Dedifferentiation Of Somatic Cells Research Articles

Appreciating animal induced pluripotent stem cells to shape plant cell reprogramming strategies.

Animals and plants have developed resilience mechanisms to effectively endure and overcome physical damage and environmental challenges throughout their life span. To sustain their vitality, both animals and plants employ mechanisms to replenish damaged cells, either directly, involving the activity of adult stem cells, or indirectly, via dedifferentiation of somatic cells that are induced to revert to a stem cell state and subsequently redifferentiate. Stem cell research has been a rapidly advancing field in animal studies for many years, driven by its promising potential in human therapeutics, including tissue regeneration and drug development. A major breakthrough was the discovery of induced pluripotent stem cells (iPSCs), which are reprogrammed from somatic cells by expressing a limited set of transcription factors. This discovery enabled the generation of an unlimited supply of cells that can be differentiated into specific cell types and tissues. Equally, a keen interest in the connection between plant stem cells and regeneration has been developed in the last decade, driven by the demand to enhance plant traits such as yield, resistance to pathogens, and the opportunities provided by CRISPR/Cas-mediated gene editing. Here we discuss how knowledge of stem cell biology benefits regeneration technology, and we speculate on the creation of a universal genotype-independent iPSC system for plants to overcome regenerative recalcitrance.

Targeted Treatment against Cancer Stem Cells in Colorectal Cancer.

The cancer stem cell (SC) theory proposes that a population of SCs serves as the driving force behind fundamental tumor processes, including metastasis, recurrence, and resistance to therapy. The standard of care for patients with stage III and high-risk stage II colorectal cancer (CRC) includes surgery and adjuvant chemotherapy. Fluoropyrimidines and their combination with oxaliplatin increased the cure rates, being able to eradicate the occult metastatic SC in a fraction of patients. The treatment for unresectable metastatic CRC is based on chemotherapy, antibodies to VEGF and EGFR, and tyrosine-kinase inhibitors. Immunotherapy is used in MSI-H tumors. Currently used drugs target dividing cells and, while often effective at debulking tumor mass, these agents have largely failed to cure metastatic disease. SCs are generated either due to genetic and epigenetic alterations in stem/progenitor cells or to the dedifferentiation of somatic cells where diverse signaling pathways such as Wnt/β-catenin, Hedgehog, Notch, TGF-β/SMAD, PI3K/Akt/mTOR, NF-κB, JAK/STAT, DNA damage response, and Hippo-YAP play a key role. Anti-neoplastic treatments could be improved by elimination of SCs, becoming an attractive target for the design of novel agents. Here, we present a review of clinical trials assessing the efficacy of targeted treatment focusing on these pathways in CRC.

In vivo reprogramming leads to premature death linked to hepatic and intestinal failure.

The induction of cellular reprogramming via expression of the transcription factors Oct4, Sox2, Klf4 and c-Myc (OSKM) can drive dedifferentiation of somatic cells and ameliorate age-associated phenotypes in multiple tissues and organs. However, the benefits of long-term in vivo reprogramming are limited by detrimental side-effects. Here, using complementary genetic approaches, we demonstrated that continuous induction of the reprogramming factors in vivo leads to hepatic and intestinal dysfunction resulting in decreased body weight and contributing to premature death (within 1 week). By generating a transgenic reprogrammable mouse strain, avoiding OSKM expression in both liver and intestine, we reduced the early lethality and adverse effects associated with in vivo reprogramming and induced a decrease in organismal biological age. This reprogramming mouse strain, which allows longer-term continuous induction of OSKM with attenuated toxicity, can help better understand rejuvenation, regeneration and toxicity during in vivo reprogramming.

