Induction Of Somatic Embryogenesis Research Articles

Physiological, Metabolic, and Transcriptomic Analyses Reveal Mechanisms of Proliferation and Somatic Embryogenesis of Litchi (Litchi chinensis Sonn.) Embryogenic Callus Promoted by D-Arginine Treatment.

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.

Synergistic effects of L-glutamine and inorganic nitrogen molar ratios enhance the induction of somatic embryogenesis of Pinus maximinoi H.E. Moore

Clonal breeding programs of Pinus maximinoi require the establishment of a robust somatic embryogenesis (SE) protocol to produce enough cell lines to accelerate the effective continuous deployment of elite planting stocks to research and commercial compartments. Somatic embryogenesis was induced from immature zygotic embryo explants enclosed in megagametophytes of P. maximinoi collected from two plantations located in different climatic conditions. Cones were collected during the winter months from July to August and the influence of seed family, cone collection date and culture medium formulation, with emphasis on the organic and inorganic nitrogen supply, were studied. Ammonium to nitrate molar ratios of 1:1 and 1:2 in modified Litvay’s medium (mLV) produced the highest numbers of extrusions, while a 1:4 ratio mostly produced unhealthy, non-embryogenic extrusions. The formation of a tissue showing a rapidly-proliferating, spiky morphotype was produced in a medium supplemented with 1.5 g/L of L-glutamine. Morphologically advanced cultures with nodular structures were produced in megagametophytes from both plantations in a 1:2 NH4+:NO3− medium regardless of L-glutamine supplementation levels. The optimal medium for P. maximinoi SE induction contained a 1:2 NH4+:NO3− molar ratio with 1.5 g/L L-glutamine. The synergy between the molar ratio of NH4+:NO3− and L-glutamine resulted in the highest numbers of extrusions. The overall inductive competence window for somatic embryogenic response in P. maximinoi was determined to be from the second week of July to the first week of August for both plantations. The “peak” period was in the fourth week of July 2022. The success of the SE technology in P. maximinoi seed families is determined by the optimal inductive competence window of the immature megagametophytes enclosing zygotic embryos and the chemical composition of the induction medium in terms of the ammonium to nitrate molar ratio and the concentration of the L-glutamine used.

Study on the induction of somatic embryogenesis and morphological structural changes during the development of Handeliodendron bodinieri L.

Study on the induction of somatic embryogenesis and morphological structural changes during the development of Handeliodendron bodinieri L.

Studies on Improving the Efficiency of Somatic Embryogenesis in Grapevine (Vitis vinifera L.) and Optimising Ethyl Methanesulfonate Treatment for Mutation Induction.

Somatic embryogenesis (SE) has many applications in grapevine biotechnology including micropropagation, eradicating viral infections from infected cultivars, mass production of hypocotyl explants for micrografting, as a continuous source for haploid and doubled haploid plants, and for germplasm conservation. It is so far the only pathway for the genetic modification of grapevines through transformation. The single-cell origin of somatic embryos makes them an ideal explant for mutation breeding as the resulting mutants will be chimera-free. In the present research, two combinations of plant growth regulators and different explants from flower buds at two stages of maturity were tested in regard to the efficiency of callusing and embryo formation from the callus produced in three white grape cultivars. Also, the treatment of somatic embryos with the chemical mutagen ethyl methanesulfonate (EMS) was optimised. Medium 2339 supplemented with β-naphthoxyacetic acid (5 μM) and 6-benzylaminopurine (BAP-9.0 μM) produced significantly more calluses than medium 2337 supplemented with 2,4-dichlorophenoxyacetic acid (4.5 µM) and BAP (8.9 µM) in all explants. The calluses produced on medium 2337 were harder and more granular and produced more SEs. Although the stage of the maturity of floral bud did not have a significant effect on the callusing of the explants, calluses produced from immature floral bud explants in the premeiotic stage produced significantly more SEs than those from more mature floral buds. Overall, immature ovaries and cut floral buds exposing the cut ends of filaments, style, etc., tested for the first time in grapevine SE, produced the highest percentage of embryogenic calluses. It is much more efficient to cut the floral bud and culture than previously reported explants such as anthers, ovaries, stigmas and styles during the short flowering period when the immature flower buds are available. When the somatic embryos of the three cultivars were incubated for one hour with 0.1% EMS, their germination was reduced by 50%; an ideal treatment considered to obtain a high frequency of mutations for screening. Our research findings will facilitate more efficient SE induction in grapevines and inducing mutations for improving individual traits without altering the genetic background of the cultivar.

