Non-embryogenic Callus Research Articles

Analysis of the plant hormone expression profile during somatic embryogenesis induction in teak (Tectona grandis).

Plant somatic embryogenesis (SE) is an efficient regeneration system for propagation. It involves the regulation of a complex molecular regulatory network encompassing endogenous hormone synthesis, metabolism, and signal transduction processes, induced through exogenous plant growth regulators. Previous studies have focused primarily on traditional propagation methods for Tectona grandis, but there is limited knowledge on SE and its hormonal regulatory mechanisms. In our study, different SE stages, including the nonembryogenic callus (NEC), embryogenic callus (EC), and globular and heart-shaped embryo (E-SEs) stages, were induced in teak cotyledons incubated on MS medium supplemented with 0.1 mg/L thidiazuron (TDZ). Morphological and histological observations indicated that EC primarily originates from the development of embryogenic cell clusters. During SE induction, the levels of six classes of endogenous hormones, IAA, CTK, ETH, ABA, SA, and JA, changed significantly. Transcriptome analysis revealed that endogenous hormones participate in SE induction in teak through various biological processes, such as biosynthesis, metabolism, and signal transduction pathways. We found that IAA biosynthesis primarily occurs through the IAM pathway during these three stages. The ETH receptor kinase gene SERF1 exhibited the highest expression levels in E-SEs. The ABA-, SA-, and JA-related signal transduction genes ABI3, NPR1, and JAZ exhibited no differential expression during different stages. Moreover, key encoding genes of SE regulators, including WUS, WOX and SERK, were differentially expressed during SE. In conclusion, this study offers insights into the roles of endogenous hormones and their interactions during SE induction.

Effects of Ethylene Inhibitors on the Long-Term Maintenance of the Embryogenic Callus of Vitis vinifera L.

Vitis vinifera is an important fruit crop which is mainly consumed fresh or used for the production of wine. Genetic improvement programs through New Genomic Techniques (NGTs) aim to develop grapevine varieties resistant to biotic and abiotic stresses or enhancing nutraceutical properties. In order to apply NGTs, maintaining embryogenic calluses from flower tissues is critical. Optimizing culture conditions—pH, gelling agents, temperature, light, growth regulators, and gas composition—is essential for inducing efficient embryogenic responses tailored to each genotype/explant. Ethylene, a pivotal gaseous plant hormone, significantly influences tissue culture by affecting organogenesis and embryogenesis processes in several plants. Modulating ethylene levels shows promise for improving tissue culture vitality. This study evaluates in Vitis vinifera the effects of silver thiosulfate (STS) and salicylic acid (SA) on embryogenic callus growth, specifically investigating their roles in maintaining and inducing embryogenic competence. STS, particularly at 40 µM and 60 µM concentrations, effectively preserved embryogenic competence in Italia and Red Globe calluses, while high SA concentrations showed varied and occasionally adverse effects. At the same time, STS markedly suppressed the non-embryogenic callus growth in recalcitrant variety Italia, potentially increasing the ratio between embryogenic to non-embryogenic calluses development.

Endogenous phytohormone analysis during sequential stages of somatic embryogenesis in Musa AAA cv. Grand Naine (subgroup Cavendish)

Abstract This is the first initiative study to provide quantified levels of four endogenous phytohormones during the induction, regeneration, maturation and germination stages of somatic embryogenesis (SE) in banana, using immature male bud explants, in the popular commercial triploid cultivar Grand Naine (AAA). Phytohormones are known to play a major role in the regulation of SE and most protocols have been developed for various cultivars of banana following empirical approaches. Although, several reports are available with regard to the impact of growth regulators exogenously supplemented to the media, the vital role of endogenous growth hormones in various developmental stages of SE remain unclear in banana. Reverse Phase High Performance Liquid Chromatography (RP-HPLC) was used to analyze endogenous status of indole acetic acid (IAA), zeatin, gibberellic acid (GA) and abscisic acid (ABA). Results from this study revealed that endogenous IAA and GA along with exogenous auxin play a major role in the induction of embryogenic competence of male bud explants. The levels of endogenous IAA and zeatin were found to be higher in embryogenic calli than non-embryogenic calli, although three different types of exogenous auxins were supplemented in callogenesis medium, but not cytokinin. Higher level of ABA and GA was observed in early and late developmental stages of somatic embryo, respectively. GA also played a predominant role in germination of somatic embryos. These findings will be applied for improvement of SE protocol in recalcitrant banana cultivars by suitably altering or supplementing exogenous growth hormones.

