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.

Efficient Transformation of Catalpa bungei Shows Crystal Genes Conferring Resistance to the Shoot Borer Omphisa plagialis

Although Catalpa bungei is a forest plant with considerable economic and ornamental value in China, its wood and decorative qualities are constrained by insect pests such as the shoot borer Omphisa plagialis (Lepidoptera). Overexpressing insect resistance genes such as crystal genes to develop an insect-resistant variety of C. bungei is an environmental and ecological approach. However, genotype limitations and low regeneration rates of embryogenic calli (EC) inhibit the development of transformation and the insect-resistant gene expression system in C. bungei. Here, we first established embryogenic callus induction and regeneration systems of five genotypes using mature seed and stem segment explants; the highest induction and regeneration rates of EC were 39.89 and 100%, respectively. Next, an efficient and stable Agrobacterium-mediated genetic transformation system was developed from EC and its positive frequency was up to 92.31%. Finally, using the transformation system, 15 and 22 transgenic C. bungei lines that expressed Cry2A and Cry9Aa-like were generated, respectively. These transgenic lines that exhibited significantly higher resistance to O. plagialis in the laboratory and field have great promise for meeting the challenge of future pest management under changing climatic conditions. Additionally, this efficient, fast, and stable transformation system could be a potential tool for gene function analysis and forest tree genetic improvement.

Temporary Immersion System Improves Regeneration of in vitro Irradiated Recalcitrant Rice Embryogenic Calli

The development of gamma rays mutant rice lines would be a solution for introducing variability in already farmer using varieties. In vitro gamma (60Co) mutagenesis reduces chimeras and allows a faster selection of desired traits but requires laboratory process optimization. The objective of the present work was the in vitro establishment of a recalcitrant rice embryogenic calli, the determination of its sensitivity to gamma radiation (Co-60), sequencing MATK and Rubisco for identification purposes, as well as generation optimization. The radiosensitivity of embryogenic calli resulted in an LD50 of 110Gy, while the 20% lethal dose was 64Gy. All sequenced genes matched perfectly with already reported MATK and Rubisco O. sativa genes with a clear SNP that identifies the local variety related to the southeast Asia Region. Callus induction improves with an MS with 2mg/L 2,4D, and the regeneration was achieved with an MS medium with 3mg/L BAP and 0,5mg/L NAA. The optimized radiation condition was 60Gy with an 83% regeneration in a semisolid medium, allowing a balance between mutation and regeneration. When increased to 80Gy, the regeneration rate falls to 29%. An immersion system (RITA®) of either 60 or 120 seconds every 8hours allowed a systematic and homogeneous total regeneration of the recalcitrant line, in contrast with the semisolid medium that resulted in positive but irregular regeneration. Other well-known recalcitrant cultivars, CR1821, CR1113 also had an improving regeneration in the immersion system, demonstrating its potential use for recalcitrant materials. To our knowledge, this is the first report on using an immersion system to allow regeneration of gamma-ray mutants from recalcitrant rice materials.

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.

Inisiasi, pertumbuhan, dan perkembangan kalus embriogenik tanaman stevia (Stevia rebaudiana)

The leaves of sweetener plant Stevia rebaudiana contain secondary metabolites of steviol glycosides which are very sweet, with no calorie and zero glycemic index. Propagation of stevia by seeds is ineffective due to its low germination rate and diverse progenies. The tissue culture of stevia can be used to mass propagate rapidly and is commonly conducted by shoot multiplication. Up to now, the technology of somatic embryogenesis (SE) in stevia has not been successful yet. SE is developed to increase the production scale, rejuvenate clonal-propagated plants, and plant genetic transformation. The research objective was to develop protocols for the initiation, proliferation, and development of embryogenic calli of stevia as potential materials for SE. The explants used were young leaves, nodes, and internodes of axenic plantlets of stevia BX clone. The explants were cultured on MS solid media containing different concentrations of auxin and cytokinin for callus initiation. Callus emerged after 2-3 weeks of culture. The calli obtained were proliferated by subculturing several times as material stocks for indirect SE. MS solid media added with 1 µM 3,4-D and 16 mM CaCl2 gave the highest callus multiplication rate (4.7 times in 3 weeks). The selection of embryogenic calli was made continuously to obtain a pure line of embryogenic calli. Three types of calli attained were friable, fast-growing, yellowish calli, shiny nodular calli, and greenish nodular calli. Histological studies revealed that cells of the nodular calli had been differentiated to potentially formed somatic embryos.

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.