Embryonic abnormalities and genotoxicity induced by 2,4-dichlorophenoxyacetic acid during indirect somatic embryogenesis in Coffea

Indirect somatic embryogenesis (ISE) is a morphogenetic pathway in which somatic cells form callus and, later, somatic embryos (SE). 2,4-dichlorophenoxyacetic acid (2,4-D) is a synthetic auxin that promotes the proliferation and dedifferentiation of somatic cells, inducing the ISE. However, 2,4-D can cause genetic, epigenetic, physiological and morphological disorders, preventing the regeneration and/or resulting abnormal somatic embryos (ASE). We aimed to evaluate the toxic 2,4-D effect during the Coffea arabica and C. canephora ISE, assessing the SE morphology, global 5-methylcytosine levels (5-mC%) and DNA damage. Leaf explants were inoculated in media with different 2,4-D concentrations. After 90 days, the friable calli were transferred to the regeneration medium, and the number of normal and abnormal SE was monthly counted. The increase of the 2,4-D concentration increased the number of responsive explants in both Coffea. At 9.06, 18.08 and 36.24 μM 2,4-D, C. arabica presented the highest values of responsive explants, differing from C. canephora. Normal and abnormal SE regeneration increased in relation to the time and 2,4-D concentration. Global 5-mC% varied at different stages of the ISE in both Coffea. Furthermore, the 2,4-D concentration positively correlated with global 5-mC%, and with the mean number of ASE. All ASE of C. arabica and C. canephora exhibited DNA damage and showed higher global 5-mC%. The allotetraploid C. arabica exhibited greater tolerance to the toxic effect of 2,4-D than the diploid C. canephora. We conclude that synthetic 2,4-D auxin promotes genotoxic and phytotoxic disorders and promotes epigenetic changes during Coffea ISE.

Targeting Breast Cancer Stem Cells.

The potential roles of breast cancer stem cells (BCSCs) in tumor initiation and recurrence have been recognized for many decades. Due to their strong capacity for self-renewal and differentiation, BCSCs are the major reasons for poor clinical outcomes and low therapeutic response. Several hypotheses on the origin of cancer stem cells have been proposed, including critical gene mutations in stem cells, dedifferentiation of somatic cells, and cell plasticity remodeling by epithelial-mesenchymal transition (EMT) and the tumor microenvironment. Moreover, the tumor microenvironment, including cellular components and cytokines, modulates the self-renewal and therapeutic resistance of BCSCs. Small molecules, antibodies, and chimeric antigen receptor (CAR)-T cells targeting BCSCs have been developed, and their applications in combination with conventional therapies are undergoing clinical trials. In this review, we focus on the features of BCSCs, emphasize the major factors and tumor environment that regulate the stemness of BCSCs, and discuss potential BCSC-targeting therapies.

An Insight into Vital Genes Responsible for β-cell Formation.

The regulation of glucose homeostasis and insulin secretion by pancreatic β-cells, when disturbed, will result in diabetes mellitus. Replacement of dysfunctional or lost β-cells with fully functional ones can tackle the problem of β-cell generation in diabetes mellitus. Various pancreatic-specific genes are expressed during different stages of development, which have essential roles in pancreatogenesis and β-cell formation. These factors play a critical role in cellular-based studies like transdifferentiation or de-differentiation of somatic cells to multipotent or pluripotent stem cells and their differentiation into functional β-cells. This work gives an overview of crucial transcription factors expressed during various stages of pancreas development and their role in β-cell specification. In addition, it also provides a perspective on the underlying molecular mechanisms.

Intermediate cells of in vitro cellular reprogramming and in vivo tissue regeneration require desmoplakin.

