Deep learning for automated segmentation and counting of hypocotyl and cotyledon regions in mature Pinus radiata D. Don. somatic embryo images.

In commercial forestry and large-scale plant propagation, the utilization of artificial intelligence techniques for automated somatic embryo analysis has emerged as a highly valuable tool. Notably, image segmentation plays a key role in the automated assessment of mature somatic embryos. However, to date, the application of Convolutional Neural Networks (CNNs) for segmentation of mature somatic embryos remains unexplored. In this study, we present a novel application of CNNs for delineating mature somatic conifer embryos from background and residual proliferating embryogenic tissue and differentiating various morphological regions within the embryos. A semantic segmentation CNN was trained to assign pixels to cotyledon, hypocotyl, and background regions, while an instance segmentation network was trained to detect individual cotyledons for automated counting. The main dataset comprised 275 high-resolution microscopic images of mature Pinus radiata somatic embryos, with 42 images reserved for testing and validation sets. The evaluation of different segmentation methods revealed that semantic segmentation achieved the highest performance averaged across classes, achieving F1 scores of 0.929 and 0.932, with IoU scores of 0.867 and 0.872 for the cotyledon and hypocotyl regions respectively. The instance segmentation approach demonstrated proficiency in accurate detection and counting of the number of cotyledons, as indicated by a mean squared error (MSE) of 0.79 and mean absolute error (MAE) of 0.60. The findings highlight the efficacy of neural network-based methods in accurately segmenting somatic embryos and delineating individual morphological parts, providing additional information compared to previous segmentation techniques. This opens avenues for further analysis, including quantification of morphological characteristics in each region, enabling the identification of features of desirable embryos in large-scale production systems. These advancements contribute to the improvement of automated somatic embryogenesis systems, facilitating efficient and reliable plant propagation for commercial forestry applications.

Real-World Experience of Metronomic Chemotherapy in Metastatic Breast Cancer: Results of a Retrospective Unicenter Study.

Introduction: Metronomic chemotherapy (MCT) is increasingly used in oncology due to its favorable therapeutic index. There is still a lack of evidence for MCT in metastatic breast cancer (MBC). In this retrospective unicenter study, we demonstrated real-word data on MCT in MBC. Methods: MBC patients who received metronomic oral cyclophosphamide (CTX) (50 mg daily) and methotrexate (MTX) (2.5 mg every other day), CTX and capecitabine (CAPE) (500 mg thrice daily), CTX, or vinorelbine (VRL) (30 mg daily) alone for at least 4 weeks between 2009 and 2021 were included. The primary endpoint was disease control rate (DCR) ≥24 weeks. Secondary endpoints were progression-free survival (PFS) and overall survival (OS). Patient characteristics and therapy response were analyzed using χ2 test. For survival analyses, Kaplan-Meier estimator and log-rank test were used. Results: Seventy-two patients were identified. Sixty-two patients received CTX/MTX, three CTX/CAPE, two CTX, and five VRL. Median age at diagnosis MBC and at start of MCT was 59.0 years and 64.5 years, respectively. 72.2% tumors were hormone receptor positive and 27.8% were triple-negative. 54.2% patients had more than two different metastases. 80.6% patients showed visceral involvement. 31.9% patients achieved DCR ≥24 weeks. Median PFS was 17.0 weeks (95% CI 14.5-19.5) and median OS was 58.0 weeks (95% CI 29.0-87.0). MCT showed similar DCR ≥24 weeks and clinically meaningful but not statistically significant shorter median PFS compared to prior therapy (31.9% versus 32.8% [p = 0.570] and 17.0 weeks versus 20.0 weeks [p = 0.093], respectively) and statistically significant higher DCR ≥24 weeks and longer median PFS compared to subsequent therapy (31.9% versus 17.4% [p = 0.038] and 17.0 weeks versus 12.0 weeks [p = 0.006], respectively). Three (4.2%) patients terminated MCT because of toxicity. Conclusion: In this real-world retrospective study, MCT was effective and well tolerated and may thus represent a valuable treatment option in selected MBC patients.

Application of Cold Storage and Short In Vitro Germination for Somatic Embryos of Pinus radiata and P. sylvestris.

