# Source paper archive - Source title: Maltose-driven carbohydrate status and early raffinose synthesis enhance maturation efficiency in hybrid fir somatic embryogenesis - Author(s): Ondřej Skala, Helena Lipavská, Terézia Salaj, Hana Konrádová - Source landing URL: https://www.biorxiv.org/content/10.64898/2026.11.05.764528v1 - PDF URL: https://www.biorxiv.org/content/10.64898/2026.11.05.764528v1.full.pdf - PDF SHA-256: 1a1c83fe36c4b2fecb022edebb7a3bdf69c0ec0d7741dc0e19c9e40d001f86ba - Converter: pymupdf4llm 0.0.27 - Limits: Automated PDF text extraction; reading order, equations, tables and formatting may be incomplete or incorrect. Images are omitted, and no OCR was performed. Markup and links are neutralized for safe downloading. Consult the original PDF for the authoritative version. --- [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 1 **Maltose-driven carbohydrate status and early raffinose synthesis enhance maturation** 2 **efficiency in hybrid fir somatic embryogenesis** 3 4 **Ondřej Skala** **[1]** **, Helena Lipavská** **[1]** **, Terézia Salaj** **[2]** **, Hana Konrádová** **[1* ]** 5 ***** corresponding author 6 1 Department of Experimental Plant Biology, Faculty of Science, Charles University, Viničná 5, Prague, 7 Czech Republic 8 2 Plant Science and Biodiversity Centre, Institute of Plant Genetics and Biotechnology, Slovak Academy 9 of Sciences, Nitra, Slovakia 10 11 ORCIDs: HL 0000-0001-8514-6049, TS 0000-0002-8649-8246, HK 0000-0003-0431-9425 12 13 **Keywords:** _Abies_ ; embryogenesis; high-humidity treatment; maltose; stachyose 14 15 **Running head:** Carbohydrates in hybrid fir somatic embryogenesis 16 17 **Corresponding author:** Hana Konrádová, hana.konradova@natur.cuni.cz, 18 Department of Experimental Plant Biology, Faculty of Science, Charles University, Viničná 5, Prague, 19 Czech Republic 20 1 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 21 **Abstract** 22 Somatic embryogenesis (SE) is a critical biotechnological tool for the large-scale propagation of the 23 _Abies_ genus, which faces significant threats from climate change and high commercial demand. While 24 sucrose is a first-choice energy source among conifers, firs often exhibit a dramatic preference for 25 maltose. Using hybrid fir ( _Abies alba_ x _numidica_ ), we have shown an order-of-magnitude higher amount 26 of mature cotyledonary embryos cultivated on maltose compared to sucrose. Determination of 27 endogenous carbohydrate balance during maturation and post-maturation phases revealed fundamental 28 difference in spectrum between sucrose (S) and maltose (M) variants, notably massive presence of 29 hexoses in S variant and, remarkably, an earlier accumulation of raffinose in M variant, pointing to 30 establishing a “mature” biochemical profile. After post-maturation desiccation treatment, though both 31 variants accumulated raffinose family oligosaccharides (RFO), their proportion of in total carbohydrate 32 content remained higher in M variant. Based on the results, we propose, that specific features of fir 33 carbohydrate processing machinery (degradation and transport) resulted in slower but steady C and 34 energy supply in M variant and represent the base of maltose superiority. 35 36 **1. Introduction** 37 The _Abies_ genus comprises more than 40 ecologically and economically indispensable species. Somatic 38 embryogenesis (SE) offers a powerful asexual propagation method allowing the rapid multiplication of 39 elite genotypes and long-term germplasm storage via cryopreservation (Hazubska-Przybył et al. 2022, 40 Tarraf et al. 2022). Importantly, besides botanical species also embryogenic cultures of interspecific 41 hybrids have been repeatedly derived (e.g., Salajová et al. 1996, Vooková and Kormuťák 2003, 2014). 42 Establishment of an embryonic culture from immature zygotic embryos enables to obtain viable 43 individuals of the crossing in which zygotic embryos abort usually several weeks after pollination 44 (Vooková and Kormuťák 2014). 45 While SE was first described in _Picea abies_ (Chalupa 1985, Hakman and von Arnold 1985), the 46 first successful protocol for the _Abies_ genus followed shortly thereafter in 1986 for silver fir ( _Abies alba_ ) 47 (Erdelský and Barančok 1986). Since then, research has successfully expanded to numerous fir 48 genotypes (for review, see Vooková and Kormuťák 2014, Jouini et al. 2025). In line with other conifer 49 models, the process of SE in firs can be divided into four steps: initiation of embryogenic suspensor 50 mass (ESM) from the primary explant (zygotic embryo), ESM proliferation, maturation of somatic 51 embryos and regeneration of plants. However, the technology still faces significant bottlenecks, 52 particularly during the transition from embryo maturation to germination and conversion into plantlet. 