Embryogenic Callus Induction Research Articles

Optimization of Key Technologies for Induction of Embryogenic Callus and Maturation of Somatic Embryos in Korean Pine (Pinus koraiensis)

Somatic embryogenesis (SE), which leads to the formation of embryonic callus (EC) tissue, is the most promising method for large-scale production and selective breeding of woody plants. However, in many species, SE suffers from low induction and proliferation rates, hindering the production of improved plant materials. We investigated the effects of the explant sterilization method, 4 °C cryopreservation, basal medium, ethylene removal, liquid medium supplementation, and a combination of PGRs on embryogenic callus (EC) induction of Korean pine, using immature embryos of Korean pine as explants. The effects of sucrose and maltose on EC proliferation and maturation were investigated. The differences in the maturation ability of EC somatic embryos before and after cryopreservation were evaluated using the induced embryonic cell lines. The results showed that zygotic embryos (ZEs) performed better than megagametophytes (MGs) as explants. The induction rate of EC was significantly increased after 28 days of cryopreservation at 4 °C. The induction rate of EC in the #5 family increased from 10.00% to 62.8%. The EC induction rate of the five families cultured with the DCR basal medium was higher than that with the mLV basal medium. Among them, the induction rate of the #5 family cultured with the mLV basal medium was 23.3%, while that with the DCR basal medium was 60.9%, an increase of 2.6 times. There was no significant difference in the maturation ability of EC somatic embryos before and after cryopreservation. In conclusion, this study provides a method to improve the EC induction rate and maturation ability of Korean pine.

Somatic embryogenesis as potential method for commercial propagation in Passiflora edulis Sims f. edulis - an important horticultural crop

Purple passion fruit (Passiflora edulis Sims f. edulis) is one of the species with high commercial value in the genus Passiflora. In this study, the somatic embryogenesis (SEs) obtained from different explants (ex vitro leaf and in vitro root) cultured on medium-supplemented auxins and cytokinins, silver nanoparticles (AgNPs) enhanced SEs and plantlets derived from SEs were evaluated for the first time. The ex vitro leaf blade (1 × 1 cm) and in vitro root fragment (0.1 × 1 cm) were used as the initial explants. The results showed that the addition of auxins and cytokinins in culture medium stimulated SEs in both explant types. Root explant only induced callus on medium supplemented with 2,4-D (1.0 - 4.0 mg L−1), however, somatic embryos were directly induced with 1.0 mg L−1 NAA. While 1.0 - 2.0 mg L−1 2,4-D stimulated embryogenic callus induction (14.81 - 25.93%), NAA stimulated direct embryogenesis from leaf explants. The medium supplemented with 2.0 mg L−1 NAA combined with 1.5 mg L−1 TDZ significantly improved the SEs rate (81.48%) and number of embryos per explant (26.67 embryos) formed directly from leaf explants. On the other hand, the synergism between 2.0 mg L−1 2,4-D and 2.0 mg L−1 AgNPs significantly enhanced callus-derived embryogenesis from leaf explant with the highest SEs rate (92.59%) and number of embryos per explant (31.67 embryos). In addition, AgNPs also had a positive effect on promoting the maturation of callus-derived embryos. The plantlets derived from SEs were acclimatized in the greenhouse with a survival rate of over 90.00%. The SEs-derived plants flowered and produced fruit normally under field conditions. The results revealed a reliable plant regeneration procedure through direct or indirect SEs derived from ex vitro leaf and in vitro root explants and AgNPs-stimulated SEs that can be applied for commercial propagation of this crop.

Insights into chlorogenic acids' efficient biosynthesis through Carthamus tinctorius cell suspension cultures and their potential mechanism as α-glucosidase inhibitors

