Zygotic Embryos Research Articles

Micropropagation of coconut (Cocos nucifera Linn var. pandan) through somatic embryogenesis technique

Coconut, also known as Cocos nucifera, is one of the most recalcitrant species for in vitro regeneration. Conventional coconut breeding has a long lifetime and high heterozygosity, making it a time-consuming, intricate, and costly technique for plant growth. As a result, it is critical to replant most of the world's agricultural land and to develop new soil. This massive undertaking will necessitate planting at least a billion coconut palm trees, which cannot be propagated by seed. Somatic embryogenesis, as a biotechnological alternative, is thus required. Work on this subject has been done in laboratories in numerous countries, with different techniques and explant shapes being investigated. The zygotic embryos are more effective in initiating the callus induction fortified on C1 and C2 formulations added with activated charcoal, gelling agent, and hormone 2, 4- dichlorophenoxyacetic acid (2,4- D). The study has observed that the C1 media successfully initiated callus formation, but the C2 media allowed embryogenic calluses and somatic embryos to develop. From this study, the seedling and regenerated callus were growing well on C3 media. Mature coconut plantlets were acclimated using a combination of sphagnum, sand, and soil mix. The plantlets, on the other hand, could not adapt to their circumstances.

Enhanced micropropagation protocol for teak (Tectona grandis) through zygotic embryo and in vitro seedling-derived explants

Enhanced micropropagation protocol for teak (Tectona grandis) through zygotic embryo and in vitro seedling-derived explants

Transcriptomic time-series analyses of gene expression profile during zygotic embryo development in Taxodium distichum × Taxodium mucronatum

Transcriptomic time-series analyses of gene expression profile during zygotic embryo development in Taxodium distichum × Taxodium mucronatum

Histological Analysis of Somatic Embryogenesis from Immature Zygotic Embryo of Wild Banana Musa acuminata ssp. malaccensis

Somatic embryogenesis, a crucial plant regeneration method, has become indispensable for crop improvement, particularly for species reliant on somatic cell manipulation techniques. Optimization of this process necessitates an understanding of the developmental stages involved. This study investigates the histological aspects of somatic embryogenesis in Musa acuminata ssp. malaccensis derived from immature zygotic embryos. Through detailed histological analysis, we aimed to elucidate the morphological changes and cellular organization occurring during the various stages of somatic embryogenesis, from induction, culture proliferation, and somatic embryo development to plantlet conversion. The initial stages of embryogenesis, characterized by nodules, were primarily composed of meristematic cells with high cell division activity. These cells contained tetrad-like structures that could develop into distinct two- and four-celled proembryoids or proembryogenic aggregates. Our histo-anatomical analysis revealed that embryogenic cultures proliferated through multiple pathways simultaneously: somatic embryo budding, proembryo formation, and pro-embryonic mass formation from both internal and peripheral cells. At the stage of somatic embryo development, embryos with a well-defined protoderm layer, containing cells with prominent nuclei and dense cytoplasm, potentially regenerate into plantlets. Furthermore, histological examination revealed the presence of procambium within mature somatic embryos, which subsequently developed into the vascular system of the complete plantlet

A transcriptome atlas of zygotic and somatic embryogenesis in Norway spruce.

Somatic embryogenesis (SE) is a powerful model system for studying embryo development and an important method for scaling up availability of elite and climate-adapted genetic material of Norway spruce (Picea abies L. Karst). However, there are several steps during the development of the somatic embryo (Sem) that are suboptimal compared to zygotic embryo (Zem) development. These differences are poorly understood and result in substantial yield losses during plant production, which limits cost-effective large-scale production of SE plants. This study presents a comprehensive data resource profiling gene expression during zygotic and somatic embryo development to support studies aiming to advance understanding of gene regulatory programmes controlling embryo development. Transcriptome expression patterns were analysed during zygotic embryogenesis (ZE) in Norway spruce, including separated samples of the female gametophytes and Zem, and at multiple stages during SE. Expression data from eight developmental stages of SE, starting with pro-embryogenic masses (PEMs) up until germination, revealed extensive modulation of the transcriptome between the early and mid-stage maturing embryos and at the transition of desiccated embryos to germination. Comparative analysis of gene expression changes during ZE and SE identified differences in the pattern of gene expression changes and functional enrichment of these provided insight into the associated biological processes. Orthologs of transcription factors known to regulate embryo development in angiosperms were differentially regulated during Zem and Sem development and in the different zygotic embryo tissues, providing clues to the differences in development observed between Zem and Sem. This resource represents the most comprehensive dataset available for exploring embryo development in conifers.