53 Even if several of the experimental conditions can be generalised and used for the most _Abies_ species, 54 other key ones must be optimised for individual species (Vooková and Kormuťák 2004). Importantly, 55 variables as a carbon source, abscisic acid (ABA) and osmotic agents have been used to increase 56 germination and conversion rates of somatic embryos (e.g. in _A. cephalonica_ ; Krajňáková et al. 2009). 57 Maltose (usually 3-6%) often gave better maturation response compared to sucrose (Nawrot-Chorabik 58 2008) and the addition of polyethylene glycol 4000 (PEG, up to 10%) to maturation medium promoted 59 the maturation of somatic embryos (Nørgaard 1997, Salajová et al. 2004, Vooková and Kormuťák 2014). 60 In addition, good germination can be achieved by partial desiccation of cotyledonary somatic embryos 61 (Nørgaard 1997, Vooková et al. 1998, Kvaalen et al. 2005). 62 The main drawback in evaluating the effectiveness of conifer embryogenic culture 63 modifications is the difficulty in identifying reliable indicators of normal development and the ultimate 64 embryo maturity (Savane et al. 2025; Eliášová et al. 2022). Morphological similarity to zygotic embryos 65 has frequently proven to be a deceptive indicator of physiological maturity and readiness for subsequent 66 development completion (Klimaszewska et al. 2004, Vooková and Kormuťák 2014). Though this 67 approach has been shown to provide some indications of the progress of embryo maturation, it reveals 68 relatively little about the embryo's developmental potential (Klimaszewska et al. 2004). Consequently, 69 research has shifted toward biochemical markers, such as the accumulation of storage proteins 70 (Kormuťák et al. 2006), lipids, and non-structural carbohydrates like raffinose family oligosaccharides 71 (RFOs; i.e., α-1,6-galactosyl extensions of sucrose) (e.g., Downie and Bewley 2000, Gösslová et al. 2 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 72 2001, Eliášová et al. 2022). Individual carbohydrate levels and their dynamics during embryo maturation 73 was repeatedly suggested as a marker of normal embryo development and metabolic maturity (Lipavská 74 et al. 2000, Iraqi and Tremblay 2001), as well as quantity and quality of reserves in general (Hakman et 75 al. 1990, Attree et al. 1992, Hakman 1993, Misra et al. 1993). Carpenter et al. (2000) suggest that the 76 appropriate marker for the maturity of SE conifers (but above all of the genus _Pinus_ ) is the level of 77 oligosaccharides of the raffinose family (RFO) and dehydrins, with increased RFO levels resulting in 78 improved sprouting process. But the fundamental difference in the conditions in which zygotic and 79 somatic embryos develop makes assessing the importance of biochemical markers much more 80 complicated. Thus, the precise determination of the role of individual compounds, including non81 structural carbohydrates, in the somatic embryogenesis of conifers remains the subject of ongoing 82 research. 83 In some conifer species, it is necessary to perform desiccation that leads to a reduction in the 84 water content of SEs (sometimes very little) and, importantly, triggers biochemical changes that support 85 further developmental processes: germination and the first steps of plantlet development (Bomal et al. 86 2002, Eliášová et al. 2022, Savane et al. 2025). The research on the desiccation treatment of conifer SEs 87 began in the 90’s and consisted of partial drying at high relative humidity (Roberts et al. 1990). For 88 _Picea_ sp., partial desiccation of SEs is a necessary prerequisite for successful germination (Roberts et 89 al. 1990, Pond et al. 2002), while in larch, on the contrary, desiccation is not required, though it improves 90 synchronisation of germination and conversion to plants (Lelu et al. 1995). In addition, desiccated SEs 91 are also interesting in terms of their long-term storage (Välimäki et al. 2022). 