Carthamus tinctorius is commonly used as food and medicine because it contains various bioactive components, particularly chlorogenic acids (CGAs). However, few research works focused on the development and application of C. tinctorius-derived CGAs. In this study, the technology system for efficient CGAs biosynthesis through C. tinctorius cells was successfully developed, and the feasibility of C. tinctorius cells-derived CGAs as α-glucosidase inhibitors was evaluated. At first, the inflorescence explant was used to induce C. tinctorius embryogenic callus (induction rate: 95%), in which a total of 10 CGAs, including rare caffeoyl and feruloyl-substituted derivatives, were identified and quantified by liquid chromatography-mass spectrometry. Secondly, an excellent C. tinctorius cell suspension culture was established. To further promote CGAs biosynthesis, parameters of elicitation combined with precursor feeding were optimized through Plackett-Burman’s Design and Central Composite Design. Under the optimal condition [cinnamic acid: 283.05 μM, methyl jasmonate: 80.98 μM and yeast extract: 666.66 mg·L−1], CGAs content could reach up to 103.234 mg·g−1 (DW), which was 2.55 times that of the cells in the initial condition. The CGAs production first time reached the highest level among plant-cultured cells in the available literature. At last, the inhibition activity test results indicated good inhibitory activities of C. tinctorius cells-derived CGAs against α-glucosidase (IC50: 0.15–1.38 g·L−1), especially di- and tri-acyl CGAs. Moreover, molecular docking simulation showed that these CGAs could interact with the active site pocket of α-glucosidase to thereby achieve the inhibitory activity. Therefore, these results provided a solid foundation for the large-scale production of CGAs and revealed the application prospects of CGAs in functional food and pharmaceutical industries as α-glucosidase inhibitor.

In vitro bioactive metabolite production and plant regeneration of medicinal plant Angelica sinensis

The root of Angelica sinensis (Oliv.) Diels is a traditional Chinese medicine (TCM) widely used for the cardio-cerebrovascular, anti-inflammatory, and anti-cancer agents, etc. Currently, the root yield is mainly limited by narrow geographical distribution, early bolting and flowering (EBF), continuous cropping obstacle, and germplasm degeneration. While two ancillary strategies to produce bioactive metabolites by bioreactor and innovate new germplasms by genome editing have not been conducted. Here is reported embryogenic callus induction, histomorphological structure identification, suspension cell culture, bioactive metabolite determination, shoot bud and root differentiation, and plantlet acclimatization at different treatments such as explants, hormones, and culture conditions. The embryogenic calli can be effectively induced (99.0%) from the root of aseptic seedlings by a combination of 2.0 mg/L IBA and 0.2 mg/L KT, and embryogenic cells including: three-cell, globular-, heart-, torpedo-, and cotyledon-shaped embryos were observed. The maximum growth rate, bioactive metabolites contents (i.e., ferulic acid, soluble sugar, flavonoids, and phenolic), and antioxidant capacities were observed at 9–12 d with inoculum density 40 g/L, 22 ℃ and 16/8 h light/dark. Robust plantlets can be obtained within 180 d after successive shoot bud and root differentiation as well as plantlet acclimation. Based on the strategies for the satisfaction of root demand, an effective and complete scheme is proposed for producing bioactive metabolites and regenerating plant of A. sinensis.

Efficient Induction of Papaya Embryogenic Callus and Plant Regeneration

Papaya ( Carica papaya  L.) has high medicinal and production value, and it is of great significance to carry out efficient induction and plant regeneration of embryogenic callus of papaya for its industrial development. In this study, Zhongbai, the main papaya cultivar from Hainan was used as explants to explore the effects of different hormones and their additive combinations on embryogenic callus induction and plant regeneration. The results showed that the most suitable medium for papaya callus induction was K5: 1/2 MS+5 mg/L 2, 4-D+0.5 mg/L KT+3% sucrose+3.6 g/L phypagel. The most suitable medium for papaya embryogenic callus induction was M13: 1/2 MS+5 mg/L 2,4-D+0.6 mg/ L-proline+3% sucrose+3.6 g/L phypagel. And the most suitable medium for papaya bud differentiation was Ci: 1/2 MS+0.2 mg/L 6-BA, 0.2 mg/L NAA+3% sucrose+3.6 g/L phypagel. The best method for rooting of papaya was treatment with 100 mg/L rooting powder and soil plantation. This ‘two-steps’ embryogenic callus induction system we created can significantly shorten the growth cycle of embryogenic callus. Furthermore, the system has a certain broad spectrum in different varieties of papaya, this study provides a solid foundation for further improving the efficiency of genetic transformation of papaya.

