morphology of embryos, osmotic stress tolerance and the ability to accumulate storage products. However, controversial or even deleterious effects of PEG on embryo structure and post-embryonic development have also been reported (Kong and Yeung 1995, Find 1997, Krajňáková et al. 2009).

Developmental sequence of embryonic structures

For AFO541, the developmental sequence was recorded previously (Svobodová et al. 1999). It has been very stable throughout an extended culture time, thus enabling us to follow biochemical characteristics with the same embryogenic line (Lipavská et al. 2000, Gösslová et al. 2001, Konrádová et al. 2002, 2003, Grigová et al. 2007, Kubeš et al. 2014). Comparison with C110 revealed the same sequence of developmental stages; however, the development was slightly faster in C110 (Table 1). In general, the developmental sequence corresponds with the zygotic embryogenesis of the same species; only the timing of the process was quite different as the development of zygotic embryos to the cotyledonary stage was accomplished within 1 week (Gösslová et al. 2001). An earlier appearance of cotyledonary embryos in PEG-treated variants might be advantageous for the following reasons: (i) timing of the process was closer to zygotic embryogenesis and (ii) speeding up of the process means lowering of both labour and cost of the procedure. However, in the poorly productive line C107, the application of PEG did not increase the proportion of regularly developing embryos.

Carbohydrate content and spectra

The morphological analyses were the basis for the precise fitting of biochemical status to certain structural developmental stages. Because of differences in the onset of particular developmental stages in the tested lines, it was important to link biochemical analysis data with morphological observations to clarify the relationship between structural development of embryos and their biochemical status. When appropriate, we compared embryonic characteristics 2 weeks after cotyledon appearance (Figures 3e, 5 and 7) rather than in a certain week of cultivation.

Overall, mature C110 cotyledonary embryos had a low total carbohydrate content and a high sucrose : hexose ratio. A very similar pattern was observed in AFO541 (Lipavská et al. 2000), and importantly in Norway spruce zygotic embryos that were nourished differently (Gösslová et al. 2001). Iraqi and Tremblay (2001) obtained similar results with P. glauca and P. mariana somatic embryos. Such a distinct carbohydrate pattern was absent in the poorly productive line, C107. Businge and Egertsdotter (2014) reported that specific metabolites appeared to be associated with either normal or aberrant embryonic development. They stressed the importance of specific carbohydrates appearing at different stages of Norway spruce SE development. The patterns of sugar levels and enzyme activities support the hypothesis that normally maturing embryos undergo a transition from metabolic sink (high invertase activity and resulting hexose prevalence) to storage (dominated by sucrose synthase and a high sucrose : hexose ratio; Konrádová et al. 2002, Lipavská and Konrádová 2004).

Thus, we propose that the changes in sugar levels described here are a robust general quality of the genus Picea. Similar patterns were also found under two contrasting situations: (i) glucose plus fructose supported maturing embryos and (ii) in embryos transferred every 2 days to fresh sucrose-containing medium (Kubeš et al. 2014). Those results strongly support the concept that endogenous carbohydrate levels and their strict control in healthy embryos were important, irrespective of the exogenous carbohydrate supply. We propose that a deviation from the described carbohydrate pattern (e.g., in C107) was a significant symptom of aberrant embryonic development.

Polyethylene glycol influence on embryo morphology and development

The imposition of changing cultivation conditions (osmotic stress induced by PEG treatment) resulted in changes in timing of the whole process. Failure to establish a functional body plan during maturation often results in poor germination and conversion frequencies; this was especially true for shoot and root apical meristem formation (Kong and Yeung 1995). In C110, the root cap was the most influenced embryo part. On PEG-free medium, loosening of its structure began 1 week after the onset of the cotyledonary stage (Figure 2d). Exposure to 3.75% PEG caused a lowering of the frequency of the above-mentioned (short root cap and its early disintegration). However, a further increase in PEG (to 5 and 7.5%) resulted in the formation of ruptures and intercellular spaces within the pericolumnar tissue (Figure 2f and g). The disintegration of embryonal tissues in vitro was not unusual. In another P. abies embryogenic line maturing on 7.5% PEG, Bozhkov and von Arnold (1998) also found ruptures and intercellular spaces in the root cap. Moreover, the cells of the root apical meristem quiescent centre were malformed and the protoplasm shrunk. The control embryos were undamaged. In AFO541, the root cap was small and loose in the absence of PEG. In 7.5% PEG, it was compact; nevertheless, ruptures and rows of dead cells appeared within the hypocotyl (Svobodová et al. 1999). Find (1997) recorded numerous intercellular spaces in the hypocotyl and subapical zones of P. abies somatic embryos treated with 5% PEG, but not in the control embryos. Many authors assume that changes in embryonic structure were responsible for the failure of germination (Kong and Yeung 1995, Find 1997, Bozhkov and von Arnold 1998). Several explanations have been suggested for the PEG-induced loss of tissue integrity, e.g., the enhanced production of ethylene (Find 1997) or an imbalance in cellular growth (different cell wall properties or differences in the ability of osmotic adjustment). In AFO541, it was shown that the negative effects can be reduced by optimizing the concentration of PEG. In both lines, AFO541 and C110, higher PEG concentrations were harmful, whereas when the PEG concentration was reduced to 3.75%, no structural aberrations appeared.