Comparison of the Development of Zygotic and Somatic Embryos of Theobroma cacao

Comparison of the Development of Zygotic and Somatic Embryos of Theobroma cacao

Establishment, Multiplication, and Biochemical Analysis of Embryogenic Lines of the Amazonian Palm Euterpe precatoria Mart. under Suspension Culture

The palm Euterpe precatoria holds great social, cultural, and environmental importance. The heart of palm and the fruit are the main products used for industrialization due to their energetic properties. Thus, the aim of this study was to establish a suspension cultivation protocol for the species using different explant sources. For this, eight lineages of E. precatoria embryogenic calluses were tested, with five in liquid medium Murashige and Skoog (MS) with 5 μM Picloram and three for comparison in semisolid medium MS with 20 μM Picloram and 5 μM 2iP. The growth curve was obtained by weighing the calli from 60 to 180 days of cultivation. The Gompertz model was applied, and growth kinetics were evaluated. At 100 days, the contents of total soluble sugars (TSSs) and total soluble proteins (TSPs) were determined. Principal components (PCA) were measured. According to the analysis of the data, the cultivation of E. precatoria lineages in liquid medium was successfully carried out, and the establishment was achieved. The model can be considered adequate since the R2 values found describe more than 90% of the growth kinetics of the lineages. In the liquid system, lineages L1 (from leaf explants and multiplied in semisolid medium—SM), L2 (from leaf explants and multiplied in SM), and L6 (from zygotic embryo explants and multiplied in liquid medium—LM) showed the shortest time to double the biomass accumulation. Multivariate analysis reveals a significant increase in masses in liquid cultures, represented by lineages L6 and L2. There was statistical difference in the amount of TSSs extracted; the highest TSS levels were observed in lineages cultivated in LM. The protein content found was very low, showing statistical differences among the lineages. In this work, the establishment and multiplication of embryogenic calli of E. precatoria are described for the first time, and they emerge as viable alternatives for the vegetative propagation of the species.

Somatic embryogenesis and plant regeneration from zygotic embryos of babassu (Attalea speciosa Mart. ex Spreng), a multi-purpose palm tree

Somatic embryogenesis and plant regeneration from zygotic embryos of babassu (Attalea speciosa Mart. ex Spreng), a multi-purpose palm tree

Functional Analysis of the PoSERK-Interacting Protein PorbcL in the Embryogenic Callus Formation of Tree Peony (Paeonia ostii T. Hong et J. X. Zhang).

SERK is a marker gene for early somatic embryogenesis. We screened and functionally verified a SERK-interacting protein to gain insights into tree-peony somatic embryogenesis. Using PoSERK as bait, we identified PorbcL (i.e., the large subunit of Rubisco) as a SERK-interacting protein from a yeast two-hybrid (Y2H) library of cDNA from developing tree-peony somatic embryos. The interaction between PorbcL and PoSERK was verified by Y2H and bimolecular fluorescence complementation analyses. PorbcL encodes a 586-amino-acid acidic non-secreted hydrophobic non-transmembrane protein that is mainly localized in the chloroplast and plasma membrane. PorbcL was highly expressed in tree-peony roots and flowers and was up-regulated during zygotic embryo development. PorbcL overexpression caused the up-regulation of PoSERK (encoding somatic embryogenesis receptor-like kinase), PoAGL15 (encoding agamous-like 15), and PoGPT1 (encoding glucose-6-phosphate translocator), while it caused the down-regulation of PoLEC1 (encoding leafy cotyledon 1) in tree-peony callus. PorbcL overexpression led to increased indole-3-acetic acid (IAA) content but decreasing contents of abscisic acid (ABA) and 6-benzyladenosine (BAPR). The changes in gene expression, high IAA levels, and increased ratio of IAA to ABA, BAPR, 1-Aminocyclopropanecarboxylic acid (ACC), 5-Deoxystrigol (5DS), and brassinolide (BL) promoted embryogenesis. These results provide a foundation for establishing a tree-peony embryogenic callus system.