92 In orthodox conifer seeds, RFO synthesis is natural part of developmental program (Downie and 93 Bewley 2000, Gösslová et al. 2001). Importantly, RFO fulfil a dual role: first, as "starter fuel," providing 94 rapidly mobilizable energy and carbon skeletons during the earliest hours of imbibition before starch or 95 lipids can be hydrolysed (Downie and Bewley 2000, Savane et al. 2025). Second, and perhaps more 96 critically, they act as osmo- and cryo-protectants (Sengupta et al. 2015). Desiccation of mature somatic 97 embryos repeatedly induced, besides other metabolic changes, the synthesis of RFOs including 98 raffinose, stachyose, and eventually higher members of the family (Konrádová et al. 2003, Businge et 99 al. 2012, Eliášová et al. 2022). RFOs (particularly stachyose) increase cytoplasmic viscosity, disrupt the 100 sucrose crystal lattice, and facilitate a vitrified, "glassy” state, which stabilizes cellular membranes and 101 prevents protein denaturation under water-limited conditions (Caffrey et al. 1988, Bernal-Lugo and 102 Leopold 1995, Hoekstra et al. 2001). 103 A characteristic feature of SE in the _Abies_ genus is its strong preference for maltose, which is 104 rather unique (Nørgaard 1997, Salaj et al. 2004, Jouini et al. 2025). It is hypothesized that the slower 105 hydrolysis of maltose compared to sucrose induces a mild "nutritional stress" that signals the embryo to 106 switch from a proliferative mode to a maturation-focused metabolic program (Scott et al. 1995, Nørgaard 107 1997). To our knowledge, there are no articles dealing with carbohydrate status dynamics during the fir 108 embryo development (both in the zygotic or the somatic ones). Therefore, this study aims to clarify how 109 the carbohydrate sources modulate carbohydrate metabolism and maturation efficiency in hybrid fir 110 ( _Abies alba_ x _numidica_ ). 111 112 **2. Materials and Methods** 113 2.1. Plant material 114 The hybrid fir line AN72 ( _Abies alba_ x _numidica_ ), derived from immature zygotic embryo, was 115 maintained in plastic Petri plates (diameter 9 cm) in the dark at 25 °C on modified DCR medium (Gupta 116 and Durzan, 1985) containing 4.4 μM benzylaminopurine, casein hydrolysate (500 mg L [−1] ), glutamine 117 (50 mg L [−1] ), _myo_ -inositol (100 mg L [−1] ) and 2% sucrose. The media were solidified with 0.3% 118 Phytagel [TM ] (Sigma-Aldrich). Transfer to fresh media occurred at 3-week intervals. 119 120 2.2. Maturation and carbohydrate treatments 3 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 121 Maturation was induced by transferring the embryonal suspensor mass (ESM) onto filter paper discs 122 placed on DCR maturation medium containing 10% PEG 4000, casein hydrolysate (500 mg L [−1] ), 123 glutamine (50 mg L [−1] ), _myo_ -inositol (100 mg L [−1] ) and 10 μM ABA in 9 cm Petri dishes. Two carbon 124 source variants were compared: maltose variant (M): 3.16% (w/v) maltose·H 2 O; and sucrose variant 125 (S): 3% (w/v) sucrose. 126 Maturation lasted 8 weeks with one subculture (transfer of the culture together with filter disc) 127 on fresh media with the same composition. This was followed by 1 week of cultivation on ABA and 128 PEG-free media supplemented with the appropriate carbon source (maturation finishing media, FM). 129 Sampling of the material was done always at the end of subcultivation periods: in week 4 (ESM as a 130 whole), in week 8 (isolated embryos, ie) and in maturation finishing phase (ie and mass remaining after 131 somatic embryo isolation, RM). 132 133 2.3. Post-maturation and germination 134 Mature SE (9 weeks old) underwent two weeks of high-humidity desiccation at 25 °C in the dark 135 (Eliášová et al. 2022). Following germination took place on DCR medium containing 2% (w/v) sucrose, 136 1% (w/v) activated carbon (Sigma) and 0.3% (w/v) Phytagel [TM] in plastic Petri dishes (9 cm diameter). 137 The quantities of the other organic supplements were the same as those in the maturation medium. The 138 first phase of germination took place in the dark at 25 °C. It was terminated once the hypocotyl had 139 elongated and the root had emerged. The cultures were then moved to a cultivation room with a 16-hour 140 photoperiod at 25 °C. 141 142 2.4. Soluble carbohydrate content determination 143 Three to six biological replicates of samples (60-200 mg fresh weight) were frozen in liquid nitrogen 144 and stored in -80 °C. Subsequently, the samples were freeze-dried and incubated in 80% methanol at 75 145 °C for 15 min. Methanol was vacuum-evaporated and the residue re-suspended in ultrapure water before 146 being purified by centrifugation and filtration. The content of soluble carbohydrates was determined 147 using high-performance liquid chromatography (HPLC) (mobile phase water, flow rate 0.5 ml min [-1], 148 temperature 80 °C) with refractometric detection (Shodex RI-101), Shodex Sugar Column SP0810 149 (Shodex, Tokio, Japan). Identification of individual carbohydrates was verified using Shodex Sugar 150 Column SC1011 (Shodex, Tokio, Japan). Standard dilutions with known concentrations of sucrose, 151 glucose, fructose, inositol, pinitol, raffinose and verbascose (all Sigma-Aldrich) were used as reference. 