Can Ethylene Inhibitors Enhance the Success of Olive Somatic Embryogenesis?

An efficient in vitro morphogenesis, specifically through somatic embryogenesis, is considered to be a crucial step for the application of modern biotechnological tools for genetic improvement in olive (Olea europaea L.). The effects of different ethylene inhibitors, i.e., cobalt chloride (CoCl2), salicylic acid (SA), and silver nitrate (AgNO3), were reported in the cyclic somatic embryogenesis of olive. Embryogenic callus derived from the olive immature zygotic embryos of the cultivar Leccino, was transferred to the expression ECO medium, supplemented with the ethylene inhibitors at 20 and 40 µM concentrations. Among these, the maximum number of somatic embryos (18.6) was obtained in media containing silver nitrate (40 µM), followed by cobalt chloride (12.2 somatic embryos @ 40 µM) and salicylic acid (40 µM), which produced 8.5 somatic embryos. These compounds interfered on callus traits: white friable embryogenic calli were formed in a medium supplemented with 40 µM cobalt chloride and salicylic acid; in addition, a yellow-compact embryogenic callus appeared at 20 µM of all the tested ethylene inhibitors. The resulting stimulatory action of silver nitrate among all the tested ethylene inhibitors on somatic embryogenesis, clearly demonstrates that our approach can efficiently contribute to the improvement of the current SE protocols for olive.

Somatic Embryogenesis In Rosa Chinensis CV. ‘Parson’s Pink China’

This study investigated the effects of explant type, plant growth regulator concentration, callis status, medium conversion time, and medium tilt on the growth of rose somatic embryos. The results showed that Rosa chinensis cv. ‘Parson’s Pink China’ leaves could induce normal embryogenic calli but petioles could not. When the 2,4-dichlorophenoxyacetic acid concentration was 3.0 mg/L, the callis induction rate was the highest in the embryo proliferation medium (EP medium) supplemented with 0.5mg/L kinetin, and white and reddish-brown translucent calli were the main type of embryogenic calli induced. As the culture time in EP medium was extended, the relative induction rate for secondary embryos and multicotyledon secondary embryos gradually increased when transferred to embryo maturation medium (EM medium), but the induction rate for somatic embryos decreased. Placing the EM medium at an angle of 45° made the somatic embryos germinate faster and the germination rate was also higher. The germination buds produced by the somatic embryos with two cotyledons showed the fastest germination and greatest survival rates. The results of this experiment will help improve somatic embryo regeneration rates and explore new ways of regeneration, and lays the foundation for further optimization of the somatic embryo genetic transformation system.

Single-Base Resolution Methylomes of Somatic Embryogenesis in Theobroma Cacao L. Reveal Epigenome Modifications Associated With Somatic Embryo Abnormalities

Propagation by somatic embryogenesis in Theobroma cacao has some issues to be solved, as many morphologically abnormal somatic embryos that do not germinate into plants are frequently observed, thus hampering plant production on a commercial scale. For the first time the methylome landscape of T. cacao somatic embryogenesis was examined, using whole-genome bisulfite sequencing technique, with the aim to understand the epigenetic basis of somatic embryo abnormalities. We identified 873 differentially methylated genes (DMGs) in the CpG context between zygotic embryos, normal and abnormal somatic embryos, with important roles in development, programmed cell death, oxidative stress, and hypoxia induction, which can help to explain the morphological abnormalities of somatic embryos. We also identified the role of ethylene and its precursor 1-aminocyclopropane-1-carboxylate in several biological processes, such as hypoxia induction, cell differentiation and cell polarity, that could be associated to the development of abnormal somatic embryos. The biological processes and the hypothesis of ethylene and its precursor involvement in the somatic embryo abnormalities in cacao are discussed.

CRISPR/Cas9 Targeted Editing of Genes Associated With Fungal Susceptibility in Vitis vinifera L. cv. Thompson Seedless Using Geminivirus-Derived Replicons

