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.

Pengaruh Jenis Eksplan dan Asam Amino pada Inisiasi dan Proliferasi Kalus Embriogenik Phalaenopsis Var. ‘Raiza Agrihorti’

<p><strong>(<em>The Effect of Explant Types and Amino Acids on Embryogenic Callus Initiation and Proliferation of Phalaenopsis Var. ‘Raiza Agrihorti’</em>)</strong></p><p>Penyiapan kalus embriogenik (KE) yang optimal memiliki peranan penting dalam menghasilkan benih bermutu Phalaenopsis skala komersial. Kendala utama yang dihadapi ialah inisiasi dan proliferasi KE yang masih rendah, serta akumulasi fenolik yang tinggi. Penelitian dilakukan di Laboratorium Kultur Jaringan Balithi dari Agustus 2019 hingga Juli 2020. Penelitian menggunakan Rancangan Acak Kelompok (RAK) pola split plot dan faktorial dengan lima ulangan. Percobaan-1: jenis eksplan (pucuk, pangkal, dan daun plantlet) sebagai petak utama dan perlakuan asam amino (tanpa asam amino, L-Proline, L-Glutamine, L-Cysteine, dan Casein-Hydrolisate) dengan konsentrasi 150 mg/l pada medium PC1 (1/2 MS + 1,0 mg/l TDZ + 0,5 mg/l BAP + 20 g/l sukrosa) sebagai anak petak. Percobaan-2: faktor-1 ialah jenis asam amino (L-Proline, L-Cysteine; L-Glutamine, dan Casein-Hydrolisate) dan faktor-2 ialah konsentrasi asam amino (0, 75, 150, 225, dan 300 mg/l). Hasil penelitian menunjukkan bahwa inisiasi KE Phalaenopsis var. ‘Raiza Agrihorti’ terbaik didapatkan dari pangkal plantlet dan 150 mg/l L-Glutamine dengan waktu inisiasi 18,3-24,0 hari, 80-100% pembentukan KE, dan ukuran KE 0,4-0,5 cm3. Proliferasi KE terbaik ditemukan pada L-Glutamine dengan konsentrasi 150 mg/l. Proliferasi KE mencapai 100% dengan penambahan berat segar sebesar 0,39 g, tingkat multiplikasi (MR) 4,55 kali dan pencokelatan 4,0%. Hasil penelitian ini berpotensi tinggi untuk diterapkan pada kultur starter Phalaenopsis hibrida lain.</p><p><strong>Keywords</strong></p><p>Phalaenopsis hibrid; Asam amino; Inisiasi; Kalus embriogenik; Proliferasi</p><p><strong>Abstract</strong></p><p>Setup of the optimum Phalaenopsis embryogenic callus (EC) is an important role in producing qualified-seedlings of Phalaenopsis in commercial scale. The main constraints that are still being faced are the low rate of culture proliferation and high phenolic accumulations. The research was carried out at the Tissue Culture Laboratory-Indonesian Ornamental Plants Research Institite, from August 2019 through July 2020. The split plot and factorial designs were arranged using a Randomized Completely Block Design (RCBD) with five replications. Experiment-1: explants type (shoot tip, basal part, and leaf of plantlet) was used as main plot and amino acids (amino acids free, L-Proline, L-Glutamine, L-Cysteine, and Casein-Hydrolisate) with 150 mg/l concentration on medium PC1 (1/2 MS + 1,0 mg/l TDZ + 0,5 mg/l BAP + 20 g/l sukrosa) as subplot. Experiment-2: the first factor was amino acids type (L-Proline, L-Cysteine; L-Glutamine, and Casein-Hydrolisate) and the second factor was amino acids concentration (0, 75, 150, 225, and 300 mg/l). Results of the studies revealed that the best EC initiation of Phalaenopsis var. ‘Raiza Agrihorti’ was produced by basal part of plantlet and PC1 medium containing 150 mg/l L-Glutamine with EC Initiation time was 18.3-24.0 days, 80-100% of EC formation and size of 0.4-0.5 cm3. The best proliferation of EC was found in L-Glutamine with 150 mg/l concentration. EC proliferation reached 100% with 4.55 EC multiplication rate, 0.39 g EC fresh weight added, and EC browning as low as 4.0%. The established method is high possibly applied for other Phalaenopsis hybrids.</p>

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.

Global Transcriptome and Coexpression Network Analyses Reveal New Insights Into Somatic Embryogenesis in Hybrid Sweetgum (Liquidambar styraciflua × Liquidambar formosana)