152 The HPLC data were evaluated using Clarity 7.8 software (DataApex, Prague, Czech Republic). 153 154 2.5. Data processing and statistics 155 Calculations and graphical data processing were performed in Microsoft Office Excel. The data are 156 presented as means ± standard deviations (SD). Statistical data evaluation was performed using NCSS 157 9 software (NCSS, LLC. Kaysville, Utah, USA). Data were checked for normality and treatment effects 158 (M vs. S variant) were evaluated using standard (equal variance) or Aspin-Welch’s (unequal variance) 159 two-sample _t_ -test. 160 161 **3. Results** 162 In this study, we investigated the effect of the type of carbohydrate supplied from the maturation medium 163 (3.16 % maltose or 3% sucrose) on development of the line AN72 of hybrid fir embryogenic culture. 164 The embryo yield and spectrum of endogenous soluble carbohydrates during maturation we examined, 165 first in the entire embryonal suspensor mass (ESM) and subsequently in the isolated developing somatic 166 embryos (SE). The embryos during post-maturation phases were also analysed. 167 168 3.1. Yield and morphology: the maltose superiority 4 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 169 The carbohydrate added to the maturation medium significantly influenced the culture behaviour. ESM 170 cultured on maltose-containing medium (M variant) was characterized by a slow but steady growth and 171 formation of numerous somatic embryos visible by the end of first subculture. In the case of the culture 172 grown on sucrose-containing medium (S variant), an initial rapid increase in ESM was followed by a 173 marked slowdown in growth and embryo formation was greatly reduced. The ESM often turned brown 174 toward the end of maturation (Fig. 1). 175 The carbohydrate source drastically influenced the productivity of the AN72 line. In a single Petri 176 dish (Ø 9 cm), the M variant yielded 133 ± 74 SE, while the S variant produced only 3 ± 2 SE/plate 177 (means ± standard deviations; n=24-27). Morphologically, maltose-grown SE were shorter but robust. 178 Due to the low SE yield in the S variant, some data points regarding the post-maturation stages of 179 development are based on lesser number of samples. Importantly, S variant SE often exhibited impaired 180 development, particularly of the root pole of the embryos, or precocious germination. 181 182 3.2. Carbohydrate status of S and M variants and maltose induced early raffinose synthesis 183 Analysis of carbohydrate status (Fig. 2a) revealed a marked decrease in total soluble carbohydrate 184 contents after transfer to the maturation medium in both variants ( _t-_ test, _p_ ≤ 0.01). However, the 185 carbohydrate profiles differed substantially between cultures grown on sucrose and maltose (Fig. 2b): 186 while hexoses significantly predominated in the sucrose variant, the maltose variant spectrum consisted 187 primarily of disaccharides with strongly prevailing maltose. Surprisingly, a considerable content of 188 raffinose was detected in the ESM of M variant as early as in the 4 [th] week of maturation (33.9 μg/mg 189 DW), while, conversely, RFOs remained undetectable in S variant at the same stage. At week 8, the total 190 soluble carbohydrate content was significantly higher in isolated embryos of M variant _(t-_ test, _p_ = 191 0.015). Moreover, M variant SE contained 22.2 μg/mg DW of total RFOs compared to 7.2 μg/mg in 192 sucrose-grown SE (significant difference according to _t-_ test, _p_ ≤ 0.01). As for the other carbohydrates 193 in the spectrum, the situation did not differ significantly: the most abundant carbohydrate in both variants 194 was sucrose, followed by smaller amounts of glucose, fructose, and inositol, with trace amounts of 195 maltose in M variant. After additional week of cultivation on the finishing maturation (MF) medium, 196 the total carbohydrate contents in the isolated embryos did not differ between variants, and no dramatic 197 change was observed when comparing with the situation after 8 weeks of maturation. However, in 198 remaining mass at this stage, the total carbohydrate levels were similar in both variants but significantly 199 higher compared to isolated embryos ( _t-_ test, _p_ ≤ 0.01). Interestingly, the carbohydrate spectra differed 200 markedly between variants: maltose dominated in M variant in contrast to S variant, where spectrum 201 consisted mainly of hexoses with small amount of sucrose. It is worth noting, however, that there was a 202 significant difference in the RFO content, as these carbohydrates were found only in the maltose variant. 