Comparative analysis of element and hormone content in zygotic embryos of Pinus elliottii and P. elliottii × P. caribaea

Somatic embryogenesis is a crucial method for achieving clonal forestry in conifers. Understanding the development of zygotic embryos is essential not only for enhancing the efficiency and quality of somatic embryogenesis, but also for advancing forestry breeding programs. This study investigated dynamic changes of element and hormone contents during ZE development of Pinus elliottii and its hybrid P. elliottii × P. caribaea. Significant differences in embryo development speed among different clones were observed. Elemental analysis was conducted using inductively coupled plasma mass spectrometry (ICP-MS) and identified 68 elements, including major, minor, and beneficial elements. In both species, the contents of potassium (K), calcium (Ca), iron (Fe), boron (B) and five beneficial elements decreased during early ZE development, while phosphorus (P) and copper (Cu) increased. Significantly higher levels of K, Ca and Fe at the initial stage, and sulfur (S) and nickel (Ni) decreased at later stages were detected in P. elliottii than in the hybrid. For the other elements, except for very few significant differences at certain stages, most differences between the two species did not reach a significant level. The contents of endogenous hormones were determined and different accumulation patterns were detected in most hormones between the two species, except abscisic acid (ABA) which simultaneously decreased with developments by stage 8. Significant differences were found in indole-3-acetic acid (IAA) contents at most stages between species, while higher levels of total cytokinin (CK) at each stage were detected in the hybrid in comparison with those in P. elliottii. As a result, lower IAA to CK ratios in the hybrid than in P. elliottii. Methyl jasmonate (JA-me) and gibberellin A3 (GA3) contents showed a similar pattern and exhibited an M-shaped fluctuation in the hybrid. Furthermore, JA-me, GA3, gibberellin A4 (GA4) and brassinolide (BR) showed significantly higher levels in the hybrid than in P. elliottii. K-means clustering and correlation analyses were used to explore relationships between elements and hormones during embryo development, revealing complex interplay in both species. These data indicate different requirement in element and hormone contents for embryogenesis and suggest species-specific media composition for each step in somatic embryogenesis. The findings provide insights into their developmental processes and informing future research and applications in somatic embryogenesis and forestry breeding.

IN VITRO REGENERATION OF STRIPE EBONY (DIOSPYROS FRUTESCENS BLUME)

The Stripe ebony tree (Diospyros frutescens Blume) is considered a species in danger of extinction due to overexploitation. Therefore, there is an urgent need to regenerate and conserve the species for future generation. In this study micropropagation protocol of Diospyros frutescens Blume was developed using nature zygotic embryos. Zygotic embryos isolated from germinated on MS medium containing 0.4mg L-1 Kinetin + 1.0mg L-1 BA. There were (83.11±0.79%) of explants induced shoots after 30 days of culture. The highest number of shoot on 1/2 MS medium supplemented with 2.5mg L-1 BA + 0.6mg L-1 IBA was 5.84±0.035 shoots per explant after 45 days of culture. The percentage of shoot produced roots the highest 85.52±0.48% after 45 days culture on 1/2 MS medium contain 0.8mg L-1 IBA.

A simple and efficient non-tissue culture method for genetic transformation of grape immature zygotic embryos via VvBBM overexpression

The process of genetic transformation of grapevine has long been hampered by the inefficiency and complexity of tissue culture-based methods, severely limiting cultivar improvement and functional genome research. In this study, we aimed to develop a novel, non-tissue culture-based method for the genetic transformation of grape immature zygotic embryos without relying on tissue culture. This approach involves the overexpression of the Vitis vinifera BABY BOOM (VvBBM) gene, a key member of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor family, which is known to enhance plant regeneration and transformation efficiency. Using ‘Cabernet Sauvignon’ immature zygotic embryos, we achieved a transformation efficiency of 42.85 % under non-tissue culture conditions, significantly reducing the cycle duration to approximately two months compared to traditional genetic transformation methods. Phenotypic and molecular analyses of the transformed grapevine plants confirmed the integration and overexpression of the VvBBM gene, resulting in improved growth metrics, including increased plant height, root length, and total root number, in a genotype-independent manner. Our results highlight the potential of VvBBM overexpression to circumvent the limitations imposed by tissue culture dependence, providing a rapid, efficient, and scalable alternative to conventional methods. Our study not only provides a practical approach to overcoming the barriers to genetic transformation in grapevine, but also opens new avenues for the application of this technology in the breeding of other economically important crops.