A protocol for the development and maintenance of Coffea arabica (L.) cell suspension cultures

Coffea spp. are remarkable sources of phytochemicals, but the lack of a well-defined culture medium aimed at the induction of non-embryogenic and friable callus hampers the establishment of plant cell suspension cultures for large-scale production of valuable compounds. In this paper, we describe a one-medium protocol suitable to obtain both callus and cell suspension cultures from leaves of two elite cultivars of C. arabica. The protocol was developed through an iterative process involving the determination of the best concentration of auxin and cytokinin, their optimal ratio, as well as the most effective molecule of either hormone class. Young leaves were found to be a good and easy-to-use explant source for callus induction and proliferation, provided that a cytokinin was present in association with a chlorinated auxin in a full strength, semi-solid MS medium. The best results were obtained by hormone concentration and combination of 1 mg/L of both kinetin and 2,4,5-trichlorophenoxyacetic acid. The same ratio of these growth regulators was conveniently used for the development and stabilization of cell suspension cultures in liquid MS medium. When grown in darkness, stabilized suspension cultures showed a fine and homogeneous texture, with a 10-fold biomass increase within 25 days and a cell viability > 90%. In addition, the phytochemical profile revealed the presence of the most widely studied coffee compounds. The protocol can be applied to obtain adequate amounts of cell biomass for use in physiological studies concerning the production of secondary metabolites.

Proteomic and Phosphoproteomic Analyses during Plant Regeneration Initiation in Cotton (Gossypium hirsutum L.).

Somatic embryogenesis (SE) is a biotechnological tool used to generate new individuals and is the preferred method for rapid plant regeneration. However, the molecular basis underlying somatic cell regeneration through SE is not yet fully understood, particularly regarding interactions between the proteome and post-translational modifications. Here, we performed association analysis of high-throughput proteomics and phosphoproteomics in three representative samples (non-embryogenic calli, NEC; primary embryogenic calli, PEC; globular embryos, GE) during the initiation of plant regeneration in cotton, a pioneer crop for genetic biotechnology applications. Our results showed that protein accumulation is positively regulated by phosphorylation during SE, as revealed by correlation analyses. Of the 1418 proteins that were differentially accumulated in the proteome and the 1106 phosphoproteins that were differentially regulated in the phosphoproteome, 115 proteins with 229 phosphorylation sites overlapped (co-differential). Furthermore, seven dynamic trajectory patterns of differentially accumulated proteins (DAPs) and the correlated differentially regulated phosphoproteins (DRPPs) pairs with enrichment features were observed. During the initiation of plant regeneration, functional enrichment analysis revealed that the overlapping proteins (DAPs-DRPPs) were considerably enriched in cellular nitrogen metabolism, spliceosome formation, and reproductive structure development. Moreover, 198 DRPPs (387 phosphorylation sites) were specifically regulated at the phosphorylation level and showed four patterns of stage-enriched phosphorylation susceptibility. Furthermore, enrichment annotation analysis revealed that these phosphoproteins were significantly enriched in endosomal transport and nucleus organization processes. During embryogenic differentiation, we identified five DAPs-DRPPs with significantly enriched characteristic patterns. These proteins may play essential roles in transcriptional regulation and signaling events that initiate plant regeneration through protein accumulation and/or phosphorylation modification. This study enriched the understanding of key proteins and their correlated phosphorylation patterns during plant regeneration, and also provided a reference for improving plant regeneration efficiency.