The woody nature of grapevine (Vitis vinifera L.) has hindered the development of efficient gene editing strategies to improve this species. The lack of highly efficient gene transfer techniques, which, furthermore, are applied in multicellular explants such as somatic embryos, are additional technical handicaps to gene editing in the vine. The inclusion of geminivirus-based replicons in regular T-DNA vectors can enhance the expression of clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) elements, thus enabling the use of these multicellular explants as starting materials. In this study, we used Bean yellow dwarf virus (BeYDV)-derived replicon vectors to express the key components of CRISPR/Cas9 system in vivo and evaluate their editing capability in individuals derived from Agrobacterium-mediated gene transfer experiments of ‘Thompson Seedless’ somatic embryos. Preliminary assays using a BeYDV-derived vector for green fluorescent protein reporter gene expression demonstrated marker visualization in embryos for up to 33 days post-infiltration. A universal BeYDV-based vector (pGMV-U) was assembled to produce all CRISPR/Cas9 components with up to four independent guide RNA (gRNA) expression cassettes. With a focus on fungal tolerance, we used gRNA pairs to address considerably large deletions of putative grape susceptibility genes, including AUXIN INDUCED IN ROOT CULTURE 12 (VviAIR12), SUGARS WILL EVENTUALLY BE EXPORTED TRANSPORTER 4 (VviSWEET4), LESION INITIATION 2 (VviLIN2), and DIMERIZATION PARTNER-E2F-LIKE 1 (VviDEL1). The editing functionality of gRNA pairs in pGMV-U was evaluated by grapevine leaf agroinfiltration assays, thus enabling longer-term embryo transformations. These experiments allowed for the establishment of greenhouse individuals exhibiting a double-cut edited status for all targeted genes under different allele-editing conditions. After approximately 18 months, the edited grapevine plants were preliminary evaluated regarding its resistance to Erysiphe necator and Botrytis cinerea. Assays have shown that a transgene-free VviDEL1 double-cut edited line exhibits over 90% reduction in symptoms triggered by powdery mildew infection. These results point to the use of geminivirus-based replicons for gene editing in grapevine and other relevant fruit species.

Formamide deionized accelerates the somatic embryogenesis of Cunninghamia lanceolata

Aim of the study: To improve the efficiency of the somatic embryogenesis (SE) in Cunninghamia lanceolata. Area of the study: The study was conducted at Nanjing Forestry University (Nanjing, China). Material and methods: Immature cones of C. lanceolata, genotype 01A1 which was planted in Yangkou State-owned Forest Farm (Fujian, China), were used to induced callus. These calli were used to induce SE, concentration gradients of 0 g/L, 0.01134 g/L, 0.1134 g/L, 1.1134 g/L and 11.34 g/L of FD was added, to explore the optimal concentration for promoting SE of C. lanceolata. Main results: Low concentration of FD promoted the maturation of somatic embryos, while high concentration of FD lead to browning of embryogenic callus. The seedling rate and rooting number of seedlings induced by different concentrations of FD were significantly different. Research highlights: This study may aid in the rapid maturation of C. lanceolata somatic embryos and is useful for accelerated C. lanceolata breeding.

Somatic embryogenesis from young inflorescences of the giant bamboo Dendrocalamus asper (Schult f.) Backer ex Heyne

Bamboos are an important worldwide non-timber forest product with current rising interest due to their environmentally friendly applications. Besides the consolidated uses of the sweet shoots and culms for structural uses, Dendrocalamus asper is an imposing ornamental bamboo for horticulture. The present work aimed to establish in vitro calli culture and plant regeneration through somatic embryogenesis starting from young inflorescences of the giant bamboo, D. asper. Pre-anthesis inflorescences were collected, disinfested, and subjected to callus induction on MS basal medium supplemented by 0 µM, 9 µM, 18 µM, 27 µM, and 36 µM of 2,4-D in combination with 9 µM of 2-iP or 9 µM Kin. The different obtained calli types were characterized and subcultured in 0 µM, 4.5 µM, 9 µM, and 18 µM of 2,4-D in combination with 9 µM of both cytokinins for multiplication and differentiation. Additionally, the explant incision and its inoculation orientation onto culture media were tested for callus induction improvement. The 2,4-D was essential for callus induction, and its combination with both cytokinins resulted in embryogenic callus induction and further somatic embryos regeneration. The subsequent reduction of this auxin to 4.5 µM resulted in somatic embryo maturation. Somatic embryos transferred to a plant growth regulator-free medium resulted in plantlet conversion. The present work showed the feasibility of using inflorescences as explants and the efficiency of using the 2-iP in combination with 2,4-D to callus induction and in vitro bamboo plant regeneration through somatic embryogenesis.

Kultur Embrio Tiga Spesies Kopi pada Umur Buah dan Formulasi Media yang Berbeda

<em>Studying the fruit age and proper media formulation is one of the important stages in embryo culture of coffee. The data is highly benefical, especially in saving embryos generated from intra- and inter-species crosses that fall prematurely or experience problems in germination. The aim of this study was to determine the suitable age and media formulation for embryo culture of Arabica, Robusta, and Liberica coffee. The study was conducted at the Tissue Culture Laboratory, Indonesian Industrial and Beverage Crops Research Institute from January 2019 to November 2020. Murashige and Skoog (MS) media with growth regulators adapted to embryonic development were used in this study. The three types of coffee divided into 5 groups, namely pinhead, immature, early mature, almost mature, mature, and used as planting material. The research was designed in a completely randomized design with 10 replications, and media formulation as a treatment. The results showed that embryo culture of the three coffee species was conducted successfully, except for pinhead fruit. The older the cultured fruit, the higher the percentage of germination. There is a difference in germination time between the three coffee species. The medium for embryo culture should be adjusted with the age of the fruit being cultured. Aside from growing embryos, the cultured mature fruit embryos on MS medium given 0.5 mg/l BA can also be used for propagation by utilizing the secondary somatic embryos formation.</em>