Regeneration and development of Coffea arabica L. plants through indirect somatic embryogenesis

Micropropagation of AS2K clones Arabica coffee (Coffea arabica L.) was attempted through indirect somatic embryogenesis by using ten different parts of the leaf such as shoot, first leaf base, second leaf base, third leaf base, first leaf middle, second leaf middle, third leaf middle, first leaf tip, second leaf tip, and third leaf tip. The influence of the part of leaf explants, combination of plant growth regulator (PGRs) in the induction of embryogenic callus and regeneration of embryo somatic were studied. Furthermore, the various protocols to induce regeneration of somatic embryo into plantlet through different step of subculture and the use of various germination medium were demonstrated. The morphological characteristics and histological analysis of embryogenic callus and embryo development were observed. In this experiment, it was observed that the M5 medium supplemented with 1 mg/L 2,4-D, 1 mg/L BAP and 4 mg/L 2-ip was closely associated with third leaf tip explants for induction of embryogenic callus. The maximum number of globular, heart-shape, torpedo and cotyledones (18, 4, 12, 4, respectively) were achieved on ERM6 medium containing 2 mg/L BAP without activated charcoal on 90th day for regeneration of embryo somatic. The length of roots is the most influence paramater on plantlet regeneration, and the 17th protocol which used B medium, large embryos and twice phase of subculture from liquid medium to solid medium is the best protocol for plantlet regeneration. The protocol developed could be useful highly for large-scale micropropagation in these commercially important Arabica coffee clones. Key words: Indirect somatic embryogenesis; 24-D; BAP; histology; anatomy.

Effects of mammalian sex hormones on regeneration capacity, retrotransposon polymorphism and genomic instability in wheat (Triticum aestivum L.)

Mammalian steroid hormones (MSH) are also inherently synthesized by several plant species. However, external application of steroid hormones further stimulates cell division, pollen germination, plant growth and development. There is little known about the effects of MSH on polymorphism and DNA damage in Triticum aestivum L under in vitro conditions. In this study, inter-primer binding site (iPBS) retrotransposon and coupled restriction enzyme digestion-iPBS (CRED-iPBS) markers were used for detection of the variation in responded embryogenic callus (REC) that were obtained from endosperm-supported mature embryo of wheat in Murashige and Skoog (MS) medium containing different concentrations [0 (control), 10–4, 10–6 and 10–8 m mol L−1] of MSH (progesterone, 17β-estradiol, estrone and testosterone). Responded embryogenic callus (REC) and regenerable callus (RE) induction from mature embryos varied with MSH type and concentrations. The highest level of genomic template stability (GTS) value (80.52%) was obtained from 10–8 mM progesterone treatments to the lowest value (68.83%) from 10–4 mM 17 β-estradiol treatments. Epigenetic changes were more frequent and variable than the genetic changes. While DNA hypermethylation was observed at higher 17 β-estradiol concentrations, DNA hypomethylation was observed in progesterone treatments. It was concluded based on iPBS and CRED-iPBS findings that high MSH concentrations caused DNA changes and methylation.

Embryogenic Callus Differentiation in Short-Term Callus Derived from Leaf Explants of Alfalfa Cultivars

Alfalfa is a forage crop that accounts for one of the best sources of protein and is commonly grown all over the world. In vitro callus and embryogenic induction of alfalfa have been investigated previously; however, most of the investigations were almost limited with callus formation. In this study, leaves of 5 Medicago sativa L. cultivars (Alsancak, Sazova, Plato Iside, and Bilensoy) have been used. Influences of culture medium contents and hormones applications on callus and embryogenic callus induction were determined to optimize in vitro culture mediums of alfalfa (0.0125 g kinetin 2,4 D 1 mg mL-1; 1 g kinetin 2,4 D 1 mg mL-1 ; 0.25 g kinetin 2,4 D 2 mg mL-1; 0.5 g kinetin 2,4 D 2 mg mL-1). Callus formation was detected at a rate of 74% in 5 different cultivars used in the experiment. The five alfalfa cultivars were classified into four categories in terms of embryogenic differentiation capacity. The tested alfalfa cultivars varied in their callus formation and embryogenic callus differentiation. Sazova, Alsancak, and Bilensoy were detected for better callus formation; similarly, the same cultivars responded with better embryogenic callus formation in the culture mediums including various hormone concentrations. The present study shows that our methods have beneficial impacts on the somatic embryo induced by alfalfa. However, it depends strongly on genotype, hormone concentrations and the other medium components.