Somatic embryogenesis (SE) is a process of somatic cells that dedifferentiate to totipotent embryonic stem cells and generate embryos in vitro. Despite recent scientific headway in deciphering the difficulties of somatic embryogenesis, the overall picture of key genes, pathways, and co-expression networks regulating SE is still fragmented. Therefore, deciphering the molecular basis of somatic embryogenesis of hybrid sweetgum remains pertinent. In the present study, we analyzed the transcriptome profiles and gene expression regulation changes via RNA sequencing from three distinct developmental stages of hybrid sweetgum: non-embryogenic callus (NEC), embryogenic callus (EC), and redifferentiation. Comparative transcriptome analysis showed that 19,957 genes were differentially expressed in ten pairwise comparisons of SE. Among these, plant hormone signaling-related genes, especially the auxin and cytokinin signaling components, were significantly enriched in NEC and EC early. The K-means method was used to identify multiple transcription factors, including HB-WOX, B3-ARF, AP2/ERF, and GRFs (growth regulating factors). These transcription factors showed distinct stage- or tissue-specific expression patterns mirroring each of the 12 superclusters to which they belonged. For example, the WOX transcription factor family was expressed only at NEC and EC stages, ARF transcription factor was expressed in EC early, and GRFs was expressed in late SE. It was noteworthy that the AP2/ERF transcription factor family was expressed during the whole SE process, but almost not in roots, stems and leaves. A weighted gene co-expression network analysis (WGCNA) was used in conjunction with the gene expression profiles to recognize the genes and modules that may associate with specific tissues and stages. We constructed co-expression networks and revealed 22 gene modules. Four of these modules with properties relating to embryonic potential, early somatic embryogenesis, and somatic embryo development, as well as some hub genes, were identified for further functional studied. Through a combination analysis of WGCNA and K-means, SE-related genes including AUX22, ABI3, ARF3, ARF5, AIL1, AIL5, AGL15, WOX11, WOX9, IAA29, BBM1, MYB36, LEA6, SMR4 and others were obtained, indicating that these genes play an important role in the processes underlying the progression from EC to somatic embryos (SEs) morphogenesis. The transcriptome information provided here will form the foundation for future research on genetic transformation and epigenetic control of plant embryogenesis at a molecular level. In follow-up studies, these data could be used to construct a regulatory network for SE; Key genes obtained from coexpression network analysis at each critical stage of somatic embryo can be considered as potential candidate genes to verify these networks.

Global Transcriptomic Analysis Reveals Differentially Expressed Genes Involved in Embryogenic Callus Induction in Drumstick (Moringa oleifera Lam.)

The plant embryogenic callus (EC) is an irregular embryogenic cell mass with strong regenerative ability that can be used for propagation and genetic transformation. However, difficulties with EC induction have hindered the breeding of drumstick, a tree with diverse potential commercial uses. In this study, three drumstick EC cDNA libraries were sequenced using an Illumina NovaSeq 6000 system. A total of 7191 differentially expressed genes (DEGs) for embryogenic callus development were identified, of which 2325 were mapped to the KEGG database, with the categories of plant hormone signal transduction and Plant-pathogen interaction being well-represented. The results obtained suggest that auxin and cytokinin metabolism and several embryogenesis-labeled genes are involved in embryogenic callus induction. Additionally, 589 transcription factors from 20 different families were differentially expressed during EC formation. The differential expression of 16 unigenes related to auxin signaling pathways was validated experimentally by quantitative real time PCR (qRT-PCR) using samples representing three sequential developmental stages of drumstick EC, supporting their apparent involvement in drumstick EC formation. Our study provides valuable information about the molecular mechanism of EC formation and has revealed new genes involved in this process.

Oil palm (Elaeis guineensis Jacq.) micropropagation via somatic embryogenesis from female inflorescences explants

The oil palm (Elaeis guineensis Jacq.) is a perennial woody oil crop in the Arecaceae family. Oil palm is well known for a long regeneration time, therefore, in vitro propagation received great enthusiasm from oil palm industries. Somatic embryogenesis (SE) has become one of the most promising clonal propagation techniques in recent times. This study was aimed to determine a protocol for micropropagation somatic embryogenesis from female inflorescences explants of oil palm. The explant used was obtained from the female inflorescences of the oil palm 2.5 years old Tenera variety. The basal media used was Y3 with the addition of the hormone 2,4-D with different concentrations (33,66,99 and 132 mg/L). Callus initiation was formed in 99 mg/L and 132 mg/L 2,4 – D concentration with the basal area and the percentage of callus formation is 31,25% and embryogenic callus was formed from primary callus development in the basal area of female florescence. Embryo somatic induction with cell suspension culture, because the liquid medium is more efficiently used in commercial-scale propagation. The embryogenic callus phase which is generally used in liquid medium is the nodular phase because it is still meristematic so that the potential for cell division is still high and can increase the percentage of embryogenic callus.

Embryogenic callus induction from immature zygotic embryos and genetic transformation of Larix kaempferi 3x Larix gmelinii 9

To date, there are few reports of the successful genetic transformation of larch and other conifers, mainly because it is difficult to transform and integrate exogenous genes. In this study, hybrid larch Larix kaempferi 3x Larix gmelinii 9 cones were collected on June 27, July 1, July 4, July 7 and July 16, 2017. Embryogenic callus induction was studied using a combination of different plant growth regulators and concentrations. The results showed that July 1 was the best stage; the highest induction rate was 10.83%, which cultured in BM medium (Button medium, which formula was listed in S1 Table) with 1.0 mg/L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.2 mg/L KT(kinetin). When cultured on a proliferation medium for 12 days, proliferation was the fastest, reaching 323.08%, which could also maintain the freshness and vitality. The suitable pre-culture medium for somatic embryogenesis was 1/4 BM medium containing 10 g/L inositol and 60 g/L sucrose. The combination of 45 mg/L ABA (abscisic acid) and 75 g/L PEG4000 (Polyethyene glycol 4000) could promote the number of somatic embryos, and reached the maximum, 210 140 per 1 g FW. The genetic transformation was carried out by the Agrobacterium-mediated transformation method with embryogenic callus cultured for 12 days. The results showed the optimal OD600 of the infection solution(suspension of A. tumefaciens) was 0.5, co-culture time was 2 days, and screening concentration of Hyg (hygromycin B) was 4 mg/L. In this study, the transformation rate of resistance callus was 32.1%. It provides a reference for low genetic transformation efficiency of larch at present. This study could be beneficial for the innovation and breeding of larch by genetic engineering and provides a certain basis for rapid propagation of excellent larch germplasm resources and genetic engineering breeding of larch and other conifers.

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.