Cryopreservation of zygotic embryos derived from BBTV resistant accessions of wild banana Musa acuminata

The wild banana species Musa acuminata encompasses numerous subspecies, some of which serve as ancestors of edible bananas. Preserving these subspecies is critical for food security, especially in regions where bananas are staple crops. While cryopreservation of M. acuminata subsp. burmanicoides zygotic embryos using air dehydration have shown high success rates, this method has not been effective for other subspecies. With at least 15 subspecies displaying various morphological traits, including seed morphology, alternative cryopreservation methods warrant exploration. In a recent study, successful cryopreservation using the droplet-vitrification method was achieved for mature zygotic embryos of accession of M. acuminata subspecies tomentosa, rutilifes, and malaccensis, all of which are resistant to the Banana bunchy top virus (BBTV). Using this approach, subjecting embryos to dehydration for 20 min in PVS2 solution and employing a regeneration medium comprising MS medium supplemented with benzyladenine and thidiazuron resulted in a success rate of 77.4 % (86 % survival rate × 90 % regrowth rate) for subsp. tomentosa and 50.4 % (70 % survival rate × 72 % regrowth rate) for subsp. malaccensis. Conversely, for subsp. rutilifes, dehydration for 10 min led to a success rate of 15.6 % (30 % survival rate × 52 % regrowth rate). Subspecies emerged as the primary factor influencing embryo regrowth, suggesting the need for protocol optimizations tailored to other subspecies. These findings highlight the potential of droplet-vitrification as a long-term preservation method for certain subspecies, while underscoring the necessity for further optimization for the other subspecies of M. acuminata.

Effect of Vitis vinifera zygotic embryo cryopreservation and post-cryopreservation on the gene expression of DNA demethylases

Grapevine (Vitis vinifera L.) crops are continuously exposed to biotic and abiotic stresses, which can cause genetic and epigenetic alterations. To determine the possible effects of grapevine cryopreservation on the regulation of DNA demethylase genes, this work studied the expression of DNA demethylase genes in cryopreserved and post-cryopreserved grapevine tissues. V. vinifera DNA demethylases were characterized by in silico analysis, and gene expression quantification was conducted by RT‒qPCR. Three DNA demethylase sequences were found: VIT_13s0074g00450 (VvDMT), VIT_08s0007g03920 (VvROS1), and VIT_06s0061g01270 (VvDML3). Phylogenetic analysis revealed that the sequences from V. vinifera and A. thaliana had a common ancestry. In the promoters of responsive elements to transcription factors such as AP-2, Myb, bZIP, TBP, and GATA, the conserved domains RRM DME and Perm CXXC were detected. These responsive elements play roles in the response to abiotic stress and the regulation of cell growth. These data helped us characterize the V. vinifera DNA demethylase genes. Gene expression analysis indicated that plant vitrification solution 2 (PVS2) treatment does not alter the expression of DNA demethylase genes. The expression levels of VvDMT and VvROS1 increased in response to cryopreservation by vitrification. Furthermore, in post-cryopreservation, VvROS1 was highly induced, and VvDML3 was repressed in all the treatment groups. Gene expression differences between different treatments and tissues may play roles in controlling methylation patterns during gene regulation in tissues stressed by cryopreservation procedures and in the post-cryopreservation period during plant growth and development.

Characterization of Citrus × Poncirus Embryo Rescued Hybrids as Rootstock Candidate using Morphological Markers