Plant regeneration from leaf mesophyll derived protoplasts of cassava (Manihot esculenta Crantz).

A high yield of isolated protoplast and reliable regeneration system are prerequisite for successful somatic hybridization and genome editing research. However, reproducible plant regeneration from protoplasts remains a bottleneck for many crops, including cassava. We evaluated several factors that influence isolation of viable protoplasts form leaf mesophyll, induction of embryogenic calli, and regeneration of plants in three cassava cultivars; Muchericheri, TMS60444 and Karibuni. A relatively higher protoplast yield was obtained with enzyme mixture containing 5 g/L Macerozyme and 10 g/L cellulase. Muchericheri recorded relatively higher protoplast yield of 20.50±0.50×106 whereas TMS60444 (10.25±0.25×106) had the least protoplast yield in 10 g/L cellulase and 4 g/L cellulase. Freshly isolated protoplast cells were plated on callus induction medium (CIM) solid medium containing MS basal salt, 60 g/L D-glucose, 30 g/L sucrose, B5 vitamins, 100 mg/L myo-inositol, 0.5 mg/L copper sulphate, 100 mg/L casein hydrolysate, 4.55 g/L mannitol, 0.1 g/L MES, 10 mg/L picloram and 3 g/L gelrite to induce protoplast growth and development. The three cultivars reached colony formation but no further development was observed in this culture method. Protoplast growth and development was further evaluated in suspension culture using varying cell densities (1, 2 and 3× 105 p/mL). Development with highest number of minicalli was observed in cell density of 3× 105 p/mL. Minicalli obtained were cultured on CIM supplemented with 10mg/L picloram. Callus induction was observed in all cell densities with the cultivars. Highest somatic embryogenesis was observed in 2× 105 p/ml while no somatic embryogenesis was observed in cell density of 1×105 p/mL. Somatic embryos were matured in EMM medium supplemented with 1 mg/L BAP, 0.02 mg/L NAA and 1.5 mg/L GA3 then germinated in hormone free medium for plant regeneration. This protocol which used simple mixture of commercial enzymes is highly reproducible and can be applied in biotechnology research on cassava.

Callus induction and transcriptomic analysis of in vitro embryos at different developmental stages of peony.

The efficient induction of peony embryogenic callus is of great significance to the improvement and establishment of its regeneration technology system. In this study, the in vitro embryos of 'Fengdanbai' at different developmental stages were selected as explants, the effects of different concentrations and types of plant growth regulator combinations on the induction and proliferation of embryonic callus at different developmental stages were investigated, and comparative transcriptome analysis of callus with different differentiation potentials were performed to explore the molecular mechanisms affecting callus differentiation. The results showed that the germination rate of 90d seed embryo was the best, which was 94.17%; the 70d and 80d cotyledon callus induction effect was the best, both reaching 100%, but the 80d callus proliferation rate was higher, the proliferation rate reached 5.31, and the optimal induction medium was MS+0.1 mg·L-1NAA+0.3 mg·L-1TDZ+3 mg·L-12,4-D, the callus proliferation multiple was 4.77. Based on the comparative transcriptomic analysis, we identified 3470 differentially expressed genes (DEGs) in the callus with high differentiation rate and low differentiation rate, including 1767 up-regulated genes and 1703 down-regulated genes. Pathway enrichment analysis showed that the "Phenylpropanoid biosynthesis" metabolic pathway was significantly enriched, which is associated with promoting further development of callus shoots and roots. This study can provide reference for genetic improvement and the improvement of regeneration technology system of peony.

Transcriptomic and physiological analyses reveal the acquisition of somatic embryogenesis potential in Agapanthus praecox