Polyethylene glycol influence on carbohydrate status

In C110 exposed to PEG treatment, changes in sugar levels reflected faster morphological development. In AFO541, the embryos showed a very similar reaction pattern when treated with 3.75 and 5% PEG. At 7.5% PEG, however, the sugar composition was severely unbalanced as the total carbohydrate content increased and the content of hexoses was very high at the end of maturation (Lipavská et al. 2000). This implies that a suitable concentration of PEG might be able to support the storage sink character of mature embryos. In mature P. glauca somatic embryos treated with 7.5% PEG, transcript levels were higher for the sucrose synthase gene and lower for glycolytic and tricarboxylic-acid-cycle genes when compared with control embryos (Stasolla et al. 2003). For the successful completion of maturation, embryos must not germinate precociously. Polyethylene glycol may contribute to this through an overall reduction in sucrose catabolism, thus withdrawing carbon and energy that would otherwise promote germination (Stasolla et al. 2003). It corresponds well with gradually increasing activity of sucrose synthase, along with decreasing invertase activity as embryo maturation proceeds (Konrádová et al. 2002).

Polyethylene glycol influence on FA composition in highly embryogenic lines

For proper embryo development during post-maturation phases, the level and quality of storage compounds are important. Attree et al. (1992) noted a significant increase in storage lipids in white spruce somatic embryos cultured on medium containing 7.5% PEG, and Misra et al. (1993) reported that PEG enhanced protein deposition in somatic embryos of white spruce. Well-developed somatic embryonic structures are necessary, but not a sufficient condition for successful post-maturation embryonic development. In soya zygotic embryos, Dahmer et al. (1991) proposed that lipid composition may be a potential diagnostic marker of somatic embryo quality. Linoleic acid (18 : 2) was the dominant FA in seeds of loblolly pine (Janick et al. 1991) and white spruce (Attree et al. 1992). In zygotic Norway spruce embryos, linoleic (18 : 2) and oleic (18 : 1) acids were the most abundant FAs. We have found a considerable difference in FA spectrum between zygotic and somatic embryos, whereas FA composition was very similar in both embryonic lines under study.

Carbohydrate levels in post-maturation phases

After completion of the ABA-induced developmental programme, embryos were fully morphologically developed. For several coniferous genera including spruce, it was reported that conversion of embryos to viable plantlets might be improved by the imposition to a desiccation period, which helped reaching ‘physiological’ maturity and positively influenced germination (Stasolla and Yeung 2003, Maruyama and Hosoi 2012). Water loss naturally occurs during later stages of seed development, where it represents an important transition point, and prepares the embryo for subsequent germination. Partial drying at high relative humidities results in a gradual and limited loss of moisture in somatic embryos (Bomal et al. 2002, Stasolla and Yeung 2003).

Several physiological and biochemical changes occurring during partial drying have been documented, including lowering of ABA content as well as reduced sensitivity to ABA (Kong and Yeung 1995, Find 1997), and changes in nucleotide and nucleic acid metabolism (Stasolla et al. 2001). Remarkable changes were also observed in the amount and spectrum of soluble carbohydrates, especially the appearance of RFO, regarded as powerful osmotic stress protectants (Bomal et al. 2002, Konrádová et al. 2003, Kubeš et al. 2014). Raffinose family oligosaccharides were reported to be a substantial part of the mature conifer zygotic embryo spectrum (Gösslová et al. 2001, Konrádová et al. 2003, Pullman and Buchanan 2008). As expected, desiccation induced the synthesis of RFO in both lines under study, and these disappeared within 1 day of germination. In the course of germination, the carbohydrate spectrum was enriched with pinitol (3-0-methyl-d-chiro-inositol), an inositol derivative. The amounts of RFO as well as of pinitol, however, were higher in AFO541. Among the soluble sugars, the roles of raffinose in plant cell protection as an osmoprotectant or antioxidant are very well known (Nishizawa-Yokoi et al. 2008, dos Santos et al. 2011). In many tree species, pinitol has been described as an important polyol, especially under stress conditions such as drought, salinity or low temperature (Ericsson 1979, Streit et al. 2013), acting as an osmolyte (Reddy et al. 2004). Variations in cyclitols such as pinitol, and soluble sugars, have been assessed in conifers (Simard et al. 2013, Streit et al. 2013) and related to active growth as well as cell protection.