Proteomic Approach during the Induction of Somatic Embryogenesis in Coffea canephora.

Plant growth regulators (PGR) are essential for somatic embryogenesis (SE) in different species, and Coffea canephora is no exception. In our study model, previously, we have been able to elucidate the participation of various genes involved in SE by using different strategies; however, until now, we have not used a proteomic approach. This research seeks to contribute to understanding the primary cellular pathways involved in developing SE in C. canephora. The process of our model consists of two stages: (1) preconditioning in MS medium with auxin (NAA) and cytokinin (KIN), and (2) induction in Yasuda liquid medium added with cytokinin (BA). Therefore, in this study, we analyzed different days of the SE induction process using shotgun label-free proteomics. An amount of 1630 proteins was found among different sampling days of the process, of which the majority were accumulated during the induction stage. We found that some of the most enriched pathways during this process were the biosynthesis of amino acids and secondary metabolites. Eighteen proteins were found related to auxin homeostasis and two to cytokinin metabolism, such as ABC, BIG, ILR, LOG, and ARR. Ten proteins and transcription factors related to SE were also identified, like SERK1, SKP1, nuclear transcription factor Y, MADS-box, and calreticulin, and 19 related to other processes of plant development, among which the 14-3-3 and PP2A proteins stand out. This is the first report on the proteomic approach to elucidate the mechanisms that operate during the induction of SE in C. canephora. So, our findings provide the groundwork for future, more in-depth research. Data are available via ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD047172.

The of Effects of Light and a Combination of Growth Regulators on the Induction of Somatic Embryogenesis in Orchid Rhynchostylis gigantea (LindI.) Ridl.

The squirrel-tail orchid (Rhynchostylis gigantea) belongs to the Orchidaceae family. This orchid is indigenous to Southeast Asia and is scented and arranged in a bouquet of dangling stems. Its uniqueness makes it commercially desirable. In vitro culture has been utilized for orchid proliferation for a very long time to aid in the development of orchid seedlings. However, not all orchid species react the same way. Through somatic embryogenesis without gamete fusion, in vitro culture techniques can produce new plants during the entire embryonic phase. Endogenous and exogenous hormones, as well as light, influence the success of embryogenesis. Determining the optimal environmental parameters (light, combination and concentration of growth regulators, and their interaction) for inducing somatic embryogenesis from leaf explants in R. gigantea is the purpose of the present study. The leaves of R. gigantea clone 19 were utilized as explants in this investigation, utilizing a completely random factorial design. The first factor is a combination of growth regulator types and concentrations, and the second factor is light. Explants that are alive and growing at a rapid rate indicate that the in vitro culture is successful. Light and the combination of growth regulators significantly affected the percentage of viable explants, the beginning date of callus formation, and the number of embryogenic calluses produced. The interaction of two components (light and a combination of growth regulators) did not affect the three characteristics, with the exception of the proportion of somatic embryogenesis. Incubation in the dark is the optimal environment for initiating somatic embryogenesis in explants. The optimal combination of plant growth regulators for inducing somatic embryogenesis was 0.5 mgL-1 TDZ and 0.1 mgL-1 BAP. Bright light and a concentration of 1.0 mgL-1 TDZ + 0.1 mgL-1 BAP were the optimal interaction conditions for the induction of somatic embryogenesis in R. gigantea.

Plant regeneration through somatic embryogenesis in cell suspensions of Cenchrus ciliaris L.