Amphibians and fish show considerable regeneration potential via dedifferentiation of somatic cells into blastemal cells. In terms of dedifferentiation, in vitro cellular reprogramming has been proposed to share common processes with in vivo tissue regeneration, although the details are elusive. Here, we identified the cytoskeletal linker protein desmoplakin (Dsp) as a common factor mediating both reprogramming and regeneration. Our analysis revealed that Dsp expression is elevated in distinct intermediate cells during in vitro reprogramming. Knockdown of <i>Dsp</i> impedes in vitro reprogramming into induced pluripotent stem cells and induced neural stem/progenitor cells as well as in vivo regeneration of zebrafish fins. Notably, reduced <i>Dsp</i> expression impairs formation of the intermediate cells during cellular reprogramming and tissue regeneration. These findings suggest that there is a Dsp-mediated evolutionary link between cellular reprogramming in mammals and tissue regeneration in lower vertebrates and that the intermediate cells may provide alternative approaches for mammalian regenerative therapy.

Reprogramming by Cytosolic Extract of Human Embryonic Stem Cells to Improve Dopaminergic Differentiation Potential of Human Adipose Tissue-derived Stem Cells.

The extract of pluripotent stem cells induces dedifferentiation of somatic cells with restricted plasticity. In this study, we used the extract of human embryonic stem cells (hESC) to dedifferentiate adipose tissue-derived stem cells (ADSCs) and examined the impact of this reprogramming event on the dopaminergic differentiation of the cells. For this purpose, cytoplasmic extract of ESCs was prepared by repeated freezing and thawing cycles. The plasma membrane of hADSCs was reversibly permeabilized by streptolysin O (SLO), exposed to hESC extract, and resealed by a CaCl2-containing medium. As revealed by qPCR analysis, expression of OCT4, SOX2, NANOG, LIN28A, and KLF4 mRNAs were downregulated in the ADSCs one week after extract incubation, while all mRNAs except for KLF4 were upregulated at the end of the second week. For dopaminergic differentiation, control and reprogrammed ADSCs were induced by a serum-free neurobasal medium containing B27 and a cocktail of sonic hedgehog (SHH), basic fibroblast growth factor (bFGF), fibroblastic growth factor 8 (FGF8), and brain-derived neurotrophic factor (BDNF) for 12 days. After differentiation, the expression levels of some neuronal and dopaminergic-related genes, including PAX6, NESTIN, NEFL, GLI1, LMXB1, EN1, NURR1, and TH, significantly increased in the reprogrammed ADSCs compared to the control group. On the whole, two weeks after reprogramming by ESC extract, ADSCs showed an improved dopaminergic differentiation potential. These findings suggest that the cytoplasmic extract of hESCs contains some regulatory factors which induce the expression of pluripotency-associated markers in somatic cells and that the exposure to ESC extract may serve as a simple and rapid strategy to enhance the plasticity of somatic stem cells for cell replacement therapy purposes. hADSCs have emerged as a valuable candidate for transplantation therapy of neurodegenerative diseases.Several studies have documented dopaminergic dedifferentiation of hADSCs.Implementing ADSCs towards a more pluripotent state using different strategies like somatic cell nuclear transfer. The extract of pluripotent stem cells induces dedifferentiation of somatic cells with restricted plasticity. In this study, we used the extract of hESC to dedifferentiate ADSCs and examined the impact of this reprogramming event on the dopaminergic differentiation of the cells. Cytoplasmic extract of ESCs was prepared by repeated freezing and thawing cycles. These cells express several neuron-specific genes, secrete several factors associated with neuroprotection, and exhibit differentiation into neural and glial cells in vitro. In recent years, several studies have documented dopaminergic differentiation of hADSCs.

Transduction of Signals during Somatic Embryogenesis.

Somatic embryogenesis (SE) is an in vitro biological process in which bipolar structures (somatic embryos) can be induced to form from somatic cells and regenerate into whole plants. Acquisition of the embryogenic potential in culture is initiated when some competent cells within the explants respond to inductive signals (mostly plant growth regulators, PRGs), and de-differentiate into embryogenic cells. Such cells, “canalized” into the embryogenic developmental pathway, are able to generate embryos comparable in structure and physiology to their in vivo counterparts. Genomic and transcriptomic studies have identified several pathways governing the initial stages of the embryogenic process. In this review, the authors emphasize the importance of the developmental signals required for the progression of embryo development, starting with the de-differentiation of somatic cells and culminating with tissue patterning during the formation of the embryo body. The action and interaction of PGRs are highlighted, along with the participation of master regulators, mostly transcription factors (TFs), and proteins involved in stress responses and the signal transduction required for the initiation of the embryogenic process.