Somatic embryogenesis (SE) is an ideal cell model for plant cell totipotency study. The poor frequency of embryogenic callus (EC) induction limits the application of SE in many plants, such as Agapanthus praecox. Herein, we performed transcriptomic and physiological analyses with different callus (Ca) differentiation directions (SE and organogenesis) and stages (initial SE and repetitive SE) to better understand the physiological and molecular characteristics of the acquisition of embryogenic potential in A. praecox. A number of differentially expressed genes (DEGs) were significantly related to plant hormonal signaling transduction, starch and sucrose metabolism, and reactive oxygen species (ROS) responses. Compared to Ca, DEGs including heat shock protein, isoamylase, histone H4, alpha-1,2-mannosyltransferase, and sucrose synthase substantially changed expression levels in EC formation. Corresponding to DEGs, physiological indicators including indole-3-acetic acid, cytokinins, brassinosteroids, ethylene, abscisic acid, ROS, hydrogen peroxide, superoxide dismutase, catalase, starch and sucrose may be responsible for the acquisition of embryogenic potential in A. praecox. Moreover, plant growth regulators, carbon source combination, osmotic regulating substance, and DNA methylation inhibitors in the culture medium significantly affected the acquisition of SE potential. Altogether, our results suggested that plant hormone signal regulation, starch and sucrose metabolism, ROS scavenging, chromatin accessibility and DNA methylation jointly contributed to the activation of embryogenic ability in A. praecox.

Optimization of high frequency plant regeneration protocol via embryogenic callus formation from diverse indica rice genotypes induced by combinations of 2,4-D and BA

A high frequency plant regeneration protocol via induction of embryogenic callus followed by somatic embryogenesis was standardized for a group of indica rice varieties consisting of land races and improved varieties, which are generally considered recalcitrant. Scutellum derived embryogenic calluses were obtained from the mature seeds inoculated into MS medium supplemented with different concentrations and combinations of 2,4-D and BA added with casein hydrolysate (500 mg/l) and proline (1 g/l). The varieties significantly differed in their response to embryogenic callusing and regeneration. Maximum embryogenic callus formation was induced in all the varieties by altering the combinations and concentrations of 2,4-D(1.0-2.0 mg/l) and BA (0.01, 0.1 and 0.5 mg/l). The land races viz., Karnel, SavuluSamba and Doddabyranellu showed higher embryogenic callus formation in all the combinations and concentrations of 2,4-D and BA. But the improved varieties showed embryogenic callus induction only in particular concentration and combinations of growth regulators. However, an increasing trend of plant regeneration was observed in regeneration media consisted of MS salts supplemented with 3.0 mg/ l BA and 1.0 mg/l NAA. We showed that maximum plant regeneration through generation of embryogenic calluses induction in indica rice varieties can be induced by altering the concentrations and combinations of 2,4-D and BA.

Development of a Highly Efficient Shoot Organogenesis System for an Ornamental Aeschynanthus pulcher (Blume) G. Don Using Leaves as Explants.

Aeschynanthus pulcher (Blume) G. Don, the “lipstick plant” is a prized ornamental plant with distinctive flowers. Here, we introduce a novel in vitro regeneration method for A. pulcher using leaf explants and an optimized combination of phytohormone plant growth regulators (PGRs). The optimal conditions for shoot regeneration included 1 mg L−1 polyvinyl pyrrolidone (PVP) plus 3 mg L−1 thidiazuron (TDZ), inducing a response rate of 82.4% and a shoot/explant ratio of 38.6. When the Murashige and Skoog (MS) medium contained indole-3-butyric acid (IBA) alone, leaves first differentiated into adventitious roots and then adventitious shoots. Leaves cultured on MS medium containing 1 g L−1 PVP, 3 mg L−1 TDZ, 5 mg L−1 casein, and 0.1 mg L−1 α-naphthaleneacetic acid (NAA) for 30 d exhibited the highest embryogenic callus (EC) induction rate (95.6%). The optimal shoot proliferation coefficient (21.5) was obtained when shoots derived from EC were cultured on the same medium as that used for EC induction for 5 weeks. The most effective medium for rooting of elongated shoots was MS medium containing 1 g L−1 PVP, 5 mg L−1 casein, 3 mg L−1 6-benzyladenine (BA), and 0.1 mg L−1 NAA, and the number of roots reached 18.8. The regenerated plants grown in a greenhouse had 100% survival following one week of hardening. Overall, our effective and efficient propagation method should result in shortened culture periods and reduced production costs, allowing for the future selective breeding and genetic improvement of A. pulcher.