BackgroundThis study aims to establish cell suspension culture, its maintenance and induction of somatic embryogenesis, and in vitro plant regeneration in Cenchrus ciliaris L. Suspension cultures are relatively homogenous cell lines facilitating uniform access to nutrition. These are ideal sources of competent cells for genetic transformation.ResultsCallus was initiated by culturing immature inflorescences of Cenchrus ciliaris cv. IGFRI-3108 on Murashige and Skoog (MS) medium containing 3 mg l−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg l−1 6-benzylaminopurine (BAP) with 30 g l−1 sucrose. Cell suspension cultures were established in liquid MS medium using an inoculum size of 10 g l−1. These were maintained to achieve embryogenic cell/regeneration competent cultures. Growth curve analysis and a subculture interval of 20 days were determined to harvest cells at the end of the exponential phase. The cell doubling time was found to be 11 days. Somatic embryogenesis was accomplished in MS medium containing 1 mg l−1 2,4-D, 1 mg l−1 BAP along with growth adjuvants, 300 mg l−1 casein hydrolysate, 400 mg l−1 glutamine and 300 mg l−1 proline. The highest number (16 ± 3.78/per inoculum) of shoots regenerated on this medium. The elongation and rooting of shoots were recorded on basal MS and ½ MS media, respectively. Rooted plants were successfully transferred to pots containing a Soilrite and cocopeat mixture in a 3:1 proportion for 3–4 weeks and later successfully acclimatized in the greenhouse with a 60% survival rate. The genetic fidelity of 12 regenerated plants was analysed using RAPD primers that were genetically identical to the mother plant.ConclusionCell suspension culture-based in vitro plant regeneration of C. ciliaris involved the establishment, maintenance and progression of somatic embryogenesis during shoot and root development. The inherent limitation of callus-mediated in vitro plant regeneration reducing the regeneration potential due to the aging of the calli has been overcome.

Callus Culture of <i>Aralia cordata</i> Thunb.: Obtaining, Selection of Cultivating Conditions, Somatic Embryogenesis Induction

Introduction. Cell cultures spreads widely in different areas of biology, biotechnology and agriculture. Aralia cordata is a perennial herbaceous plant, which has been listed in Red book of the Russian Federation. Pharmaceuticals which are based on raw materials of Aralia ssp. have valuable types of pharmacological activity and are widely used in oriental medicine. A serious obstacle for resumption of the natural populations is the presence of the period of morphophysiological dormancy in the seeds of the plants, which requires a long-time stratification process. Limitation of the natural geographic range and the combination of biological activities useful for humans make Aralia cordata Thunb. prospective object for in vitro introduction.Aim. The aim of the study is obtaining of the viable cell culture of Aralia cordata Thunb., investigation of the somatic embryogenesis conditions.Materials and methods. Pieces of the leaves of Aralia cordata intact plant from Komarov Botanical Institute of the Russian Academy of Sciences were used as a primary explants. Pieces of leaves were sterilized in a 2 % benzalkonium chloride solution for 5 minutes, induction of primary callogenesis was carried out on Murasige – Skoog medium. Nutrient media with different constituents were discovered for choosing one for long-time cultivation of calli. Induction of somatic embryogenesis was carried out on the nutrient media with high auxins content. Ethanol extracts from the intact plant and calli cultures were assayed with HPTLC PRO SYSTEM (CAMAG AG, Switzerland).Results and discussion. After two weeks of cultivation, the formation of primary callus was observed on the surface of the explants. The Linsmaier – Skoog medium with a reduced amount of sucrose (20 g/l) was recognized as the most suitable medium for long-term maintenance of cultures. Embryoid structures of Aralia cordata have been obtained, now we are continuing to collect analytics data about this process. Qualitative analysis of the extracts showed that callus cultures accumulate triterpene glycosides, their composition is close to that of the intact plant.Conclusion. A viable strain of callus culture of Aralia cordata Thunb. was obtained, a nutrient medium for long-term cultivation of calli was established. Somatic embryoids have been obtained, and their further development is currently being monitored. A preliminary phytochemical study showed that the composition of the chemical components of calli is close to that of an intact plant.

Features of the expression of breeding traits in Linum usitatissimum L. somaclones

Aim. Determining the level of expression, variability and features of inheritance of the main breeding traits of flax somaclones (Linum usitatissimum L.) in connection with the possibility of their use as initial selection material was the aim of our research. Methods. Hypocotyl and epicotyl segments for the induction of callusogenesis and somatic embryogenesis were cultured in vitro on the nutrient medium of Murashige and Skoog with the addition of 1.00 mg/l 6-benzylaminopurine and 0.05 mg/l 1-naphthylacetic acid, 30 g/l sucrose, a photoperiod of 16 h, illumination 2500 lux, relative air humidity 60–80 %, air temperature 22–24°C. Somaclones were adapted in vivo, and their generations were studied in the field. Results. Somaclones of the Hlinum variety were tested in the field according to the main selection traits and were not significantly inferior to the control variant; however, among them it is possible to single out some valuable individual plants for further breeding. Conclusions. Peculiarities of correlations between traits indicate the possibility of creating breeding material with a high level of fibrousness and seed productivity at the same time, high fiber mass and its content, allow selection for fibrousness by indirect traits – total and/or technical stem length, stem mass. A promising direction is the use of somaclones as sources of a high level of manifestation of the trait of the number of capsules on a plant (up to 17 pcs.).