Somatic embryogenesis (SE) is an advanced vegetative propagation technology that, when used in combination with breeding and cryopreservation, offers the forest industry a powerful tool for the deployment of elite genotypes. Germination and acclimatization are critical and cost-intensive phases in the production of somatic plants. The efficient conversion of somatic embryos into robust plants is a necessity if a propagation protocol is to be successfully adopted by the industry. In this work, these late phases of the SE protocol of two pine species were investigated. A shortened germination protocol and more controlled acclimatization were investigated for Pinus radiata, testing embryos from 18 embryogenic cell lines. A more simplified protocol, including a cold storage phase, was also compared among 10 of these cell lines. A shortened germination period and more controlled protocols significantly improved the acclimatization of somatic embryos directly from the lab to the glasshouse. When results for all cell lines were pooled, there were significant improvements in all growth characteristics (shoot height, root length, root collar diameter, and root quadrant score). When the more simplified protocol involving cold storage was tested, improvements were seen in the root architecture. For Pinus sylvestris, the late phases of somatic embryogenesis were investigated on seven cell lines in a set of two trials (four to seven cell lines per trial). During the germination phase, a shortened and simplified in vitro period, a cold storage option and basal media were explored. Viable plants were obtained from all treatments. However, there is still the need to improve germination and related protocols together with growing regimes for Pinus sylvestris. The improvements to protocols presented here, particularly for Pinus radiata, result in greater survival and quality of somatic emblings, leading to reduced costs and increased confidence in the technology. Simplified protocols using a cold storage option show great promise and, with some further research, could lead to reductions in the cost of the technology.

Different Responses in Vascular Traits between Dutch Elm Hybrids with a Contrasting Tolerance to Dutch Elm Disease.

The ascomycetous fungus Ophiostoma novo-ulmi is the causative agent of the current Dutch elm disease (DED) pandemic, which has ravaged many tens of millions of European and North American elm trees. Host responses in vascular traits were studied in two Dutch elm hybrids, 'Groeneveld' and 'Dodoens', which show different vascular architecture in the secondary xylem and possess contrasting tolerances to DED. 'Groeneveld' trees, sensitive to DED, possessed a high number of small earlywood vessels. However, these trees showed a poor response to DED infection for the earlywood vascular characteristics. Following infection, the proportion of least vessels with a vessel lumen area less than 2500 µm2 decreased from 65.4% down to 53.2%. A delayed response in the increasing density of vessels showing a reduced size in the latewood prevented neither the rapid fungal spread nor the massive colonisation of the secondary xylem tissues resulting in the death of the infected trees. 'Dodoens' trees, tolerant to DED, possessed a low number of large earlywood vessels and showed a prominent and fast response to DED infection. Vessel lumen areas of newly formed earlywood vessels were severely reduced together with the vessel size : number ratio. Following infection, the proportion of least vessels with a vessel lumen area less than 2500 µm2 increased from 75.6% up to 92.9%. A trend in the increasing density of vessels showing a reduced size was maintained not only in the latewood that was formed in the year of infection but also in the earlywood that was formed in the consecutive year. The occurrence of fungal hyphae in the earlywood vessels that were formed a year following the infection was severely restricted, as revealed by X-ray micro-computed tomography imaging. Possible reasons responsible for a contrasting survival of 'Groeneveld' and 'Dodoens' trees are discussed.

Somatic Embryogenesis of Abies cephalonica Loud.

Greek fir (Abies cephalonica Loudon) belongs to the Mediterranean fir species and is widely distributed in the mountains of Central and Southern Greece. Considering a climatic scenario, infestation by pathogens or insects and fire episodes, it has been proposed that Mediterranean firs could be in danger in some parts of their present range but, on the other hand, could also replace other species in more northern zones with temperate humid climates (e.g., silver fir, Abies alba Mill.). As fir species are generally highly productive and therefore important for commercial forestry, they have traditionally been involved in conventional tree improvement programs. A lot of effort has been put into the development of vegetative propagation methods for firs, in order to rapidly gain the benefits of traditional breeding to be utilized in reforestation. The present paper provides up to date information on protocols for somatic embryogenesis (i.e., the most promising in vitro method for vegetative propagation) of Greek fir. Moreover, the protocols for cryopreservation and long-term storage of embryogenic material are described as well.

Host responses and metabolic profiles of wood components in Dutch elm hybrids with a contrasting tolerance to Dutch elm disease.