Although the pattern of carbohydrates was very similar for both highly embryogenic lines during maturation, this resemblance disappeared during desiccation and germination. In both lines, hydrolysis of stored sucrose and the resulting rise in hexose content increased with germination. However, the trend was much more pronounced in C110, where only negligible amounts of sucrose were preserved, while in AFO541, sucrose represented half of the total soluble carbohydrates at the end of 3-week germination. Businge et al. (2013) ascribed the improved germination of somatic embryos to the high levels of sucrose, raffinose and late embryogenesis abundant proteins that promoted acquisition of desiccation tolerance. Similarly, we suggest that higher germination efficiency of AFO541 embryos may be related to their higher content of RFO, pinitol and sucrose during post-maturation phases.

Effect of PEG on carbohydrates during desiccation and germination

We posed the question whether the application of PEG during maturation might have any impact on carbohydrate status during subsequent post-maturation phases (desiccation and germination). Embryos cultured at different PEG concentrations were subjected to different osmotic stress levels, and thus, the biochemical processes during desiccation might be influenced differently as well. Notably, there was no clear effect of previous PEG treatments on carbohydrate content and changes during post-maturation phases.

Polyethylene glycol effect on embryo yields

The overall efficiency of SE can be defined as the final number of normally germinating embryos per unit weight of initial ESM. It integrates two features: (i) the number of mature embryos and (ii) embryo quality. In C110, both of these parameters were strongly affected by PEG: the number of somatic embryos formed increased with increasing PEG concentrations from 3.75 up to 7.5% (no significant difference between 5 and 7.5% PEG; Figure 7a). The proportion of Class 1 embryos, however, decreased sharply at 3.75% PEG, with no further significant decrease with increasing PEG concentration (Figure 7b). These results correspond well with the literature data reporting PEG-induced increasing number of somatic embryos along with decreasing germinability (Kong and Yeung 1995, Find 1997, Bozhkov and von Arnold 1998, Jones and Van Staden 2001). The authors attributed problems with germination to abnormal embryo structure. As in C110, disintegration of the root cap at 3.75% PEG did not interfere with radicle protrusion (note 0% PEG with root cap disintegration, but exhibiting the highest germination); we suggest that in addition to structural quality, other factors play important roles in successful germination. Businge et al. (2013) demonstrated that 7.5% PEG had a positive effect on embryo maturation. Their data seem to support a positive effect of PEG, but this conclusion might be questioned as the sources of carbon and energy were changed (sucrose was in PEG-treated variant replaced by maltose).

In AFO541, the nature of the reaction to PEG was similar to C110, but the pattern of response was different: in AFO541, the optimum PEG concentration proved to be 5%, and a further increase in PEG to 7.5% caused a decrease in somatic embryo numbers; in C110, the significant increase was also observed up to 5%. Therefore, the overall efficiency of the process was much more positively influenced by PEG application in AFO541 than in C110, although the optimum concentrations were the same for both lines (between 3.75 and 5%). In both highly embryogenic lines, maturation was faster in PEG-treated cultures. The application of PEG in C107, however, did not influence substantially the number of high-quality embryos. Therefore, we cannot propose PEG application as a means for improvement of poorly productive lines.

Conclusion

Comparisons between two unrelated highly embryogenic Norway spruce lines (different geographical origins, ages and previous histories) have identified common features that correlate with high embryogenic and germination capacity. They were as follows: (i) morphogenic events leading to formation of all embryonic organs and completion of histo-differentiation; (ii) a gradual decrease in total carbohydrates during maturation, accompanied by a progressive increase in the sucrose : hexose ratio; and (iii) the ability to react to desiccation treatment by RFO deposition, and their immediate consumption at the start of germination. In the poorly productive line, the above-mentioned traits accompanying regular maturation were disturbed. The efficiency of SE in both highly embryogenic lines was influenced by PEG, in a concentration-dependent manner, with positive effects on mature embryo yield and negative effects on germination. Importantly, it appears necessary to precisely optimize the PEG concentration, representing a compromise between positive (enhanced maturation) and negative (impaired germination) effects of PEG for each particular line.

Conflict of interest

None declared.

Funding

This work was supported by the Czech Ministry of Education, Youth and Sports—Project LO1417.

Acknowledgments

The authors are very thankful to Dr Martin Vágner for providing us with lines C110 and C107 derived in his laboratory, Mrs Magdalena Cvečková and Dr Tomáš Řezanka for the help with fatty acid determination and Prof. John D. Brooker for valuable text revision.

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