Scanning electron microscopic and morphological studies on in-vitro derived callus of low-chill peach (Prunus persica L. Batsch)

In this investigation the effect of collection seasons of explants (winter, spring and summer), type of explants (leaf disc and intermodal segments) and length of explants (0.5, 1.0 and 1.5 cm) for callusing in low-chill peach were standardized. The maximum callus induction (97.78%) in the low-chill peach was obtained from the intermodal segments of 0.5 cm in length used as an explant collected during spring season. The structural changes on the surface of the callus (5–7 weeks old yellowish green compact callus) during the progress of somatic embryogenesis of low-chill peach from the both intermodal segment as well as leaf disc derived callus were also examined with the use of scanning electron microscope (SEM). The SEM studies indicated that callus derived from internodal segment explant had the highest frequency of somatic embryos than callus from leaf discs. The SEM investigation, also demonstrated the sequential events/steps leading to low-chill peach somatic embryogenesis which was originating from somatic embryo mother cells through one unicellular pathway. Two types of calli were morphologically distinguished in both leaf disc and intermodal segment generated callus and these were the compact, well organized yellowish green embryogenic callus, containing large number of small, rich cytoplasmic, starch containing meristematic cells and soft and unorganized non-embryogenic callus containing sparsely cytoplasmic, vacuolated, and large cells devoid of metabolic reserves. The present SEM studies clearly demonstrated that somatic cells from peach explants generated callus could develop into fully differentiated somatic embryos through the characteristic embryological patterns of differentiation.

Biochemical Changes in Embryogenic and Nonembryonic Callus of Glycyrrhiza glabra L. during Somatic Embryogenesis

The continuous extraction of glycyrrhizin from Glycyrrhiza glabra L. has led to the critical endangerment of this therapeutic plant. Glycyrrhizin, a triterpenoid saponin, is a vital secondary metabolite renowned for its diverse pharmacological benefits, including anti-inflammatory, immunomodulatory, antiallergic, antiulcer properties, and even efficacy against HIV. However, the relentless harvesting of roots for high-value glycyrrhizin results in the obliteration of the entire plant, posing a severe threat to its existence and causing a detrimental impact on biodiversity. To counteract this challenge and conserve the species, sustainable approaches are imperative. One such approach involves the utilization of in vitro regeneration through somatic embryogenesis for clonal proliferation and genetic modification. This report presents findings on SE-associated proteins in Glycyrrhiza glabra L., accomplished through the implementation of the discontinuous gel electrophoresis technique. The study focused on a comparative isozyme analysis employing non-embryogenic and embryogenic calli of G. glabra L. Remarkably, these isozymes were exclusively identified and exhibited affiliations with a spectrum of cellular functions. A substantial portion of these proteins are engaged in a diverse range of metabolic and stress-related functions. This study utilized crude enzyme/total protein extracts as a basis to evaluate the genetic stability of in vitro cultivated plant tissue. Acid phosphatase, esterase, and peroxidase were selected as indicators for this assessment. Intriguingly, esterase and acid phosphatase exhibited significant polymorphism between the segregated embryogenic callus (EC) and non-embryogenic callus (NEC) derived from the same G. glabra L. genotype. Comparative analyses encompassing esterase, acid phosphatase, and peroxidase activities, alongside the expression profiles of these isozymes throughout somatic embryogenesis (SE), were conducted using polyacrylamide gel electrophoresis. Notably, the isoenzyme patterns of calluses were assessed. The activity of isozymes within an embryogenic line of callus surpassed that within a non-embryogenic counterpart. However, no conspicuous alterations in quality were observed between the non-embryogenic and embryogenic calluses.

Single-cell transcriptome atlas reveals somatic cell embryogenic differentiation features during regeneration.

Understanding somatic cell totipotency remains a challenge facing scientific inquiry today. Plants display remarkable cell totipotency expression, illustrated by single-cell differentiation during somatic embryogenesis (SE) for plant regeneration. Determining cell identity and exploring gene regulation in such complex heterogeneous somatic cell differentiation have been major challenges. Here, we performed high-throughput single-cell sequencing assays to define the precise cellular landscape and revealed the modulation mode of marker genes during embryogenic differentiation in cotton (Gossypium hirsutum L.) as the crop for biotechnology application. We demonstrated that nonembryogenic calli (NEC) and primary embryogenic calli (PEC) tissues were composed of heterogeneous cells that could be partitioned into four broad populations with six distinct cell clusters. Enriched cell clusters and cell states were identified in NEC and PEC samples, respectively. Moreover, a broad repertoire of new cluster-specific genes and associated expression modules were identified. The energy metabolism, signal transduction, environmental adaptation, membrane transport pathways, and a series of transcription factors were preferentially enriched in cell embryogenic totipotency expression. Notably, the SE-ASSOCIATED LIPID TRANSFER PROTEIN (SELTP) gene dose-dependently marked cell types with distinct embryogenic states and exhibited a parabolic curve pattern along the somatic cell embryogenic differentiation trajectory, suggesting that SELTP could serve as a favorable quantitative cellular marker for detecting embryogenic expression at the single-cell level. In addition, RNA velocity and Scissor analysis confirmed the pseudo-temporal model and validated the accuracy of the scRNA-seq data, respectively. This work provides valuable marker-genes resources and defines precise cellular taxonomy and trajectory atlases for somatic cell embryogenic differentiation in plant regeneration.