The Chemical Environment at Maturation Stage in Pinus spp. Somatic Embryogenesis: Implications in the Polyamine Profile of Somatic Embryos and Morphological Characteristics of the Developed Plantlets

Changes in the chemical environment at the maturation stage in Pinus spp. somatic embryogenesis will be a determinant factor in the conversion of somatic embryos to plantlets. Furthermore, the study of biochemical and morphological aspects of the somatic embryos could enable the improvement of somatic embryogenesis in Pinus spp. In the present work, the influence of different amino acid combinations, carbohydrate sources, and concentrations at the maturation stage of Pinus radiata D. Don and Pinus halepensis Mill. was analyzed. In P. radiata, the maturation medium supplemented with 175 mM of sucrose and an increase in the amino acid mixture (1,100 mgL–1 of L-glutamine, 1,050 mgL–1 of L-asparagine, 350 mgL–1 of L-arginine, and 35 mgL–1 of L-proline) promoted bigger embryos, with a larger stem diameter and an increase in the number of roots in the germinated somatic embryos, improving the acclimatization success of this species. In P. halepensis, the maturation medium supplemented with 175 mM of maltose improved the germination of somatic embryos. The increase in the amount of amino acids in the maturation medium increased the levels of putrescine in the germinated somatic embryos of P. halepensis. We detected significant differences in the amounts of polyamines between somatic plantlets of P. radiata and P. halepensis; putrescine was less abundant in both species. For the first time, in P. radiata and P. halepensis somatic embryogenesis, we detected the presence of cadaverine, and its concentration changed according to the species.

Genomic Methylated Cytosine Level during the Dedifferentiation and Cellular Competence in Coffea arabica Lines: Insights about the Different In Vitro Responses

Coffea arabica genotypes present distinct responses in vitro, and somaclonal variation occurrence has been reported. Global cytosine methylation is one of the epigenetic mechanisms that influences the Coffea in vitro responses. We aimed to establish the indirect somatic embryogenesis in C. arabica ‘Catuaí Vermelho’, ‘Caturra’ and ‘Oeiras’, associate the distinct responses to the methylated cytosine genomic level, and check the ploidy stability. Leaf explants were cultured in callus induction and proliferation medium. The resulted calli were transferred to the regeneration medium, and the mature cotyledonary somatic embryos were transferred to the seedling medium. ‘Oeiras’ exhibited the highest number of responsive leaf explants, followed by ‘Caturra’ and ‘Catuaí Vermelho’. Global methylated cytosine level increased over time in the ‘Catuaí Vermelho’ and ‘Caturra’ friable calli, remaining constant in ‘Oeiras’. ‘Oeiras’ did not regenerate somatic embryos, while ‘Catuaí Vermelho’ exhibited the highest number. Somatic embryo regeneration was associated with the increase of the methylated cytosine level. However, the ‘Catuaí Vermelho’ embryogenic calli showed a lower methylated cytosine level than ‘Caturra’. Recovered plantlets exhibited the same 2C value and chromosome number to the explant donors. Therefore, cytosine hypermethylation occurred during C. arabica indirect somatic embryogenesis, influencing cell competence and somatic embryos regeneration.

Morpho-Anatomical and Biochemical Characterization of Embryogenic and Degenerative Embryogenic Calli of Phoenix dactylifera L.

The study of morpho-anatomical aspects, metabolic changes of proteins, antioxidant substances, as well as phenolic compounds in embryogenic callus (EC) and degenerative embryogenic callus (DEC) was the aim of the present investigation. Ability to form somatic embryos (SEs) was associated with the softness of the EC, which exhibited a white or creamy color and was composed of isodiametric cells containing dense cytoplasm, conspicuous nuclei and minimal vacuoles with observed mitotic activity. Furthermore, protein, reduced glutathione (GSH) and ascorbic acid (ASC) concentrations and the ratio between ASC and dehydroascorborbic acid (DHA) were increased significantly in the EC in comparison to the DEC. In addition, the phenolic extract of the EC was proved to have higher scavenging activity than the extract from the DEC. A loss of embryogenic competence in the DEC was correlated with the presence of more rigid clumps and such calli had a yellowish to brown color and no cell division could be observed in the cells of such aggregates as the cells had large vacuoles and they have very thick walls. Moreover, these morphological and anatomical observations of the DEC were accompanied by accumulations of the oxidized form of ascorbic acid (DHA), H2O2, total soluble phenolic compounds and overaccumulation of naringenin. Alternations in cellular metabolism can affect and regulate the morphogenesis of somatic embryos.