Structure-activity relationship of 2,4-D correlates auxinic activity with the induction of somatic embryogenesis in Arabidopsis thaliana.

2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic analogue of the plant hormone auxin that is commonly used in many in vitro plant regeneration systems, such as somatic embryogenesis (SE). Its effectiveness in inducing SE, compared to the natural auxin indole-3-acetic acid (IAA), has been attributed to the stress triggered by this compound rather than its auxinic activity. However, this hypothesis has never been thoroughly tested. Here we used a library of forty 2,4-D analogues to test the structure-activity relationship with respect to the capacity to induce SE and auxinic activity in Arabidopsis thaliana. Four analogues induced SE as effectively as 2,4-D and 13 analogues induced SE but were less effective. Based on root growth inhibition and auxin response reporter expression, the 2,4-D analogues were classified into different groups, ranging from very active to not active auxin analogues. A halogen at the 4-position of the aromatic ring was important for auxinic activity, whereas a halogen at the 3-position resulted in reduced activity. Moreover, a small substitution at the carboxylate chain was tolerated, as was extending the carboxylate chain with an even number of carbons. The auxinic activity of most 2,4-D analogues was consistent with their simulated TIR1-Aux/IAA coreceptor binding characteristics. A strong correlation was observed between SE induction efficiency and auxinic activity, which is in line with our observation that 2,4-D-induced SE and stress both require TIR1/AFB auxin co-receptor function. Our data indicate that the stress-related effects triggered by 2,4-D and considered important for SE induction are downstream of auxin signalling.

Transcriptome and physiological analysis highlights the hormone, phenylpropanoid, and photosynthesis effects on early somatic embryogenesis in Ginkgo biloba

The somatic embryogenesis (SEs) system in Ginkgo biloba L. has not been fully established due to the recalcitrance of this species, and it has seriously limited the application of SEs technology, such as elite varieties large-scale production, gene editing, or transformation. Recently, we have induced the cotyledon-shaped somatic embryos (SE) of G. biloba, which was so far observed the closest to mature somatic embryos. Here, we performed the physiological analysis in six cultured stages during SEs formation, including d-NEC (non-embryogenic callus), d-EC (embryogenic callus), d-SE, d-WHC (while hairy callus), L-EC (EC in light), and L-SE (SE in light). The transcriptomic analysis in d-NEC, d-EC, L-EC, and d-WHC was also carried out. We found that the higher (IAA+ZR+iPA)/ABA and the lower IAA/(iPA+ZR) levels were critical in the somatic embryogenesis of G. biloba. The higher levels of soluble sugar, total flavonoid, terpene lactone, PAL, F3'H, and CCR were beneficial to somatic embryogenesis. In addition, the peroxidase (POD) might be related to the cell wall formation of somatic embryos in G. biloba. A total of 36517 unigenes were identified in four groups by transcriptome analysis, and genes differentially expressed (DEGs) were related to hormone homeostasis i.e., SAUR50, Jasmonic acid synthesis i.e., OPR3, phenylpropanoid biosynthesis i.e., CCR1, POD43 and C4H, terpenoid backbone biosynthesis i.e., HMGS, starch and sucrose metabolism, i.e., coniferin β-glucosidase, and photosynthesis, i.e., D1 protein. Furthermore, the majority of identified up-regulated transcription factors (TFs) such as MYB4, WRKY43, EFR8, and WUS might be responsible for the acquisition of SEs. The light was helpful to the early formation of somatic embryos, and the gene GbWUS1 and GbCLV3 were detected with higher expression in early SE induction of G. biloba. This finding will provide important theoretical and technical support for gene editing, cell totipotency research, and rapid propagation in G. biloba.