In generally, there are both of nucellar and zygotic embryos in the citrus seeds after cross breeding. Since genetic variations are very important for success of plant breeding, morphological traits of individuals are evaluated to identify diversity. The present study aimed to characterization of citrus hybrids for new rootstock genotypes based on its morphological characters at seedling stage. A total of 335 putative interspecies hybrids, derived from 3 crosses [Common sour orange (Citrus aurantium L.) × Troyer citrange (Citrus sinensis L. × Poncirus trifoliata L.), Common mandarin (Citrus deliciosa Ten.) × Troyer citrange (Citrus sinensis L. × Poncirus trifoliata L.) and King mandarin (Citrus nobilis L.) × Carrizo citrange (Citrus sinensis L. × Poncirus trifoliata L.)], were observed by their plant morphology. The eight qualitative and five quantitative characteristics of hybrid plants such as seedling growth, leaf and thorniness characteristics were evaluated. The average plant height of the population was found between 70.2 cm and 133.2 cm. The average stem diameter varied between 5.9 mm and 8.0 mm. Hybrid seedlings were separated on the basis of dominant trifoliate leaf marker. There was wide diversity among the accessions with respect to quantitative leaf characters. In terms of leaf division, 268 genotypes have bifoliate and 67 were trifoliate in all combinations, and many intermediate forms were also observed. In addition 66 of the genotypes were thornless while 269 of the genotypes were thorny. The genotype No. 4, has been assessed as triploid, from Common mandarin × Troyer citrange combination, has the longest and dense spines. Morphological markers data were analysed by clustering method to compare similarities of hybrids. The dissimilarity index was observed between 0.004 and 17.318 within three hybridization combinations. The hybrids obtained at 110 days after pollination were more distant relative to each other all hybridization combination.

Proteomic Profiling of Cocos nucifera L. Zygotic Embryos during Maturation of Dwarf and Tall Cultivars: The Dynamics of Carbohydrate and Fatty Acid Metabolism.

There is a limited number of studies analyzing the molecular and biochemical processes regulating the metabolism of the maturation of Cocos nucifera L. zygotic embryos. Our research focused on the regulation of carbohydrate and lipid metabolic pathways occurring at three developmental stages of embryos from the Mexican Pacific tall (MPT) and the Yucatan green dwarf (YGD) cultivars. We used the TMT-synchronous precursor selection (SPS)-MS3 strategy to analyze the dynamics of proteomes from both embryos; 1044 and 540 proteins were determined for the MPT and YGD, respectively. A comparison of the differentially accumulated proteins (DAPs) revealed that the biological processes (BP) enriched in the MPT embryo included the glyoxylate and dicarboxylate metabolism along with fatty acid degradation, while in YGD, the nitrogen metabolism and pentose phosphate pathway were the most enriched BPs. Findings suggest that the MPT embryos use fatty acids to sustain a higher glycolytic/gluconeogenic metabolism than the YGD embryos. Moreover, the YGD proteome was enriched with proteins associated with biotic or abiotic stresses, e.g., peroxidase and catalase. The goal of this study was to highlight the differences in the regulation of carbohydrate and lipid metabolic pathways during the maturation of coconut YGD and MPT zygotic embryos.

ABA exerts a promotive effect on the early process of somatic embryogenesis in Quercus aliena Bl

Quercus aliena, a native Chinese tree species, is significant in industry and landscaping. However, it is traditionally propagated by seeds with many limitations, such as pest infestations, seed yield and quality. Thus, this study firstly introduces a somatic embryogenesis (SE) system for Q. aliena, enhancing its cultivation prospects. Thereinto, the development stage of zygotic embryo had a significant effect on SE, only immature embryos in 10–11 weeks after full bloom (WAF), rich in endogenous abscisic acid (ABA), could induce SE. Exogenous application ABA had positive roles in the early development process of both primary and secondary SE, while its antagonist had opposite roles. Transcriptome analysis showed that transcription regulation occupied the major position. Mfuzz cluster and WGCNA co-expression analysis showed that 24 candidate genes were involved in the SE process. The expression of the 24 genes were also affected by exogenous ABA signals, among which QaLEC2, QaCALS11 and QaSSRP1 occupied the important roles. Additionally, the callose content were also affected by exogenous ABA signals, which had significantly positive correlations with the expression of QaLEC2 and QaCALS11. This study not only established an efficient reproduction system for Q. aliena, but also revealed the difference in embryogenic ability of zygotic embryos from the aspects of transcriptome and endogenous hormone content, and lay a foundation for clarifying the molecular mechanism of SE, and provided a reference for exploring the vital roles of ABA in SE.