Effect of Picloram and Desiccation on the Somatic Embryogenesis of Lycium barbarum L.

An efficient and reproducible in vitro method for indirect somatic embryogenesis was optimized by culturing leaf and leaf with petiole explants of Lycium barbarum L. Murashige and Skoog (MS) medium, supplemented with various concentrations of Picloram and 2,4-Dichlorophenoxyacetic acid (2,4-D), individually and in combinations, were tested. Picloram (1.0 µM) showed a better response compared to 2,4-D and results indicate it to be a better auxin for induction of somatic embryos for Goji berry. It was seen that the leaf explants were more responsive in callus and somatic embryo induction than the leaf with petiole explant when incubated in the dark for 5 weeks. Embryogenic callus, after being transferred to MS medium containing Benzyl amino purine (BAP) in 1.0 µM, 5.0 µM and 10.0 µM, began to differentiate in light after one week. MS medium with 1.0 µM Picloram + 10 µM BAP resulted as the most favorable treatment for somatic embryogenesis in Lycium barbarum L. Removal of plant growth regulators from MS medium and culturing induced calluses under 16 h photoperiod resulted in globular, heart, torpedo, cotyledons, and further development into plantlets. Well-developed plants have been obtained and are capable of acclimatizing in ex vitro conditions. In addition, the effects of desiccation treatments (0, 1, 3, 6, 9 h, and 12 h) on embryogenic callus for somatic embryo induction were found to be directly proportional to the length of desiccation treatment at room temperature. After 9 h and 12 h of desiccation treatments, 60% and 90% of plated calluses resulted in somatic embryos, respectively. In a L. barbarum callus mass, Acetocarmine and Evans blue double staining differentiated between embryogenic and non-embryogenic callus. These findings will help Goji berry improvement by elite clone production, ex situ conservation projects, scaling up plant production, and agronomy for the commercial production of this superfruit in the future.

Somatic embryogenesis from mature sorghum seeds: An underutilized genome editing recipient system

Somatic embryogenesis is a process of cell totipotency in vitro, whereby an embryogenic cell develops from vegetative tissues rather than from zygotes after double fertilization. Sorghum is a recalcitrant crop in genetic transformation; previous recipient systems have usually been from immature zygotic embryos, which needed more time and labors to prepare. Here, an efficient 2,4-dichlorophenoxyacetic acid (2,4-D)-induced somatic embryogenesis system from mature sorghum seeds was introduced. 2,4-D can induce two types of calli from a plumular axis section. Low-concentration 2,4-D (e.g., 2 mg/L) induces white and loose non-embryogenic calli (type 1), while high-concentration 2,4-D (e.g., 8 mg/L) induces yellow and compact embryogenic calli (type 2), which can be clearly distinguished by Sudan red staining. Germinating seeds have a long 2-day window for SE induction. Somatic embryogenesis can be enhanced by HDAC inhibitor, trichostatin A (TSA), a histone deacetylase treatment, which shows more SE productivity and a bigger size. Importantly, this easily prepared protocol does not show obvious genotype dependency in sorghum hybrids. In this study, a high-concentration 2,4-D-induced SE system was established from mature sorghum seeds. This finding provides a technical option for the genome editing recipient in sorghum.