BACKGROUND AND AIMS: Changes occurring in the macromolecular traits of cell wall components in elm wood following attack by Ophiostoma novo-ulmi, the causative agent of Dutch elm disease (DED), are poorly understood. The purpose of this study was to compare host responses and the metabolic profiles of wood components for two Dutch elm (Ulmus) hybrids, 'Groeneveld' (a susceptible clone) and 'Dodoens' (a tolerant clone), that have contrasting survival strategies upon infection with the current prevalent strain of DED. METHODS: Ten-year-old plants of the hybrid elms were inoculated with O. novo-ulmi ssp. americana × novo-ulmi. Measurements were made of the content of main cell wall components and extractives, lignin monomer composition, macromolecular traits of cellulose and neutral saccharide composition. KEY RESULTS: Upon infection, medium molecular weight macromolecules of cellulose were degraded in both the susceptible and tolerant elm hybrids, resulting in the occurrence of secondary cell wall ruptures and cracks in the vessels, but rarely in the fibres. The (13)C nuclear magnetic resonance spectra revealed that loss of crystalline and non-crystalline cellulose regions occurred in parallel. The rate of cellulose degradation was influenced by the syringyl:guaiacyl ratio in lignin. Both hybrids commonly responded to the medium molecular weight cellulose degradation with the biosynthesis of high molecular weight macromolecules of cellulose, resulting in a significant increase in values for the degree of polymerization and polydispersity. Other responses of the hybrids included an increase in lignin content, a decrease in relative proportions of d-glucose, and an increase in proportions of d-xylose. Differential responses between the hybrids were found in the syringyl:guaiacyl ratio in lignin. CONCLUSIONS: In susceptible 'Groeneveld' plants, syringyl-rich lignin provided a far greater degree of protection from cellulose degradation than in 'Dodoens', but only guaiacyl-rich lignin in 'Dodoens' plants was involved in successful defence against the fungus. This finding was confirmed by the associations of vanillin and vanillic acid with the DED-tolerant 'Dodoens' plants in a multivariate analysis of wood traits.

Changes in ATP, glucose-6-phosphate and NAD(P)H cellular levels during the proliferation and maturation phases of Abies alba Mill. embryogenic cultures.

The aim of the present study was to evaluate the adenosine triphospate (ATP), glucose-6-phosphate (glu-6P) and reduced form of nicotinamide adenine dinucleotide phosphate (NAD(P)H) cellular levels during the proliferation and maturation phases of Abies alba Mill. somatic embryos. For a better understanding of the dynamics of these parameters during the proliferation cycle, four embryonic cell lines were tested. During the maturation period, three independent experiments were conducted, focused on the effects of PEG-4000 (5 or 10% (w/v)) and abscisic acid (16, 32 or 64 µM) applied together (Experiments A and B) or with addition of gibberellic acid (Experiment C) on the dynamics of bio-energetic molecules and on the mean number of cotyledonary somatic embryos. Our results demonstrated that the cellular levels of bio-energetic molecules strongly depended on the composition of maturation media. Generally, the higher the number of cotyledonary embryos produced, the higher the level of ATP observed after a 2-week maturation period. The cellular level of ATP, glu-6P and NAD(P)H increased, particularly after the transition from the proliferation to the maturation phase when the differentiation and growth of somatic embryos occurred.

Leaf trait dissimilarities between Dutch elm hybrids with a contrasting tolerance to Dutch elm disease.

BACKGROUND AND AIMS: Previous studies have shown that Ophiostoma novo-ulmi, the causative agent of Dutch elm disease (DED), is able to colonize remote areas in infected plants of Ulmus such as the leaf midrib and secondary veins. The objective of this study was to compare the performances in leaf traits between two Dutch elm hybrids 'Groeneveld' and 'Dodoens' which possess a contrasting tolerance to DED. Trait linkages were also tested with leaf mass per area (LMA) and with the reduced Young's modulus of elasticity (MOE) as a result of structural, developmental or functional linkages. METHODS: Measurements and comparisons were made of leaf growth traits, primary xylem density components, gas exchange variables and chlorophyll a fluorescence yields between mature plants of 'Groeneveld' and 'Dodoens' grown under field conditions. A recently developed atomic force microscopy technique, PeakForce quantitative nanomechanical mapping, was used to reveal nanomechanical properties of the cell walls of tracheary elements such as MOE, adhesion and dissipation. KEY RESULTS: 'Dodoens' had significantly higher values for LMA, leaf tissue thickness variables, tracheary element lumen area (A), relative hydraulic conductivity (RC), gas exchange variables and chlorophyll a fluorescence yields. 'Groeneveld' had stiffer cell walls of tracheary elements, and higher values for water-use efficiency and leaf water potential. Leaves with a large carbon and nutrient investment in LMA tended to have a greater leaf thickness and a higher net photosynthetic rate, but LMA was independent of RC. Significant linkages were also found between the MOE and some vascular traits such as RC, A and the number of tracheary elements per unit area. CONCLUSIONS: Strong dissimilarities in leaf trait performances were observed between the examined Dutch elm hybrids. Both hybrids were clearly separated from each other in the multivariate leaf trait space. Leaf growth, vascular and gas exchange traits in the infected plants of 'Dodoens' were unaffected by the DED fungus. 'Dodoens' proved to be a valuable elm germplasm for further breeding strategies.