203 204 3.3. Post-Maturation Carbohydrate Shift. 205 Standard desiccation at high-humidity resulted in a qualitative change in the RFO pool. While raffinose 206 was dominant RFO in isolated SE during late maturation, higher RFO member, stachyose, appeared in 207 S variant and slightly increased _(t-_ test, _p_ = 0.09) in M variant after the desiccation. The desiccation led 208 to the significant increase in the proportion of RFO in the carbohydrate profile of both variants ( _t-_ tests, 209 _p_ ≤ 0.01; compared with 1t FM of the respective variants). In contrast to the situation observed during 210 maturation, the standard two weeks of slow desiccation led to higher increase of RFO accumulation in 211 SE derived from S variant compared to M variant. Nevertheless, the proportion of RFO in the total 212 carbohydrate content remained higher in the maltose variant ( _t_ -test, _p_ = 0.043). As for the total amount 213 of carbohydrates, the amount found after desiccation was significantly higher in the S variant. 214 Subsequently, the somatic embryos of both variants were transferred to germination media. Figure 215 3 shows 3-week-old fir seedlings; the SE of M variant did not exhibit normal development instead we 216 observed abnormalities in them, particularly poorly developed roots. In both variants, the endogenous 217 carbohydrate status of harvested seedlings was characterized by the complete disappearance of RFO and 218 presence of considerable proportion of sequoitol along with lower amount of pinitol (both are _O_ -methyl 5 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 219 inositol isomers). Nevertheless, M variant contains significantly higher sum of methyl-inositols 220 compared to S variant ( _t_ -test, _p_ = 0.030). 221 222 **4. Discussion** 223 In fir somatic embryogenesis, numerous studies have demonstrated frequent maltose superiority to 224 support maturation efficiency. However, to our knowledge, there is no sound experimental basis for 225 explaining this phenomenon. In spruce, the dynamics of the endogenous carbohydrate content proved 226 to be a good indicator of favourable embryo development when evaluating the effects of protocol 227 modifications (Lipavská and Konrádová 2004, Businge et al. 2014, Hudec et al. 2016, Eliášová et al. 228 2022). In this study, we made use of this experience and tried to reveal the physiological basis of the 229 phenomenon by monitoring endogenous carbohydrate dynamics as affected by the exchange of 230 exogenous carbohydrate supply. Surprisingly, there is a complete lack of information on carbohydrate 231 status during the somatic embryogenesis of the fir. 232 233 4.1. Maltose as a yield-enhancing factor acting through nutritional modification 234 With the use of hybrid fir embryogenic line AN72 ( _Abies alba_ x _numidica)_ ; Salaj et al. 2004) we 235 confirmed the dramatic disparity in embryo yield between the application of maltose. The advantage of 236 using this line stems from the possibility to perform analysis even on sucrose-supported embryos, though 237 the yield of mature embryos was very low. The data indicated that sucrose´s failure to promote high238 quality maturation in line AN72 likely resulted from its rapid hydrolysis by wall invertases in the 239 apoplast, which led to a surplus of hexoses (glucose and fructose) and potentially induced hypoxia or 240 ethanol toxicity (Nørgaard 1997, Iraqi et al. 2005). In the early stages of maturation (after 4 weeks on 241 maturation medium), the culture was rich in hexoses, particularly fructose, which was probably due to 242 the high activity of hexose transporters and also the lower activity of enzymes capable of o processing 243 fructose in the cytosol. The pronounced accumulation of fructose in our sucrose-grown cultures, 244 accompanied by tissue browning and impaired embryo development, closely mirrors the fructose245 induced developmental block reported in _Picea abies_ (Businge et al. 2012, Businge and Egertsdotter 246 2014). Fructose is a highly reactive reducing keto-sugar making it an extraordinarily potent initiator of 247 non-enzymatic glycation via the Maillard reaction (Suarez et al. 1989, Semchyshyn et al. 2011). Fructose 248 accumulation therefore triggers protein carbonylation, DNA damage, and glutathione depletion, 249 ultimately causing tissue necrosis and cellular death (Baynes 2002, Businge and Egertsdotter 2014, Tola 250 et al. 2021). Thus, the low quality and quantity of embryos in this variant can be attributed to fructose 251 toxicity and associated oxidative stress. This is further consistent with the finding that browning in _Picea_ 252 _abies_ was caused not by the accumulation of phenolic compounds, but rather by their increased oxidation 253 in an environment with a reduced content of low-molecular-weight ROS scavengers (Švorc Štepánová 254 et al. 2026). Moreover, high level of fructose was reported to down-regulate the expression of 255 _WUS/WOX_, a stem cell-promoting transcription factor, via the fructose activated sensor FINS1 (fructose 256 1,6-bisphosphatase) which acts as a bifunctional factor (Li et al. 2023). On top of all that, hexoses can 257 interact with abscisic acid (ABA), auxin, and ethylene pathways to modulate cell division and 258 development (Li et al. 2023). 