The search for inhibitors of somatic embryogenesis in Medicago truncatula

The success of protocols for the genetic transformation of legumes is limited by their low ability to regenerate. Plant regeneration can occur both along the path of shoot regeneration and their further rooting, and along the path of somatic embryogenesis (SE). SE is similar to zygotic embryogenesis (ZE). It is a method of asexual reproduction, in which the somatic cell, due to its totipotency, switches on the embryogenesis program.
 SE and ZE involve common participants in transcriptional, hormonal, and epigenetic control. Like many processes in the plant organism, SE is controlled by the activity of various stimulants or repressors.
 As a result of transcriptional analysis of embryogenic and non-embryogenic calli ofMedicago truncatulaat different stages of development, putative genes-inhibitors of SE were found. Using the Golden Gate system, we created vectors for agrobacterial transformation to overexpress genes of interest in embryogenic calli and evaluate their effect on SE. Overexpression of two genes encoding the transcription factors from WRKY and Homobox-WOX families had a significant inhibitory effect on SE (the average number of somatic embryos per callus decreased).
 TheMtCLE16,a previously found SE inhibitor from the CLE peptides group, was edited and heterozygous frameshift mutants were obtained. The impact of loss of its function on the SE capacity remains to be analyzed.
 This research was supported by the Sirius University of Science and Technology, project PBB-RND-2243.

Molecular mechanism of somatic embryogenesis in paeonia ostii ‘Fengdan’ based on transcriptome analysis combined histomorphological observation and metabolite determination

BackgroundTree peony (Paeonia sect. Moutan DC.) is a famous flower native to China with high ornamental, medicinal, and oil value. However, the low regeneration rate of callus is one of the main constraints for the establishment of a genetic transformation system in tree peony. By histomorphological observation, transcriptomic analysis and metabolite determination, we investigated the molecular mechanism of somatic embryogenesis after the establishment of a culture system and the induction of somatic embryo(SE) formation.ResultsWe found that SE formation was successfully induced when cotyledons were used as explants. A total of 3185 differentially expressed genes were screened by comparative transcriptomic analysis of embryogenic callus (EC), SE, and non-embryogenic callus (NEC). Compared to NEC, the auxin synthesis-related genes GH3.6 and PCO2 were up-regulated, whereas cytokinin dehydrogenase (CKX6) and CYP450 family genes were down-regulated in somatic embryogenesis. In SE, the auxin content was significantly higher than the cytokinin content. The methyltransferase-related gene S-adenosylmethionine synthase (SAMS) and the flavonoid biosynthesis-related gene (ANS and F3’5’H) were down-regulated in somatic embryogenesis. The determination of flavonoids showed that rhoifolin and hyperoside had the highest content in SE. The results of transcriptome analysis were consistent with the relative expression of 8 candidate genes by quantitative polymerase chain reaction analysis.ConclusionThe results revealed that auxin and cytokinin may play a key role in ‘Fengdan’ somatic embryogenesis. The genes related to somatic embryogenesis were revealed, which has partly elucidated the molecular mechanism of somatic embryogenesis in ‘Fengdan’.

Callus stage and morphology affect the DNA yield on the DNA extraction from the sugarcane in vitro callus

The main concern with the micropropagation of sugarcane (Saccharum officinarum) with a complex polyploid genome is the frequent occurrence of somaclonal variations. Therefore, genetic stability analysis is often required to ensure the uniformity of the micropropagated sugarcane, as early as in the callus stage. DNA extraction is the initial and important step of the genetic stability analysis. In this experiment, we compared the yield of DNA extraction from the sugarcane embryogenic and non-embryogenic calli. The sugarcane non-embryogenic callus was initiated from immature rolled leaf explants of the 6 months-old plant cane in the callus induction media, that were incubated in the dark room. The embryogenic calli were obtained by routine subcultures of the non-embryogenic calli every six weeks. The results showed that the extraction yield from the non-embryogenic calli was far lower than that from the embryogenic calli, most likely due to the high-water content of the non-embryogenic calli. Furthermore, histological analysis using Safranin and Fast Green staining revealed the low lignin content and nuclei in the non-embryogenic calli cell mass, causing the transparent and watery appearance of the calli and might influence the low DNA extraction yield.

Transcriptomic Profiling of Embryogenic and Non-Embryogenic Callus Provides New Insight into the Nature of Recalcitrance in Cannabis.