Detection of <i>AtRKD4</i> Gene and Induction of Somatic Embryo in Transformant of <i>Phalaenopsis amabilis</i> Carrying 35S::GR::<i>AtRKD4</i>

Phalaenopsis amabilis (L.) Blume is a native Indonesian orchid that plays an important role in the breeding of orchid’s hybrid worldwide. The high consumer demand causes a decline in the population of orchids for commercial trade. Plant propagation through induction of somatic embryogenesis will be very beneficial, because plants can be obtained in large numbers and uniform. AtRKD4 gene is an important gene in the model plant Arabidopsis thaliana which functions very early in development stage to initiate embryo formation. The AtRKD4 gene has been inserted into the P. amabilis orchid and several transformants have been obtained. This study aims to determine the stability integration of the AtRKD4 gene in the transformant P. amabilis genome and induce somatic embryo formation on transformant orchids. Plantation of leaf explants from P. amabilis transformants on hormone-free New Phalaenopsis basic medium induced somatic embryo formation by 20%.  Anatomical analysis showed that there is no difference stage between anatomy of somatic embryo development pattern in P. amabilis transformant and somatic embryo development pattern of monocot plants in general, PCR analysis using AtRKD4 specific primers showed that the embryos still contained 198 bp fragments of the AtRKD4 gene. In conclusion, the AtRKD4 gene is stably integrated in the P. amabilis orchid genome and can continuously induce the formation of somatic embryo from somatic cells of orchid transformants.

Establishment of an Efficient Somatic Embryogenesis Protocol for Giant Reed (Arundo donax L.) and Multiplication of Obtained Shoots via Semi-Solid or Liquid Culture

This study developed an efficient protocol for the in vitro propagation of giant reed (Arundo donax L.) biomass, defining a complete cycle of the induction of somatic embryogenesis from immature inflorescences, followed by the maturation of somatic embryos and the subsequent multiplication of the derived shoots in liquid culture in a temporary immersion system (TIS). The best explants were found to be 30 cm long immature inflorescences, preferably collected in spring. Such an explant type was easy to decontaminate, and the spikelets isolated from it provided over 100 embryogenic callus lines. Among the callus induction media tested, gelled MS medium supplemented with 1.1 mg/L 2,4-D provided the highest percentage of responsive spikelets and the highest density of embryogenic callus. Maturation of the embryogenic callus was easily triggered on gelled MS medium devoid of plant growth regulators. The obtained shoots could be further multiplied on previously optimized gelled DKW medium supplemented with 30 g/L sucrose, 5 mg/L BA, 0.1 mg/L IBA, and 6.8 g/L plant agar. Subsequent high multiplication of the developed shoots was achieved in liquid culture in TIS using a Plantform™ bioreactor, with an immersion cycle of 12 min every 8 h.

Induction of direct somatic embryogenesis and genetic stability of somatic embryo-derived plants of broccoli

Induction of direct somatic embryogenesis and genetic stability of somatic embryo-derived plants of broccoli

Recipient Somatic Embryogenesis System Promotes CRISPR/Cas Genome Editing

Somatic embryogenesis (SE) is an important type of plant regeneration, which can be used as a recipient system of genome editing. It is vital to optimize the SE system to improve the mutated somatic cells regenerated into fertile plants after transformation. 2,4-D was used as inducer of somatic embryogenesis from mature seeds. Arabidopsis wild type Col-0 and ABA biosynthesis double mutant <i>nced6 / 9 </i>were grown in 15°C and 25°C respectively, to investigate the effect of sampled plants growth conditions on the SE rate of their seeds and to optimize SE protocol. On the other hand, construct Pbi121-U6-26-Cas9-sgRNA-ELF6-FastR was used for genome editing. The positive seeds after floral dip were separated into two parts: some for sowing and the rest seeds went though SE induction. The efficiency of gene editing was compared by sequencing. Our results show that the SE rate of <i>nced6 / 9</i> seeds from 25°C was significantly lower than that from 15°C. When transgenic positive seeds were sown directly, the target sequence was not found to be edited; However, when seeds went though somatic embryogenesis protocol, gene editing were found around PAM sequence, suggesting that SE system could improve gene editing after transformation. The sampled plants grown at low temperature (15°C) may affect SE ability of their seeds by ABA level. The mature seed somatic embryogenesis system can improve the efficiency of gene editing.

Using Oxygen Bioavailability and Somatic Embryogenesis Techniques for Regeneration of Old Seeds of Common Bean (Phaseolus vulgaris L.)

