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.

D-arginine (D-Arg) can promote embryogenic callus (EC) proliferation and increase the rate of somatic embryo induction of litchi (Litchi chinensis Sonn.), yet the mechanism underlying the processes is incompletely understood. To investigate the mechanism, physiological responses of polyamines (PAs) [putrescine (Put), spermidine (Spd), and spermine (Spm)] were investigated for D-Arg-treated litchi EC and enzyme activity related to polyamine metabolism, plant endogenous hormones, and polyamine- and embryogenic-related genes were explored. Results showed that the exogenous addition of D-Arg reduces the activity of diamine oxidase (DAO) and polyamine oxidase (PAO) in EC, reduces the production of H2O2, promotes EC proliferation, and increases the (Spd + Spm)/Put ratio to promote somatic embryo induction. Exogenous D-Arg application promoted somatic embryogenesis (SE) by increasing indole-3-acetyl glycine (IAA-Gly), kinetin-9-glucoside (K9G), and dihydrozeatin-7-glucoside (DHZ7G) levels and decreasing trans-zeatin riboside (tZR), N-[(-)-jasmonoyl]-(L)-valine (JA-Val), jasmonic acid (JA), and jasmonoyl-L-isoleucine (Ja-ILE) levels on 18 d, as well as promoting cell division and differentiation. The application of exogenous D-Arg regulated EC proliferation and somatic embryo induction by altering gene expression levels of the WRKY family, AP2/ERF family, C3H family, and C2H2 family. These results indicate that exogenous D-Arg could regulate the proliferation of EC and the SE induction of litchi by changing the biosynthesis of PAs through the alteration of gene expression pattern and endogenous hormone metabolism.

Somatic embryogenesis of Korean pine (Pinus koraiensis Sieb. Et Zucc.), an ecologically and econimically very important conifer species, was hindered by the gradually weakens and fast runaway of the embryogenicity and embryo competence of the embryogenic callus. Brassinolide (BL) has shown the enhancing capability of somatic embryo regeneration. For checking the function of BL in this issue, we applied different concentrations of BL to Korean pine callus materials exhibiting different embryogenic capacities and subsequently monitored the physiological alterations and hormone dynamics of the embryogenic callus. Our study revealed that calli with different embryogenic strengths responded differently to different concentrations of BL, but the effect after the addition of BL was very uniform. The addition of BL during the proliferation phase of embryogenic callus may help to stimulate the biological activity of callus during the proliferation process and improve the level of cell metabolism, which is accompanied by a reduction in storage substances. BL could reduce the level of endogenous auxin IAA in embryogenic callus and increase the level of abscisic acid to regulate cell division and differentiation. In addition, the MDA content in the callus was significantly decreased and the activity of antioxidant enzymes was significantly increased after the addition of BL. During the proliferation of embryogenic callus, BL was added to participate in the metabolism of phenylpropane in the cells and to increase the activity of phenylalanine ammonia-lyase and the content of lignin in the cells. We deduced that the proper doses of BL for Korean pine embryogenic callus culture were as follow: calli with low, high and decreasing embryogenicity were subcultured after the addition of 0.75 mg/L, 0.35 mg/L, 2.00 mg/L BL, respectively, during proliferation culture stage.

GATA transcription factors, which are DNA-binding proteins with type IV zinc finger binding domains, have a role in transcriptional regulation in biological organisms. They have an indispensable role in the growth and development of plants, as well as in improvements in their ability to face various environmental stresses. To date, GATAs have been identified in many gene families, but the GATA gene in longan (Dimocarpus longan Lour) has not been studied in previous explorations. Various aspects of genes in the longan GATA family, including their identification and classification, the distribution of their positions on chromosomes, their exon/intron structures, a synteny analysis, their expression at different temperatures, concentration of PEG, early developmental stages of somatic embryos and their expression levels in different tissues, and concentrations of exogenous hormones, were investigated in this study. This study showed that the 22 DlGATAs could be divided into four subfamilies. There were 10 pairs of homologous GATA genes in the synteny analysis of DlGATA and AtGATA. Four segmental replication motifs and one pair of tandem duplication events were present among the DlGATA family members. The cis-acting elements located in promoter regions were also found to be enriched with light-responsive elements, which contained related hormone-responsive elements. In somatic embryos, DlGATA4 is upregulated for expression at the globular embryo (GE) stage. We also found that DlGATA expression was strongly up-regulated in roots and stems. The study demonstrated the expression of DlGATA under hormone (ABA and IAA) treatments in embryogenic callus of longan. Under ABA treatment, DlGATA4 was up-regulated and the other DlGATA genes did not respond significantly. Moreover, as demonstrated with qRT-PCR, the expression of DlGATA genes showed strong up-regulated expression levels under 100 μmol·L-1 concentration IAA treatment. This experiment further studied these and simulated their possible connections with a drought response mechanism, while correlating them with their expression under PEG treatment. Overall, this experiment explored the GATA genes and dug into their evolution, structure, function, and expression profile, thus providing more information for a more in-depth study of the characteristics of the GATA family of genes.