Callus Development from Potato (Solanum tuberosum) Stem at Various Concentrations of Benzylaminopurine

Potato has the potential for food diversification.The propagation method in a short period is needed. One of the methods used is plant tissue culture. Benzyl Amino Purine (BAP), determine the success of propagation by tissue culture method. This research aimed to study the use of different BAP concentrations for callus development from stem explants. The explants put on culture medium added by 0, 1, 2, 3 ppm BAP. The development of the stem explant observed every week for a month. The results showed that callus formed in all media. Without BAP treatment, callus were formed after 2 weeks and got browning, then stopped growing. Callus grew and showed differentiation by application of all the BAP concentration. Callus greowth was optimally at 2 ppm BAP treatment. The callus from 1 ppm BAP produced the most number of roots, shoots and leaves than another concentration. This experiment showed that different BAP concentrations affected callus development of S. tuberosum from stem explant. The conclusion was callus growth has obtained by the treatment of 2 ppm BAP, while the development of callus has obtained on addition of 1 ppm BAP. The novelty of this research is the callus induction method from potato sprout stems grown from potato seeds with plant growth regulators Benzyl Amino Purine. Callus induction method from potato sprout stems grown from potato seeds can be a guide for embryogenic callus induction.

Combined QTL Mapping across Multiple Environments and Co-Expression Network Analysis Identified Key Genes for Embryogenic Callus Induction from Immature Maize Embryos.

The ability of immature embryos to induce embryogenic callus (EC) is crucial for genetic transformation in maize, which is highly genotype-dependent. To dissect the genetic basis of maize EC induction, we conducted QTL mapping for four EC induction-related traits, the rate of embryogenic callus induction (REC), rate of shoot formation (RSF), length of shoot (LS), and diameter of callus (DC) under three environments by using an IBM Syn10 DH population derived from a cross of B73 and Mo17. These EC induction traits showed high broad-sense heritability (>80%), and significantly negative correlations were observed between REC and each of the other traits across multiple environments. A total of 41 QTLs for EC induction were identified, among which 13, 12, 10, and 6 QTLs were responsible for DC, RSF, LS, and REC, respectively. Among them, three major QTLs accounted for >10% of the phenotypic variation, including qLS1-1 (11.54%), qLS1-3 (10.68%), and qREC4-1 (11.45%). Based on the expression data of the 215 candidate genes located in these QTL intervals, we performed a weighted gene co-expression network analysis (WGCNA). A combined use of KEGG pathway enrichment and eigengene-based connectivity (KME) values identified the EC induction-associated module and four hub genes (Zm00001d028477, Zm00001d047896, Zm00001d034388, and Zm00001d022542). Gene-based association analyses validated that the variations in Zm00001d028477 and Zm00001d034388, which were involved in tryptophan biosynthesis and metabolism, respectively, significantly affected EC induction ability among different inbred lines. Our study brings novel insights into the genetic and molecular mechanisms of EC induction and helps to promote marker-assisted selection of high-REC varieties in maize.

Plant regeneration via callus-mediated organogenesis in commercial variety of Chuanbeichai No. 1 in Bupleurum chinense DC.

Bupleurum chinense DC is an important medicinal plant with many active ingredients that are used for the treatment of different types of diseases and valued in pharmaceutical markets. In vitro shoot regeneration can efficiently contribute to the improvement of B. chinense. In the present study, we investigated the effects of the explant type and plant growth regulators (PGRs) on embryogenic callus induction and plant regeneration in B. chinense. Our investigation demonstrated that 2 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) combined with 1 mg/L thidiazuron (TDZ) played a major role in promoting callus induction from leaf, hypocotyl and stem 2 explants, whereas the most effective treatment for stem 1 callus formation was Murashige and Skoog (MS) medium supplemented with 1 mg/L 2,4-D, 0.5 mg/L 6-benzyladenine (BA) and 0.5 mg/L kinetin (Kin). The highest shoot regeneration rate (57.14%) was obtained from hypocotyl-induced calli in MS medium with 0.5 mg/L Kin after 12 weeks of cultivation. This regeneration protocol can be used in large-scale cultivation and may be useful for future genetic modifications of B. chinense.