259 In contrast, the slower utilization of exogenously provided maltose very likely ensures a 260 moderate, but more stable, carbon supply to nourish maturing embryos. We can base this assumption on 261 the observed high proportion of maltose in the carbohydrate spectrum of ESM after 4 weeks of 262 maturation, as well as in the residual mass remaining after the isolation of embryos upon finishing 263 maturation. We suggest that this is the result of very inefficient apoplastic degradation of maltose, and 264 low ability of transporters to help maltose to cross cell membranes. Together it might mimic the slow265 release nutritional environment of a developing seed (Scott et al. 1995, Nørgaard 1997). This probably 266 brought about only mild and balanced „nutritional stress" which acts as a biochemical signal, steering 267 the tissue away from simple proliferation toward the SE maturation program, including the early 268 synthesis of RFO, with capacity to act as protective compounds (Scott et al. 1995, Nørgaard 1997, 269 Sengupta et al. 2015). 6 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 270 271 4.2. RFO accumulation: a developmental program vs. stress response 272 Another striking difference between the two treatments was the presence of raffinose in the ESM after 273 4 weeks of maturation exclusively in the maltose variant. If we consider that RFO synthesis is often 274 associated with stress exposure and the stress load is due to high endogenous hexoses levels higher in 275 sucrose variant, then the stress-based idea faces a problem. However, it is necessary to keep in mind that 276 the synthesis of RFO is a standard part of developmental program of conifers, and thus we have to search 277 for another signal that strengthens or triggers embryonic development ((Downie and Bewley 2000, 278 Gösslová et al. 2001). This assumption is reinforced by the finding that the few embryos that formed on 279 sucrose medium also synthesized RFO in later stages of development (after 8 weeks of maturation) even 280 though the amount of RFO remained higher in maltose supported ones. It should be noted that the 281 carbohydrate profiles were already similar in both variants at this stage of development, with sucrose 282 clearly predominating; sucrose apparently originated from resynthesis even in the sucrose variant 283 (Kubeš et al. 2014). We can further speculate that the individual components of the carbohydrate 284 spectrum, specific in sucrose and maltose variants, especially at the early stages of maturation, have 285 entirely different signalling potentials for the plant cells. Therefore, we propose that the carbohydrate 286 signalling background established in maltose variant may be the key factor not only for RFO synthesis 287 but also for promoting embryo formation. 288 In this context, it may be interesting to note that the carbohydrate status fundamentally differs 289 from the Norway spruce model ( _Picea abies_ ), where RFOs are almost entirely absent until triggered by 290 post-maturation stressors like cold or drying (Gösslová et al. 2001, Konrádová et al. 2003, Businge et 291 al. 2013, Eliášová et al. 2022). On the other hand, there is an interesting similarity between these two 292 conifer models: as maturation progresses, the proportion of endogenous sucrose in the carbohydrate 293 profile increases in both species, even in fir cultures grown on maltose. In fir, the early detection of 294 raffinose in the ESM by week 4 suggests that RFO synthesis is a part of the developmental program and 295 indicates the embryos’ readiness for further development, which is triggered, in this case, by maltose 296 signalling. In _Picea_, even synergistic stressors have to be applied to reach peak RFO levels, whereas in 297 _Abies_, the metabolic machinery for RFO production is already activated while the embryos are still in 298 contact with the high-water-potential maturation medium. This highlights that RFO metabolism in 299 conifers is strictly species-specific, and no general model for "biochemical maturity" can be applied 300 across the Pinaceae (Lipavská and Konrádová 2004, Jouini et al. 2025, Stojičić et al. 2026). 