Long-term cryopreservation of Greek fir embryogenic cell lines: recovery, maturation and genetic fidelity.

In coniferous species, including Greek fir (Abies cephalonica Loud), the involvement of somatic embryo plants in breeding and reforestation programs is dependent on the success of long-term cryostorage of embryogenic cultures during clonal field testing. In the present study on Greek fir, we assayed the recovery, morphological characteristics and genetic fidelity of embryogenic cell lines 6 and 8 during proliferation and maturation after long-term cryostorage. Our results indicate successful recovery of both cell lines after 6 years in cryostorage. In the maturation phase, both cell lines were capable of producing somatic embryos although some differences were detected among experiments. However, these changes were more dependent on the differences in the components of the maturation media or in the experimental set-up than on the long-term cryostorage. During both proliferation and maturation phases, the morphological fidelity of the embryogenic cultures as well as of the somatic embryos were alike before and after cryopreservation. The genetic fidelity of the cryopreserved cell line 6 that was assayed by random amplified polymorphic DNA (i.e. RAPD) markers demonstrated some changes in the RAPD profiles. The results indicate possible genetic aberrations caused by long-term cryopreservation or somaclonal variation during the proliferation stage. However, in spite of these changes the embryogenic cultures did not lose their proliferation or maturation abilities.

Fulvic acid affects proliferation and maturation phases in Abies cephalonica embryogenic cells.

Embryogenic cell masses (ECM) of Abies cephalonica were grown on proliferation media in the presence and absence of fulvic acid (FA), whose molecular composition and conformational rigidity were evaluated by CPMAS-¹³C NMR spectroscopy. To assess the physiological effects of this humic material during proliferation and maturation stages of somatic embryogenesis (SE), proliferation rate, proportion of consecutive developmental stages of pro-embryogenic masses (PEM), cellular ATP and glucose-6-phosphate were evaluated at regular intervals. FA increased the proliferation rate, especially during the early sampling days, and the percentage of PEM in their advanced developmental stage. Cellular ATP and glucose-6-phospahte were increased by FA pre-treatment during the maturation phase. Furthermore, the effects of the anti-auxin p-chlorophenoxyisobutyric acid (PCIB), such as a decrease of growth and the enhancement of PEM III induction, were inverted by FA. Proton pumping ATPase and PPase activities were decreased in microsomes from PCIB-treated ECM, while they increased in the presence of FA. This fulvic matter also induced a delay in somatic embryo formation during the maturation phase. Both the improvement of the PEM proliferation and the reduction of the subsequent maturation process of A. cephalonica are explained by a release from the complex humic structure of low molecular-weight molecules, which may interact with the plant hormonal signaling pathway. These effects appear to be related to the hydrophilic and conformationally labile nature of FA. The structure-activity relationship observed here suggests that the influence of FA on ECM may be attributed to specific bioactive molecules that are preferentially released from the FA loose superstructure.

Mitochondrial bioenergetics linked to the manifestation of programmed cell death during somatic embryogenesis of Abies alba.

The present work reports changes in bioenergetic parameters and mitochondrial activities during the manifestation of two events of programmed cell death (PCD), linked to Abies alba somatic embryogenesis. PCD, evidenced by in situ nuclear DNA fragmentation (TUNEL assay), DNA laddering and cytochrome c release, was decreased in maturing embryogenic tissue with respect to the proliferation stage. In addition, the major cellular energetic metabolites (ATP, NAD(P)H and glucose-6-phosphate) were highered during maturation. The main mitochondrial activities changed during two developmental stages. Mitochondria, isolated from maturing, with respect to proliferating cell masses, showed an increased activity of the alternative oxidase, external NADH dehydrogenase and fatty-acid mediated uncoupling. Conversely, a significant decrease of the mitochondrial K (ATP)(+) channel activity was observed. These results suggest a correlation between mitochondrial activities and the manifestation of PCD during the development of somatic embryos. In particular, it is suggested that the K (ATP)(+) channel activity could induce an entry of K(+) into the matrix, followed by swelling and a release of cytochrome c during proliferation, whereas the alternative pathways, acting as anti-apoptotic factors, may partially counteract PCD events occurring during maturation of somatic embryos.