Conservation analysis of core cell cycle regulators and their transcriptional behavior during limb regeneration in Ambystoma mexicanum

Ambystoma mexicanum (axolotl) has been one of the major experimental models for the study of regeneration during the past 100 years. Axolotl limb regeneration takes place through a multi-stage and complex developmental process called epimorphosis that involves diverse events of cell reprogramming. Such events start with dedifferentiation of somatic cells and the proliferation of quiescent stem cells to generate a population of proliferative cells called blastema. Once the blastema reaches a mature stage, cells undergo progressive differentiation into the diverse cell lineages that will form the new limb. Such pivotal cell reprogramming phenomena depend on the fine-tuned regulation of the cell cycle in each regeneration stage, where cell populations display specific proliferative capacities and differentiation status. The axolotl genome has been fully sequenced and released recently, and diverse RNA-seq approaches have also been generated, enabling the identification and conservatory analysis of core cell cycle regulators in this species. We report here our results from such analyses and present the transcriptional behavior of key regulatory factors during axolotl limb regeneration. We also found conserved protein interactions between axolotl Cyclin Dependent Kinases 2, 4 and 6 and Cyclins type D and E. Canonical CYC-CDK interactions that play major roles in modulating cell cycle progression in eukaryotes.

An Artificial Conversion of Roots into Organs with Shoot Stem Characteristics by Inducing Two Transcription Factors.

SummarySomatic plant cells can regenerate shoots and/or roots or adventitious embryonic calluses, which may induce organ formation under certain conditions. Such regenerations occur via dedifferentiation of somatic cells, induction of organs, and their subsequent outgrowth. Despite recent advances in understanding of plant regeneration, many details of shoot induction remain unclear. Here, we artificially induced shoot stem-like green organs (SSOs) in Arabidopsis thaliana roots via simultaneous induction of two transcription factors (TFs), ARABIDOPSIS THALIANA HOMEOBOX PROTEIN 25 (ATHB25, At5g65410) and the B3 family transcription factor REPRODUCTIVE MERISTEM 7 (REM7, At3g18960). The SSOs exhibited negative gravitropism and differentiated vascular bundle phenotypes. The ATHB25/REM7 induced the expression of genes controlling shoot stem characteristics by ectopic expression in roots. Intriguingly, the restoration of root growth was seen in the consecutive and adjacent parts of the SSOs under gene induction conditions. Our findings thus provide insights into the development and regeneration of plant shoot stems.

The Evolution of Stem Cells, Disease Modeling, and Drug Discovery for Neurological Disorders.

Human neurological disorders are among the most challenging areas of translational research. The difficulty of acquiring human neural samples or specific representative animal models has necessitated a multifaceted approach to understanding disease pathology and drug discovery. The dedifferentiation of somatic cells to human induced pluripotent stem cells (hiPSCs) for the generation of neural derivatives has broadened the capability of biomedical research to study human cell types in neurological disorders. The initial zeal for the potential of hiPSCs for immediate biomedical breakthroughs has evolved to more reasonable expectations. Over the past decade, hiPSC technology has demonstrated the capacity to successfully establish "disease in a dish" models of complex neurological disorders and to identify possible novel therapeutics. However, as hiPSCs are used more broadly, an increased understanding of the limitations of hiPSC studies is becoming more evident. In this study, we review the challenges of studying neurological disorders, the current limitations of stem cell-based disease modeling, and the degrees to which hiPSC studies to date have demonstrated the capacity to fill essential gaps in neurological research.

Autophagy mediates temporary reprogramming and dedifferentiation in plant somatic cells.