Effect of culture media, auxins and genotypes on plantlet regeneration from oil palm (Elaeis guineensis Jacq.) zygotic embryos through somatic embryogenesis

Aim: This study evaluated efficient culture media for the regeneration of elite material through somatic embryogenesis from oil palm zygotic embryos. Methodology: For callus induction, zygotic embryos of four elite genotypes (G1-264T, G2-238DX17P, G3-37DX17P and G4-237T) were cultured on three basal media (Y3, MS and N6) with different auxin 2 mg l-1 (Picloram, 2,4-D and Dicamba) combinations. Subculture was made every month for three passages. It evaluated various callus characters. The embryogenic calli from callus induction media were transferred to the embryo maturation medium and subcultured until the polyembryoids formed. For shoot and root formation, somatic embryo clumps were transferred into regeneration media. In-vitro plantlets with well-grown roots were hardened in pots for six weeks and assessed clonal fidelity using polymorphic SSR primers. Results: Among the treatments, calli from N6+2,4-D, Y3+2,4-D and N6+Picloram showed the highest embryogenic callus potential. G4-237T induced more embryogenic calli (32.982) among genotypes, which was on par with G1-264T (24.196). Embryogenic calli grown on N6 media with Dicamba showed the highest proliferation rate (1.141). After 60 days of culture on regeneration media, the highest number of plantlets per somatic embryogenic clump was obtained from G1-264T on N6 media supplemented with Dicamba. Interpretation: Culture media salt concentration showed a significant difference among media by causing perturbations of auxin flow during somatic embryogenesis affecting callus induction, proliferation and plantlet regeneration. This may be useful for standardizing the genotype-specific regeneration media in oil palm.

Cryopreservation of Arecanut (Areca catechu L.) Embryogenic Calli by V Cryo-plate Method

Aims: Arecanut, a perennial palm species of Arecaceae family, has huge commercial value, and is grown mainly for its masticatory nuts. The ever-increasing demand for uniform quality plantlets from growers necessitates putting in place In vitro mass multiplication and other crop improvement programmes. The present study was carried out to standardize the procedure for cryopreservation of embryogenic calli of arecanut, derived from immature inflorescence cultures, by vitrification based cryo-plate technique. Study Design: Completely randomized design (CRD) with three replications. Place and Duration of Study: ICAR-Central Plantation Crops Research Institute, Kerala, India during 2019. Methodology: The embryogenic calli were precultured in Eeuwen's Y3 basal medium supplemented with sucrose (0.2, 0.3 and 0.4 M) for three days. Explants were affixed on cryo-plates and later dehydrated using plant vitrification solution 3 (PVS3) for 30 min. Cryoplates were inserted in cryovials and cryopreserved. Explants with no cryostorage served as control. Explants were rewarmed quickly in a water bath (40ºC) for 2 min and treated with unloading solution and cultured on recovery medium. Results: The results showed 8-10 % recovery of embryogenic calli that resulted in normal plantlet production. The clonal fidelity studies, using Start Codon Targeted (SCoT) marker, showed no variation of cryopreserved calli in comparison to the original calli. Conclusion: This preliminary study demonstrated the successful use of vitrification (V) cryo-plate technique in cryopreservation of embryogenic calli of arecanut. With better recovery percentage, the optimal concentration of sucrose in the preculture medium was found to be 0.3 M. Desiccation in PVS3 solution for 30 min had no adverse effect.

Embryogenic Calli Induction and Salt Stress Response Revealed by RNA-Seq in Diploid Wild Species Gossypium sturtianum and Gossypium raimondii

Wild cotton species can contribute to a valuable gene pool for genetic improvement, such as genes related to salt tolerance. However, reproductive isolation of different species poses an obstacle to produce hybrids through conventional breeding. Protoplast fusion technology for somatic cell hybridization provides an opportunity for genetic manipulation and targeting of agronomic traits. Transcriptome sequencing analysis of callus under salt stress is conducive to study salt tolerance genes. In this study, calli were induced to provide materials for extracting protoplasts and also for screening salt tolerance genes. Calli were successfully induced from leaves of Gossypium sturtianum (C1 genome) and hypocotyls of G. raimondii (D5 genome), and embryogenic calli of G. sturtianum and G. raimondii were induced on a differentiation medium with different concentrations of 2, 4-D, KT, and IBA, respectively. In addition, embryogenic calli were also induced successfully from G. raimondii through suspension cultivation. Transcriptome sequencing analysis was performed on the calli of G. raimondii and G. sturtianum, which were treated with 200 mM NaCl at 0, 6, 12, 24, and 48 h, and a total of 12,524 genes were detected with different expression patterns under salt stress. Functional analysis showed that 3,482 genes, which were differentially expressed in calli of G. raimondii and G. sturtianum, were associated with biological processes of nucleic acid binding, plant hormone (such as ABA) biosynthesis, and signal transduction. We demonstrated that DEGs or TFs which related to ABA metabolism were involved in the response to salt stress, including xanthoxin dehydrogenase genes (ABA2), sucrose non-fermenting 1-related protein kinases (SnRK2), NAM, ATAT1/2, and CUC2 transcription factors (NAC), and WRKY class of zinc-finger proteins (WRKY). This research has successfully induced calli from two diploid cotton species and revealed new genes responding to salt stress in callus tissue, which will lay the foundation for protoplast fusion for further understanding of salt stress responses in cotton.