301 302 4.3. Post maturation embryo behaviour as affected by maltose supported maturation 303 The observed synthesis of RFO during maturation raised the question of what effect the application of 304 a post-maturation desiccation treatment might have on the subsequent content and spectrum of 305 carbohydrates. The desiccation exposure (more precisely high-humidity treatment) significantly 306 enhanced the proportion of RFO in both variants. Moreover, besides raffinose, the stachyose proportion 307 also increased in the maltose variant and stachyose appeared in sucrose one as well. To our knowledge, 308 in fir, there is no information on carbohydrate balance even during post-maturation phases. However, 309 desiccation treatment is frequently reported as beneficial for further successful development (Nørgaard 310 1997, Vooková and Kormuťák 2003, 2014, Salaj et al. 2020, Jouini et al. 2025). In spruce, where 311 desiccation treatment is critical condition for embryo germination, it is accompanied by RFO synthesis, 312 including stachyose (Gösslová et al. 2001, Konrádová et al. 2003, Eliášová et al. 2022). Stachyose is 313 reported to be more effective than raffinose at increasing cytoplasmic viscosity and raising the transition 314 temperature for the vitrified glassy state (Bernal-Lugo and Leopold 1995, Sun 1997, Buitink and 315 Leprince 2008, Sengupta et al. 2015). Based on data presented here, it is unfortunately not possible to 316 establish a clear relationship between the beneficial effect of desiccation and RFO accumulation in fir 317 embryogenesis. This is further illuminated by recent transcriptomic findings in desiccation-treated 318 conifer embryos showing that RFO synthesis (via PaGOLS and PaRFS) is co-regulated with cell wall 319 remodelling under PaMYB12 transcription factor control (Ling et al. 2025, Savane et al. 2025). Anyway, 320 our previous data alongside broader literature - even in the case of well-studied spruce model - clearly 7 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 321 show that this relationship is more complex, as the induction of RFO synthesis e.g. by cold, though 322 beneficial, proved to be insufficient on their own to guarantee high-quality germination (Konrádová et 323 al. 2003, Businge et al. 2013, Eliášová et al. 2022, Välimäki et al. 2022). During germination, the RFO 324 fraction disappeared completely from the spectrum, suggesting that this carbohydrate plays a role in 325 promoting germination as an immediately mobilizable carbon and energy source (Downie and Bewley 326 2000, Gangl and Tenhaken 2016). Interestingly, differences in the carbohydrate spectrum caused by 327 maltose versus sucrose supply in maturation can even be traced up to the germination phase, particularly 328 a significantly higher proportion of _O-_ methylated inositol derivatives in the embryos of the maltose 329 variant. However, the significance of this difference, if any, remains unclear. 330 Recent findings suggest that the environmental conditions during SE - such as temperature or 331 osmotic stress - can induce an epigenetic memory that influences the future phenology and physiology 332 of the emblings (Lobo et al. 2022; Trontin et al. 2025). We propose that similarly, maltose as a carbon 333 source in fir maturation likely influences the "metabolic memory" of the embryo. While our study has 334 focused on the carbohydrate markers of this process, the broader implication is that optimizing the 335 maturation environment not only improves yield but also defines the future fitness and adaptive potential 336 of the resulting somatic plants in forest environments. 337 338 **. Conclusions** 339 - Maltose is the essential driver for hybrid fir ( _Abies alba_ x _numidica_ ) SE, ensuring massive 340 productivity of heathy embryos. 341 - The endogenous carbohydrate profiles of the maltose- and sucrose-supported variants differed 342 significantly: low hexose levels and the early synthesis of raffinose were most noteworthy 343 characteristic of the maltose variant, indicating a distinct signalling network status. 344 - Desiccation enhanced total RFO accumulation in both variants, while stimulating stachyose 345 synthesis; germination caused complete RFO consumption. Notably, the carbohydrate profile 346 differences between variants, manifested in their _O_ -methyl-inositol derivative levels during 347 germination) persisted until the end of the monitored phase. 