Sex segregation increases the cost of Carica papaya production through seed-based propagation. Therefore, in vitro techniques are an attractive option for clonal propagation, especially of hermaphroditic plants. Here, we performed a temporal analysis of the proteome of C. papaya calli aiming to identify the key players involved in embryogenic callus formation. Mature zygotic embryos used as explants were treated with 20 μM 2,4-dichlorophenoxyacetic acid to induce embryogenic callus. Total proteins were extracted from explants at 0 (zygotic embryo) and after 7, 14, and 21 days of induction. A total of 1407 proteins were identified using a bottom-up proteomic approach. The clustering analysis revealed four distinct patterns of protein accumulation throughout callus induction. Proteins related to seed maturation and storage are abundant in the explant before induction, decreasing as callus formation progresses. Carbohydrate and amino acid metabolisms, aerobic respiration, and protein catabolic processes were enriched throughout days of callus induction. Protein kinases associated with auxin responses, such as SKP1-like proteins 1B, accumulated in response to callus induction. Additionally, regulatory proteins, including histone deacetylase (HD2C) and argonaute 1 (AGO1), were more abundant at 7 days, suggesting their role in the acquisition of embryogenic competence. Predicted protein-protein networks revealed the regulatory role of proteins 14-3-3 accumulated during callus induction and the association of proteins involved in oxidative phosphorylation and hormone response. Our findings emphasize the modulation of the proteome during embryogenic callus initiation and identify regulatory proteins that might be involved in the activation of this process.

Oats (Avena sativa) hold immense economic and nutritional value as a versatile crop. They have long been recognized as an exceptional choice for human consumption and animal feed. Oats\' unique components, including proteins, starches, and β-glucans, have led to its widespread use in various food products such as bread, noodles, flakes, and milk. The popularity of oat milk as a vegan alternative to dairy milk has soared due to the increasing number of vegetarians/vegans and growing environmental awareness. Oat milk offers a sustainable option with reduced greenhouse gas emissions during its production, rendering it an appropriate choice for individuals who are lactose-intolerant or have dairy allergies. To ensure improved adaptability and enhanced nutrition, the development of new oat varieties is crucial, considering factors like cultivation, climate, and growing conditions. Plant cell culture plays a crucial role in both traditional and contemporary breeding methods. In classical breeding, plant cell culture facilitates the rapid production of double haploid plants, which can be employed to accelerate the breeding process. In modern breeding methods, it enables genetic manipulation and precise genome editing at the cellular level. This review delves into the importance of oats and their diverse applications, highlighting the advantages of plant cell culture in both classical and modern breeding methods. Specifically, it provides an overview of plant tissue culture, encompassing genetic transformation, haploid technology, protoplast technology, and genome editing.