In Vitro Propagation of Aconitum violaceum Jacq. ex Stapf through Seed Culture and Somatic Embryogenesis

Aconitum violaceum Jacq. ex Stapf is a threatened medicinal plant with restricted global distribution. The highest frequency of seed germination was recorded on Murashige and Skoog’s (MS) basal medium, supplemented with 0.5 mg L−1 kinetin with a germination rate of 77.32% and mean germination time of 27 days. Among the various plant growth regulators examined, 0.1 mg L−1 kinetin (Kn) + 0.5 mg L−1 indole-3-acetic acid (IAA) proved to be effective for maximum embryogenic callus production (51.0%) within 31 days of inoculation. The conversion rate of somatic embryos into complete plantlets was highest in the MS medium augmented with 0.1 mg L−1 Kn + 0.5 mg L−1 IAA (68.00%), with an average root initiation time of 25 days. The rooted plantlets were subsequently hardened into jiffy pots with a combination of loamy soil, coco-peat, and vermicompost (1:1:1 v/v), and then transplanted into a greenhouse with a 60% survival rate. To our knowledge, this is the first study on direct in vitro propagation and embryogenic callus induction from seeds. The established regeneration protocol could be employed to propagate A. violaceum on a large scale in a short time. This would contribute significantly to its rapid propagation and germplasm conservation, and establish a framework for the domestication of this highly valued threatened medicinal plant.

Integrated Proteomic and Metabolomic Analyses Provide Insights Into Acquisition of Embryogenic Ability in Agapanthus praecox.

Somatic embryogenesis (SE) is an ideal model for plant cell totipotency. Transition from somatic cells to embryogenic cells is the key to SE. The poor frequency of embryogenic callus (EC) induction has limited the application of SE in many plants, such as Agapanthus praecox. We performed large-scale, quantitative proteomic and metabolomic analyses with different callus differentiation directions (SE and organogenesis) and stages (initial SE and repetitive SE) to better understand the morphological, physiological, and molecular characteristics of the acquisition of embryogenic ability in A. praecox. Integrated proteomic and metabolomic analyses suggested that callus differentiation direction was potentially regulated by pathways related to carbohydrate and energy metabolism (fatty acid metabolism, pyruvate metabolism, glycolysis/gluconeogenesis, pentose and glucuronate interconversions, starch and sucrose metabolism, galactose metabolism, carbon fixation pathways in prokaryotes, carbohydrate digestion and absorption, and fructose and mannose metabolism), chromatin accessibility and DNA methylation, reactive oxygen species responses and resistance (ascorbate and aldarate metabolism), and plant hormonal signaling. As a validation, we found that carbon source combination and plant hormone regulation in the culture medium significantly affected the acquisition of embryogenic ability, thereby inducing EC. Interestingly, plant hormonal signaling-related genes showed different expression patterns significantly when callus cultured with different carbon sources. Thus, our results suggested that energy supply and hormone signal transduction seemed to cooperatively contribute to the activation of embryogenic ability. Altogether, this study revealed valuable information regarding the molecular and biochemical changes that occurred during EC induction and provided valuable foundation for comprehensive understanding of the mechanisms associated with SE and organogenesis in A. praecox.

English

Sorghum is a tropical grass used for various purposes including food, animal feed, and biofuels source, but its in vitro propagation still found an obstacle due to tissue browning. This study aims to propagate and regenerate callus in the Indonesian sorghum hybrid, named Numbu cultivar from seed and meristematic leaf whorl explants. The combined effect of plant growth regulators (PGRs), namely 2,4-D and proline as well as NAA (naphthalene acetic acid) and BAP (6-benzyl amino purine) was investigated on embryogenic callus induction and regeneration, respectively. The results showed that callus induction was significantly faster in the seed explants compared to leaf whorl. The optimal medium used to obtain a large induction in the seed explants was MS salt containing 3 mg L-1 2,4-D and 5000 mg L-1 proline, which gave a 68% yield. Furthermore, the best medium used to obtain a high plant regeneration frequency was the MS medium containing 0.1 mg L-1 NAA and 3 mg L-1 BAP, which regenerated 78% of the callus into plantlet. The addition of 560 mg L-1 proline to the combination medium increased doubled the regeneration into the planlet. Based on the results, PGRs combinations are required to improve embryogenic callus induction and regeneration into planlet in sorghum. © 2022 Friends Science Publishers

Genetic architecture of embryogenic callus induction in maize from the perspective of population genomics

Genetic architecture of embryogenic callus induction in maize from the perspective of population genomics