Differential gene expression profiles of various cannabis calli including non-embryogenic and embryogenic (i.e., rooty and embryonic callus) were examined in this study to enhance our understanding of callus development in cannabis and facilitate the development of improved strategies for plant regeneration and biotechnological applications in this economically valuable crop. A total of 6118 genes displayed significant differential expression, with 1850 genes downregulated and 1873 genes upregulated in embryogenic callus compared to non-embryogenic callus. Notably, 196 phytohormone-related genes exhibited distinctly different expression patterns in the calli types, highlighting the crucial role of plant growth regulator (PGRs) signaling in callus development. Furthermore, 42 classes of transcription factors demonstrated differential expressions among the callus types, suggesting their involvement in the regulation of callus development. The evaluation of epigenetic-related genes revealed the differential expression of 247 genes in all callus types. Notably, histone deacetylases, chromatin remodeling factors, and EMBRYONIC FLOWER 2 emerged as key epigenetic-related genes, displaying upregulation in embryogenic calli compared to non-embryogenic calli. Their upregulation correlated with the repression of embryogenesis-related genes, including LEC2, AGL15, and BBM, presumably inhibiting the transition from embryogenic callus to somatic embryogenesis. These findings underscore the significance of epigenetic regulation in determining the developmental fate of cannabis callus. Generally, our results provide comprehensive insights into gene expression dynamics and molecular mechanisms underlying the development of diverse cannabis calli. The observed repression of auxin-dependent pathway-related genes may contribute to the recalcitrant nature of cannabis, shedding light on the challenges associated with efficient cannabis tissue culture and regeneration protocols.

Plant Regeneration via Somatic Embryogenesis and Indirect Organogenesis in Blue Honeysuckle (Lonicera caerulea L.)

Blue honeysuckle (Lonicera caerulea L.), which belongs to the Caprifoliaceae family, is an emerging fruit crop worldwide. For the development of a transgenic system and multipurpose tissue culture, this study for the first time established an in vitro regeneration system via somatic embryogenesis, as well as improving the previously established indirect organogenesis-based regeneration system. For embryogenesis, Murashige and Skoog (MS) medium supplemented with 1.0 mg/L 2,4-dichlorophenoxyacetic acid (2,4-D) showed the highest induction rate of the embryogenic callus (97.6%), and MS supplemented with 0.1 mg/L 6-benzyladenine (6-BA), 0.1 mg/L α-naphthaleneacetic acid (NAA), and 0.5 g/L activated carbon (AC) achieved the highest somatic embryo rate (28.3%). For indirect organogenesis, MS medium supplemented with 1.0 mg/L 6-BA and 0.1 mg/L NAA resulted in the highest non-embryogenic callus induction rate (98.9%) and adventitious shoot induction rate (51.6%). For adventitious root induction, MS supplemented with 1.0 mg/L indole-3-butyric acid (IBA) achieved the highest root induction rate (96.0%) and average root length (4.6 cm), whereas MS supplemented with 0.5 mg/L indole-3-acetic acid (IAA) resulted in the highest average regenerated root number (8.8). The total time for the regeneration from explants to soil-planted seedlings (10 euphylla) was 105 and 150 days with an efficiency of 44.1% and 23.9% through organogenesis and somatic embryogenesis, respectively. This study provides a powerful tool for rapid propagation, proliferation, and transformation, as well as laying a technological foundation for gene function research and genetic improvement of blue honeysuckle.

Primary Metabolite Screening Shows Significant Differences between Embryogenic and Non-Embryogenic Callus of Tamarillo (Solanum betaceum Cav.).

Tamarillo is a solanaceous tree that has been extensively studied in terms of in vitro clonal propagation, namely somatic embryogenesis. In this work, a protocol of indirect somatic embryogenesis was applied to obtain embryogenic and non-embryogenic callus from leaf segments. Nuclear magnetic resonance spectroscopy was used to analyze the primary metabolome of these distinct calli to elucidate possible differentiation mechanisms from the common genetic background callus. Standard multivariate analysis methods were then applied, and were complemented by univariate statistical methods to identify differentially expressed primary metabolites and related metabolic pathways. The results showed carbohydrate and lipid metabolism to be the most relevant in all the calli assayed, with most discriminant metabolites being fructose, glucose and to a lesser extent choline. The glycolytic rate was higher in embryogenic calli, which shows, overall, a higher rate of sugar catabolism and a different profile of phospholipids with a choline/ethanolamine analysis. In general, our results show that a distinct primary metabolome between embryogenic and non-embryogenic calli occurs and that intracellular levels of fructose and sucrose and the glucose to sucrose ratio seem to be good candidates as biochemical biomarkers of embryogenic competence.