BACKGROUND: Tree peony (Paeonia sect. Moutan DC.) is a famous flower native to China with high ornamental, medicinal, and oil value. However, the low regeneration rate of callus is one of the main constraints for the establishment of a genetic transformation system in tree peony. By histomorphological observation, transcriptomic analysis and metabolite determination, we investigated the molecular mechanism of somatic embryogenesis after the establishment of a culture system and the induction of somatic embryo(SE) formation.
RESULTS: We found that SE formation was successfully induced when cotyledons were used as explants. A total of 3185 differentially expressed genes were screened by comparative transcriptomic analysis of embryogenic callus (EC), SE, and non-embryogenic callus (NEC). Compared to NEC, the auxin synthesis-related genes GH3.6 and PCO2 were up-regulated, whereas cytokinin dehydrogenase (CKX6) and CYP450 family genes were down-regulated in somatic embryogenesis. In SE, the auxin content was significantly higher than the cytokinin content. The methyltransferase-related gene S-adenosylmethionine synthase (SAMS) and the flavonoid biosynthesis-related gene (ANS and F3\'5\'H) were down-regulated in somatic embryogenesis. The determination of flavonoids showed that rhoifolin and hyperoside had the highest content in SE. The results of transcriptome analysis were consistent with the relative expression of 8 candidate genes by quantitative polymerase chain reaction analysis.
CONCLUSIONS: The results revealed that auxin and cytokinin may play a key role in \'Fengdan\' somatic embryogenesis. The genes related to somatic embryogenesis were revealed, which has partly elucidated the molecular mechanism of somatic embryogenesis in \'Fengdan\'.

Sinopodophyllum hexandrum (Royle) T. S. Ying, an important source of podophyllotoxin (PTOX), has become a rare and endangered plant because of over-harvesting. Somatic embryogenesis (SE) is the main way of seedling rapid propagation and germplasm enhancement, but the regeneration of S. hexandrum has not been well established, and the PTOX biosynthesis abilities at different SE stages remain unclear. Therefore, it is extremely important to elucidate the SE mechanism of S. hexandrum and clarify the biosynthesis variation of PTOX. In this study, the transcriptomes of S. hexandrum at different SE stages were sequenced, the contents of PTOX and 4\'-demethylepipodophyllotoxin were assayed, and the transcript expression patterns were validated by qRT-PCR. The results revealed that plant hormone (such as auxins, abscisic acid, zeatin, and gibberellins) related pathways were significantly enriched among different SE stages, indicating these plant hormones play important roles in SE of S. hexandrum; the expression levels of a series of PTOX biosynthesis related genes as well as PTOX and 4\'-demethylepipodophyllotoxin contents were much higher in embryogenic callus stage than in the other stages, suggesting embryogenic callus stage has the best PTOX biosynthesis ability among different SE stages. This study will contribute to germplasm conservation and fast propagation of S. hexandrum, and facilitate the production of PTOX.

The development of a cell suspension culture system for the scaling up of coconut embryogenic callus (EC) production would drastically improve efforts to achieve the large-scale production of high-quality clonal plantlets. To date, the hard nature of coconut EC appeared to be the main constraint for developing cell suspension cultures. Hence, this study attempted to acquire friable EC through the following approaches: The manipulation of (1) medium type and subculture frequency, (2) a reduced 2,4-dichlorophenoxy acetic acid concentration during subculture, (3) the nitrate level and the ammonium-to-nitrate ratio, and the addition of amino acid mixture, (4) the addition of L-proline, and (5) the reduction of medium nutrients. Unfortunately, none of these culture conditions produced friable coconut EC. Even though friable EC was not achieved via these approaches, some of the conditions were found to influence the formation of compact EC, therefore these results are important for further studies focused on somatic embryogenesis in coconut and other species.

Lily (Lilium spp.) has elegant flowers and beautiful colors, which makes it popular among people. However, the poor stress resistance and self-propagation ability of lily limit its application in landscaping to a great extent. In addition, transgenic technology is an important means to improve plant characteristics, but the lack of a stable and efficient genetic transformation system is still an important factor restricting the development of lily transgenic technology. Therefore, this study established a good lily regeneration system by screening different explants and plant growth regulators of different concentrations. Then, the genetic transformation system of lily was optimized by screening the critical concentration of antibiotics, the concentration of bacterial solution, and the infection time. Finally, the homologous lily cold resistance gene LlNAC2 and bulblet generation gene LaKNOX1 were successfully transferred to \'Siberia\' and \'Sorbonne\' to obtain lily transgenic lines. The results showed that when the stem axis was used as explant in \'Siberia\', the induction rate was as high as 87%. The induction rate of \'Sorbonne\' was as high as 91.7% when the filaments were used as explants. At the same time, in the optimized genetic transformation system, the transformation rate of \'Siberia\' and \'Sorbonne\' was up to 60%. In conclusion, this study provides the theoretical basis and technical support for improving the resistance and reproductive ability of Oriental lily and the molecular breeding of lily.