Activity of a KATP+ channel in Arum spadix mitochondria during thermogenesis.

This report demonstrates that mitochondria isolated from thermogenic Arum spadices possess an ATP-sensitive potassium channel--responsible for electrical potential (DeltaPsi) collapse and mitochondrial swelling--whose characteristics are similar to those previously described in pea and wheat mitochondria. In order to study the relationship between this K(ATP)(+) channel and the uncoupled respiration, linked to thermogenesis, K(+) transport activities were compared with those of mitochondria that were isolated from pea stems, soybean suspension cell cultures and Arum tubers. The channel from Arum spadices is highly active and its major features are (i) potassium flux is performed primarily in an inward-rectifying manner; (ii) the influx of K(+) is associated with a matrix volume increase in both energized and non-energized mitochondria; and (iii) its activity depends on the redox state of electron transport chain (ETC) and oxygen availability. In particular, this paper shows that the K(ATP)(+) channel is inwardly activated in parallel with the alternative oxidase (AO). The activation is linked to an ETC-oxidized state and to high oxygen consumption. The putative role of this K(ATP)(+) channel is discussed in relation to flowering of thermogenic Arum spadices.

Isolation of mitochondria from embryogenic cultures of Picea abies (L.) Karst. and Abies cephalonica Loud.: characterization of a K+(ATP) channel.

A valuable method to isolate and purify mitochondria from embryonal masses of two coniferous species (Picea abies [L.] Karst. and Abies cephalonica Loud.) is described. Crude mitochondria from both species were shown to be intact, oxygen consuming (with malate plus glutammate, succinate and NADH as substrates) and well coupled (respiratory control ratio ca. 4). The oxidation of the substrates was only partially KCN-insensitive (alternative oxidase) in some cases. However, these fractions were contaminated by membranes (e.g. plasmalemma, tonoplast, Golgi and endoplasmic reticulum). After purification by a discontinuous Percoll gradient (18, 23, 40%, v/v), three mitochondrial populations were separated. The 0/18 interface fraction was composed mainly of broken and uncoupled mitochondria, while the other two (18/23 and 23/40 interface fractions) contained intact and coupled mitochondria, but only 23/40 interface fraction revealed to be better purified starting from both coniferous embryonal masses. In the latter purified fraction, the presence of a cyclosporin A-sensitive K (ATP) (+) channel was demonstrated. These findings were discussed in the light of the potential use of these mitochondrial fractions in bioenergetic studies, or in the involvement of these organelles to stress response in conifers.

Involvement of the mitochondrial K(+)ATP channel in H2O2- or NO-induced programmed death of soybean suspension cell cultures.

Soybean suspension cell cultures were treated by H2O2 or nitric oxide (NO), to assess the mechanism leading to programmed cell death (PCD). Hydrogen peroxide (5 mM) induced PCD. Cells become necrotic at 20 mM H2O2, with cells exhibiting intermediate hallmarks before that (necrapoptotic cells). The level of ATP and of glucose-6-phosphate remained constant in cells undergoing PCD, while it decreased significantly in the necrotic ones. Mitochondria, isolated from 5 mM H2O2-treated (apoptotic) cells, showed that succinate-dependent oxygen consumption was slightly uncoupled, and the electrical potential difference (delta psi) weakly decreased. The addition of KCl to the delta psi formed determined a partial dissipation, which was higher than the dissipation observed in mitochondria from control cells. The addition of cyclosporin A (CsA) to de-energized mitochondria also induced delta psi formation, due to a K+ efflux from the matrix, which was decreased in mitochondria from treated cells. The same pattern of response was also observed in mitochondria isolated from 1 mM sodium nitroprusside (NO)-treated cells, exhibiting apoptotic symptoms. In mitochondria isolated from 20 mM H2O2-treated (necrotic) cells, succinate-dependent oxygen consumption was completely uncoupled, delta psi generation significantly inhibited, and CsA-dependent delta psi formation prevented. In addition, mitochondria isolated from control cells still underwent swelling, which was partially or completely prevented in mitochondria isolated from apoptotic or necrotic cells, respectively. The moderate swelling was accompanied by a slight rupture of the outer membrane and by a release of cytochrome c. These results point to the involvement of a K(+)ATP channel during the manifestation of PCD induced by H2O2 or NO in plants.