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.

Biochemical changes in embryogenic and non-embryogenic callus of Bambusa nutans Wall. during somatic embryogenesis

Biochemical changes in embryogenic and non-embryogenic callus of Bambusa nutans Wall. during somatic embryogenesis

Comparative Transcriptome Analysis between Embryogenic and Non-Embryogenic Callus of Davidia involucrata

Davidia involucrata Baill. (D. involucrata), a rare and endangered wild plant, is native to China and is globally recognized as an ornamental tree species. However, D. involucrata exhibits inherent biological characteristics that contribute to its low reproductive efficiency. To address this challenge, somatic embryogenesis, a biotechnological method, offers numerous advantages, including enhanced reproductive efficiency, a large reproductive coefficient, and a complete structural composition. Consequently, somatic embryogenesis holds significant value in the propagation and genetic improvement of this particular tree species. In a previous study, we utilized immature zygotic embryos of D. involucrata as explants and induced somatic embryogenesis from embryogenic callus, thereby establishing a rapid propagation and plant regeneration scheme. In this study, we utilized Illumina RNA sequencing to compare the transcriptomes of the embryogenic callus (EC) and non-embryogenic callus (NEC) of D. involucrata. The analysis revealed 131,109 unigenes assembled from EC and NEC, and 12,806 differentially expressed genes (DEGs) were identified. To verify the authenticity of the transcriptome sequencing results, qRT-PCR was performed and 16 DEGs were screened, with the stable reference gene UBQ being selected. Our analysis focused on genes related to plant growth regulators and somatic embryogenesis, such as the Aux, IAA, ARF, GH3, AHP, ARR, CYCD, BBM, WUS, GRF, SERK, and WOX gene families. We found that certain genes in these families were significantly upregulated in EC induction compared to NEC, indicating that they play crucial roles in D. involucrata cell proliferation, differentiation, and cell totipotency. These results offer new insights into the role of these gene families in EC, and may guide efforts to improve the somatic embryo induction, culture conditions, and genetic transformation efficiency of D. involucrata.

De Novo Sequencing and Transcriptome Analysis Reveal Genes’ Specific Expression in Chinese Fir (Cunninghamia lanceolata) Callus

While the progress made in vitro culture of Chinese fir has produced satisfactory results, further improvements are warranted. To understand the mechanism of somatic embryogenesis (SE) in Chinese fir, we conducted phenotypic observations, physiological and biochemical measurements, and transcriptome analysis of embryonic (EC) and non-embryogenic callus (NEC) to provide a scientific basis for SE in this species. We found that EC and NEC showed significant morphological and physiological-biochemical indicators differences. Compared with NEC, EC had higher levels of soluble protein and proline and lower levels of malondialdehyde (MDA), peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT). Callus transcriptome sequencing assembled 152,229 unigenes, and 438 differentially expressed genes (DEGs) were screened, including transcription factor-related (TFs), DNA methylation-related, cell wall component protein, signal transduction-related, and stress response-related. GO and KEGG enrichment analyses of DEGs identified starch and sucrose, glutathione, and cysteine and methionine metabolism as the most representative pathways significantly enriched in EC and NEC genes and were associated with cell proliferation and embryogenesis. For the first time, the specific patterns of gene expression in Chinese fir callus were found through transcriptome comparison between callus, 16-year-old Chinese fir cambium, and drought-stressed tissue culture seedlings. In Chinese fir callus, 75.1% of genes were co-expressed in 16-year-old Chinese fir cambium and drought-stressed tissue culture seedlings, and 24.9% were only specifically expressed in callus. DEGs from EC and NEC indicated that 68.2 and 31.8% were co-expressed and specifically expressed, respectively. These results provided a basis for Chinese fir rapid propagation, which is expected to have theoretical and practical significance.