Somatic cells acclimate to changes in the environment by temporary reprogramming. Much has been learned about transcription factors that induce these cell-state switches in both plants and animals, but how cells rapidly modulate their proteome remains elusive. Here, we show rapid induction of autophagy during temporary reprogramming in plants triggered by phytohormones, immune, and danger signals. Quantitative proteomics following sequential reprogramming revealed that autophagy is required for timely decay of previous cellular states and for tweaking the proteome to acclimate to the new conditions. Signatures of previous cellular programs thus persist in autophagy-deficient cells, affecting cellular decision-making. Concordantly, autophagy-deficient cells fail to acclimatize to dynamic climate changes. Similarly, they have defects in dedifferentiating into pluripotent stem cells, and redifferentiation during organogenesis. These observations indicate that autophagy mediates cell-state switches that underlie somatic cell reprogramming in plants and possibly other organisms, and thereby promotes phenotypic plasticity.

High Concentration of Spermine Induces the Dedifferentiation of Somatic Cells into Pluripotent Stem Cells

Cancer tissues contain cancer stem cells (CSCs), which play important roles in cancer metastasis. However, the mechanisms through which cancer cells dedifferentiate into stem cells have not yet been elucidated. In this study, the effects of high concentrations of polyamines produced in cancer cells on dedifferentiation were examined. The results showed that when normal human fibroblasts were cultured with high concentrations of spermine, the obtained polyamine-induced cells expressed alkaline phosphatase and marker proteins of pluripotent stem cells, although apoptosis occurred in most cells. In contrast, another polyamine-induced stem (PIS) cell line (Spe-2 PIS cells), obtained by culture in medium containing Rock, p53, and Bax inhibitors plus spermine, did not show signs of apoptosis. These Spe-2 PIS cells expressed marker proteins of pluripotent stem cells and differentiated into cardiomyocytes, brown adipocytes, and nerve cells. These results suggest that a high concentration of spermine, which often induces apoptosis in normal cells, has the capacity to dedifferentiate somatic cells into pluripotent stem cells and may be associated with the dedifferentiation of cancer cells, which continuously produce high concentrations of spermine. Moreover, the procedure to obtain Spe-2 PIS cells, which is simple and efficient, may have potential applications in regenerative medicine.

The dualistic origin of human tumors.

Life starts with a zygote, which is formed by the fusion of a haploid sperm and egg. The formation of a blastomere by cleavage division (nuclear division without an increase in cell size) is the first step in embryogenesis, after the formation of the zygote. Blastomeres are responsible for reprogramming the parental genome as a new embryonic genome for generation of the pluripotent stem cells which then differentiate by Waddington’s epigenetic landscape to create a new life. Multiple authors over the past 150 years have proposed that tumors arises from development gone awry at a point within Waddington’s landscape. Recent discoveries showing that differentiated somatic cells can be reprogrammed into induced pluripotent stem cells, and that somatic cell nuclear transfer can be used to successfully clone animals, have fundamentally reshaped our understanding of tumor development and origin. Differentiated somatic cells are plastic and can be induced to dedifferentiate into pluripotent stem cells. Here, I review the evidence that suggests somatic cells may have a previously overlooked endogenous embryonic program that can be activated to dedifferentiate somatic cells into stem cells of various potencies for tumor initiation. Polyploid giant cancer cells (PGCCs) have long been observed in cancer and were thought originally to be nondividing. Contrary to this belief, recent findings show that stress-induced PGCCs divide by endoreplication, which may recapitulate the pattern of cleavage-like division in blastomeres and lead to dedifferentiation of somatic cells by a programmed process known as “the giant cell cycle”, which comprise four distinct but overlapping phases: initiation, self-renewal, termination and stability. Depending on the intensity and type of stress, different levels of dedifferentiation result in the formation of tumors of different grades of malignancy. Based on these results, I propose a unified dualistic model to demonstrate the origin of human tumors. The tenet of this model includes four points, as follows. 1. Tumors originate from a stem cell at a specific developmental hierarchy, which can be achieved by dualistic origin: dedifferentiation of the zygote formed by two haploid gametes (sexual reproduction) via the blastomere during normal development, or transformation from damaged or aged mature somatic cells via a blastomere-like embryonic program (asexual reproduction). 2. Initiation of the tumor begins with a stem cell that has uncoupled the differentiation from the proliferation program which results in stem cell maturation arrest. 3. The developmental hierarchy at which stem cells arrest determines the degree of malignancy: the more primitive the level at which stem cells arrest, the greater the likelihood of the tumor being malignant. 4. Environmental factors and intrinsic genetic or epigenetic alterations represent the risk factors or stressors that facilitate stem cell arrest and somatic cell dedifferentiation. However, they, per se, are not the driving force of tumorigenesis. Thus, the birth of a tumor can be viewed as a triad that originates from a stem cell via dedifferentiation through a blastomere or blastomere-like program, which then differentiates along Waddington’s landscape, and arrests at a developmental hierarchy. Blocking the PGCC-mediated dedifferentiation process and inducing their differentiation may represent a novel alternative approach to eliminate the tumor occurrence and therapeutic resistance.