The deterioration of the viability of seeds of old varieties (heirloom varieties) by exposure to long storage periods is one of the main problems facing plant breeding programmes; Thus, oxygen bioavailability and somatic embryogenesis techniques can overcome this problem to some extent. The goals of this study were to: 1) define the optimal oxygen bioavailability concentration for increasing the germination rates of old and waterlogged seeds; 2) define the relationship between the concentration of bioavailable oxygen in the liquid ingested by the germinating seed and seed vigor; and 3) use somatic embryogenesis techniques as a method for recovering plant germplasm from old or improperly stored seeds for use in plant breeding programs. This study was conducted in the tissue culture laboratory of the Potato and Vegetatively Propagated Vegetables Department, Horticulture Research Institute, Agricultural Research Centre, Egypt. This research was conducted to regenerate perennial seeds (8 years) of common bean (Phaseolus vulgaris L.) variety Giza 6 (local variety) and compared with 1 year old seeds as control treatment of the same cultivar. This study established that prolonged hypoxia throughout a protracted period of storage can inhibit germination. Hypoxia during the impregnation and germination of bean seeds can be overcome by supplying the seeds with hydrogen peroxide (20 mM) throughout the germination process. Catalase enzyme (10.5 units/mL) is needed to convert hydrogen peroxide into oxygen. Also, to release oxygen from hydrogen peroxide, other catalytic agents, such as transition metal ions like Fe+2, Cu+2, and Mn+2, can be used at the following concentrations: 0.001M, 0.001M, and 0.0001M, respectively. A protective layer of CaSO4 (0.5 mM) was applied since aged seeds have very fragile biological membranes. The test of tetrazolium gave results for some seeds that it non-viable or some parts of the seed are damaged or defective; However, 2,4-D at a concentration of 5 mg/l + supplements (0.1 µM GA3/L + 2 mg kintien/L + 1.0 mM glutamine) was able to produce somatic embryos of the old seeds tissues for common bean cv. Giza 6 (a local variety). The study of the morphology and induction of somatic embryogenesis was done used scanning electron microscopy. In its early phases, the callus appeared as a scattering of elongated cells. And after being transferred to the DCR medium, the cells became tightly packed together, and globular-shaped embryos began to emerge between the tightly packed together cells.

RWP-RK Domain 3 (OsRKD3) induces somatic embryogenesis in black rice

BackgroundPlants have the unique capability to form embryos from both gametes and somatic cells, with the latter process known as somatic embryogenesis. Somatic embryogenesis (SE) can be induced by exposing plant tissues to exogenous growth regulators or by the ectopic activation of embryogenic transcription factors. Recent studies have revealed that a discrete group of RWP-RK DOMAIN-CONTAINING PROTEIN (RKD) transcription factors act as key regulators of germ cell differentiation and embryo development in land plants. The ectopic overexpression of reproductive RKDs is associated with increased cellular proliferation and the formation of somatic embryo-like structures that bypass the need for exogenous growth regulators. However, the precise molecular mechanisms implicated in the induction of somatic embryogenesis by RKD transcription factors remains unknown.ResultsIn silico analyses have identified a rice RWP-RK transcription factor, named Oryza sativa RKD3 (OsRKD3), which is closely related to Arabidopsis thaliana RKD4 (AtRKD4) and Marchantia polymorpha RKD (MpRKD) proteins. Our study demonstrates that the ectopic overexpression of OsRKD3, which is expressed preferentially in reproductive tissues, can trigger the formation of somatic embryos in an Indonesian black rice landrace (Cempo Ireng) that is normally resistant to somatic embryogenesis. By analyzing the transcriptome of induced tissue, we identified 5,991 genes that exhibit differential expression in response to OsRKD3 induction. Among these genes, 50% were up-regulated while the other half were down-regulated. Notably, approximately 37.5% of the up-regulated genes contained a sequence motif in their promoter region, which was also observed in RKD targets from Arabidopsis. Furthermore, OsRKD3 was shown to mediate the transcriptional activation of a discrete gene network, which includes several transcription factors such as APETALA 2-like (AP2-like)/ETHYLENE RESPONSE FACTOR (ERF), MYB and CONSTANS-like (COL), and chromatin remodeling factors associated with hormone signal transduction, stress responses and post-embryonic pathways.ConclusionsOur data show that OsRKD3 modulates an extensive gene network and its activation is associated with the initiation of a somatic embryonic program that facilitates genetic transformation in black rice. These findings hold substantial promise for improving crop productivity and advancing agricultural practices in black rice.