Effect of abiotic and biotic elicitors on vincristine accumulation in endosperm derived in vitro cultures in Catharanthus roseous (L.) G. Don

Catharanthus roseus (L.) G. Don (Apocynaceae) is a well-studied herb renowned for its in vitro culture as a source of the anti-cancer alkaloid, vincristine. However, despite the recognized advantages of triploid cells over diploid cells in terms of productivity, the triploid endosperm tissue of this important medicinal plant has not been utilized for in vitro culture initiation. In this investigation, zygotic embryos and endosperm tissues were cultured on Murashige and Skoog (MS) medium supplemented with various combinations of auxins and cytokinins. The medium containing 2.50 µM 6-Benzyladenine (BA) and 1.25 µM 2,4-Dichlorophenoxyacetic acid (2,4-D) proved to be the most effective for callus and cell culture formation. Ploidy analysis using ploidy analyzer confirmed that the endosperm-derived callus exhibited mixoploid, while the embryo-derived callus remained diploid. Liquid Chromatography Mass Spectrometry (LC–MS) analysis of callus and cell cultures grown on MS media with different combinations of auxins, cytokinins, elicitors, and precursors (both biotic and abiotic) revealed the accumulation of vincristine. Notably, treatment with a biotic elicitor derived from Aspergillus niger (300 mg/l) demonstrated superior efficacy in promoting the maximum accumulation of vincristine in endosperm-derived callus and cell biomass. These findings hold promise for the sustainable production of the anti-cancer alkaloid vincristine from endosperm-derived callus and cell cultures of Catharanthus roseus.

Mining GATA Transcription Factor Encoding Genes in The Cocoa Tree (<i>Theobroma cacao</i> L.) Suggests Their Potential Roles in Embryo Development and Biotic Stress Response

GATA transcription factors (TFs) are widely recognized as significant regulators, characterized by a DNA-binding domain that consists of a type IV zinc finger motif. This TF family has been widely investigated in numerous higher plant species. The purpose of the present work was to comprehensively analyze the GATA TF in cocoa plant (Theobroma cacao L.) by using various bioinformatics tools. As a result, a total of 24 members of the GATA TFs have been identified and annotated in the assembly of the cocoa plant. According to phylogenetic analysis, these TcGATA proteins were classified into four distinct groups, including groups I (10 members), II (seven members), III (five members), and IV (two members). Next, our investigation indicated that the TcGATA proteins in different groups exhibited a high variation in their physic-chemical features due to their different protein lengths, gene structures, and conserved motif distributions, whereas the TcGATA proteins in the same clade might share the common conserved motifs. Additionally, the gene duplication of the TcGATA genes in the cocoa plant was also investigated. Of our interest, the relative expression levels of the TcGATA genes were investigated according to available transcriptome databases. The results exhibited differential expression patterns of all TcGATA genes in various developmental stages of zygotic and somatic embryogenesis, indicating that these TcGATA genes divergently function during various developmental stages of the zygotic and somatic embryos. Moreover, TcGATA genes were differently expressed under Phytophthora megakarya treatment across different points of treatment and cocoa varieties. To sum up, our findings could provide a basis for a further deep understanding of the GATAs in the cocoa plant.

Long-Term Conservation for the Safeguard of Abies nebrodensis: An Endemic and Endangered Species of Sicily.

The combined approaches between ex situ and in situ conservation are of great importance for threatened species in urgent need of protection. This study aims to develop concrete actions to preserve the relic of 30 adult trees of the Sicilian fir (Abies nebrodensis) from extinction using long-term germplasm conservation in liquid nitrogen (LN, -196 °C). Pollen grains were collected, and their moisture content (MC) was measured. Then, viability (2,3,5-tryphenyl tetrazolium chloride, TTC), in vitro germinability, and enzymatic antioxidant activity (ascorbate peroxidase, APX; catalase, CAT) were evaluated before and after cryopreservation. Seeds collected from mature cones underwent X-ray analysis, and only full seeds were used to excise the zygotic embryos (ZEs) for cryopreservation. The MC percentage of ZEs was determined, and then they were plunged in LN with (+PVS2) or without (-PVS2) Plant Vitrification Solution 2; untreated ZEs were used as a control. Viability (TTC test) and in vitro germination were assessed for all ZEs (+PVS2, -PVS2, and control). Embryogenic callus (EC) lines obtained from mature ZEs were cryopreserved applying the 'encapsulation-dehydration' technique. This study has allowed, after optimizing cryopreservation protocols for pollen, ZEs, and EC of A. nebrodensis, to establish the first cryobank of this endangered species in Polizzi Generosa (Palermo, Italy), inside the 'Madonie Regional Park'. The strategy developed for Sicilian fir conservation will pave the way for similar initiatives for other critically endangered conifer species.