348 - The generic application of conifer sucrose-based maturation strategies is incompatible with the 349 unique metabolic requirements of particular _Abies_ species. 350 351 **Acknowledgements** 352 This work is based upon work from COST Action CA21157 COPYTREE (European Network for 353 Innovative Woody Plant Cloning), COST (European Cooperation in Science and Technology). 354 355 **Authors’ Contributions** 356 Ondřej Skala: Investigation, Visualization, Formal analysis. Helena Lipavská: Writing – review and 357 editing, Methodology, Supervision. Terézia Salaj: Writing – review and editing, Conceptualization, 358 Methodology. Hana Konrádová: Writing - original draft, Writing – review and editing, Data curation, 359 Project administration, Conceptualization. All authors read and approved the final manuscript. 360 361 **Funding** 362 This work was supported by the Czech Ministry of Education, Youth and Sports, Project No. LUC23150. 363 364 **Conflict of Interest** 365 The authors have no relevant financial or non-financial interests to disclose. 366 367 **Data and Materials Availability** 368 The data generated during the study are available from the corresponding author on reasonable request. 8 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 369 370 **References** 371 Attree SM, Pomeroy MK, Fowke LC. 1992. Manipulation of conditions for the culture of somatic embryos of 372 white spruce for improved triacylglycerol biosynthesis and desiccation tolerance. 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Frontiers in Plant Science. 13:1031686. 531 [https://doi.org/10.3389/fpls.2022.1031686](https://doi.org/10.3389/fpls.2022.1031686) 532 533 **Legends to figures** 534 **Figure 1** The appearance of the hybrid fir embryogenic cultures grown on the medium with maltose (M 535 variant) and sucrose (S variant) after 8 weeks of maturation and 1 additional week on maturation 536 finishing (MF) medium (for details see section 2.2.). 537 **Figure 2** Endogenous soluble carbohydrate contents and spectra in embryogenic line AN72, effect of 538 maltose (Malt) or sucrose (Suc) during maturation. P: proliferation; 4tM, 8tM: 4 [th] and 8 [th] week of 539 maturation; 1tMF: after 1 week of cultivation on maturation-finishing medium. ESM: embryogenic 540 culture analysed as a whole; ie: isolated somatic embryos; RM: mass remaining after embryo isolation. 541 Stach: stachyose; raf: raffinose; glu: glucose; fru: fructose. Total content of soluble carbohydrates (a) 542 and proportion of individual carbohydrates (b). Bars in (a) indicate standard deviations for total 543 carbohydrates, n = 6; for 1tMF Suc n = 3. Asterisk denotes statistical difference in total soluble 544 carbohydrates according to the two-sample _t_ -test, _p_ ≤ 0.05. 545 **Figure 3** Appearance of 3-week-old germinating seedlings (emblings) matured on medium with maltose 546 (a) or sucrose (b). 547 **Figure 4** Endogenous soluble carbohydrate contents and spectra in embryogenic line AN72, effect of 548 maltose (Malt) or sucrose (Suc) after 2 weeks of desiccation at high humidity and subsequent 3 weeks 549 of germination. Isolated embryos; 2wD: 2 weeks of desiccation; 3wG: 3 weeks of germination. Stach: 550 stachyose; raf: raffinose; glu: glucose; fru: fructose. Total content of soluble carbohydrates (a) and 551 proportion of individual carbohydrates (b), n = 5. Bars in (a) indicate standard deviations for total 552 carbohydrates. Asterisk denotes statistical difference in total carbohydrates according to the two-sample 553 _t_ -test, _p_ ≤ 0.05. 12 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 554 **Figures:** 555 556 557 558 **Figure 1** 559 13 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 560 561 562 563 564 |Col1|Col2|Col3|Col4|Col5|Col6|Col7|Col8|Col9|Col10|Col11|Col12|Col13|Col14|Col15|Col16|Col17|Col18|Col19| |---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---| |||||||||||||||||||| |||||||||||||||||||| |||~~*~~|~~*~~|~~*~~|~~*~~|~~*~~|~~*~~|~~*~~|~~*~~|~~*~~|~~*~~|~~*~~|~~*~~|~~*~~|~~*~~|||| |||||||||||||||||||| |||||||||||||||||||| |||||||||||||||||||| |||||||||||||||||||| |||||||||||||||||||| |||||||||||||||||||| 565 **Figure 2** 566 14 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 567 568 569 570 571 572 573 574 575 576 577 578 579 **Figure 3** 580 15 581 582 583 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 584 **Figure 4** 585 586 587 16 588 [bioRxiv preprint doi: https://doi.org/10.64898/2026.11.05.764528; this version posted November 6, 2026. The copyright holder for this](https://doi.org/10.64898/2026.11.05.764528) preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 17