Optimization of TDZ Hormone on the Formation of Somatic Embryogenesis in Dendrobium Orchids (D.50TH Stage Beauty X D. Bobby Mesina)

The Dendrobium variety orchid is a plant that has rare potential and growth. This requires large amounts of seeds that can be propagated in vitro. This study aims to determine the effect of TDZ on the direct induction of somatic embryogenesis. The study was conducted using a completely randomized design (CRD) with one factor, namely media consisting of basal Murashige and Skoog (MS) media with growth regulator TDZ. Each treatment out of a total of seven treatments was repeated four times. The research was carried out from March 2022 to August 2022. The results showed that leaf explants (clonal) could only be induced in 2 treatments (1 mg/L and 1.5 mg/L) out of 28 experimental media treatment units. The earliest callus appeared in the 1 mg/L treatment with a growth time of 90 days after planting (DAP) and the earliest PLB appeared in the 1.5 mg/L treatment with a growing time of 45 days after planting (DAP), the percentage of explant growth that became PLB with 1.5 mg/L treatment resulted in a percentage of 20 % and the percentage of callus growth at 1 mg/L yields a percentage of 20%.

Dynamics of Endogenous Auxin and Its Role in Somatic Embryogenesis Induction and Progression in Cork Oak

Somatic embryogenesis (SE) is a feasible in vitro regeneration system with biotechnological applications in breeding programs, although, in many forest species, SE is highly inefficient, mainly due to their recalcitrance. On the other hand, SE represents a valuable model system for studies on cell reprogramming, totipotency acquisition, and embryogenic development. The molecular mechanisms that govern the transition of plant somatic cells to embryogenic cells are largely unknown. There is increasing evidence that auxins mediate this transition and play a key role in somatic embryo development, although data on woody species are very limited. In this study, we analyzed the dynamics and possible role of endogenous auxin during SE in cork oak (Quercus suber L.). The auxin content was low in somatic cells before cell reprogramming, while it increased after induction of embryogenesis, as revealed by immunofluorescence assays. Cellular accumulation of endogenous auxin was also detected at the later stages of somatic embryo development. These changes in auxin levels correlated with the expression patterns of the auxin biosynthesis (QsTAR2) and signaling (QsARF5) genes, which were upregulated after SE induction. Treatments with the inhibitor of auxin biosynthesis, kynurenine, reduced the proliferation of proembryogenic masses and impaired further embryo development. QsTAR2 and QsARF5 were downregulated after kynurenine treatment. Our findings indicate a key role of endogenous auxin biosynthesis and signaling in SE induction and multiplication, as well as somatic embryo development of cork oak.

Calcium dynamics and modulation in carrot somatic embryogenesis.

Free calcium (Ca2+) is a pivotal player in different in vivo and in vitro morphogenic processes. In the induction of somatic embryogenesis, its role has been demonstrated in different species. In carrot, however, this role has been more controversial. In this work, we developed carrot lines expressing cameleon Ca2+ sensors. With them, Ca2+ levels and distribution in the different embryogenic structures formed during the induction and development of somatic embryos were analyzed by FRET. We also used different chemicals to modulate intracellular Ca2+ levels (CaCl2, ionophore A23187, EGTA), to inhibit calmodulin (W-7) and to inhibit callose synthesis (2-deoxy-D-glucose) at different times, principally during the first stages of embryo induction. Our results showed that high Ca2+ levels and the development of a callose layer are markers of cells induced to embryogenesis, which are the precursors of somatic embryos. Disorganized calli and embryogenic masses have different Ca2+ patterns associated to their embryogenic competence, with higher levels in embryogenic cells than in callus cells. The efficiency of somatic embryogenesis in carrot can be effectively modulated by allowing, within a range, more Ca2+ to enter the cell to act as a second messenger to trigger embryogenesis induction. Once induced, Ca2+-calmodulin signaling seems related with the transcriptional remodeling needed for embryo progression, and alterations of Ca2+ or calmodulin levels negatively affect the efficiency of the process.