PRC2 Represses Hormone-Induced Somatic Embryogenesis in Vegetative Tissue of Arabidopsis thaliana.

Many plant cells can be reprogrammed into a pluripotent state that allows ectopic organ development. Inducing totipotent states to stimulate somatic embryo (SE) development is, however, challenging due to insufficient understanding of molecular barriers that prevent somatic cell dedifferentiation. Here we show that Polycomb repressive complex 2 (PRC2)-activity imposes a barrier to hormone-mediated transcriptional reprogramming towards somatic embryogenesis in vegetative tissue of Arabidopsis thaliana. We identify factors that enable SE development in PRC2-depleted shoot and root tissue and demonstrate that the establishment of embryogenic potential is marked by ectopic co-activation of crucial developmental regulators that specify shoot, root and embryo identity. Using inducible activation of PRC2 in PRC2-depleted cells, we demonstrate that transient reduction of PRC2 activity is sufficient for SE formation. We suggest that modulation of PRC2 activity in plant vegetative tissue combined with targeted activation of developmental pathways will open possibilities for novel approaches to cell reprogramming.

New Insight Into Early Somatic Embryogenesis of Mangosteen (Garcinia mangostana) Through de Novo and Comparative Transcriptome Analyses

Somatic embryogenesis in mangosteen (Garcinia mangostana L.), an apomictic and recalcitrant-seeded species, allows stock improvement through genetic manipulation as well as mass propagation in a shortened time. To understand molecular events and gene expression during early somatic embryogenesis of mangosteen, we used RNA-Seq technology and assembled the mangosteen transcriptome de novo. Using the Trinity package, 186,203 transcripts were assembled with a mean size of 767 bp, 34.8 % of which showed significant similarities to known sequences in the GenBank non-redundant protein database. A total of 4001 transcripts were differentially expressed during somatic embryogenesis, while transcripts encoding signalling components of plant hormones such as auxin and cytokinin were significantly enriched. All the cytokinin-related genes were up-regulated, while some auxin-related genes were down-regulated and others were up-regulated. Somatic embryogenesis candidate genes such as SERK2, AGL15, CLVs, WUS and PIN1 were up-regulated in somatic embryos. Zinc finger and MADS family transcription factor genes were among the most presented differentially expressed transcripts in early somatic embryogenesis. Further qRT-PCR analysis confirmed the expression levels of the candidate genes. This study reports a de novo transcriptome assembly and analysis of gene expression during early somatic embryogenesis of mangosteen. Differentially expressed genes reveal that cytokinin has critical role in triggering somatic cell dedifferentiation and induction of somatic embryogenesis in mangosteen. The present work can serve as an important resource for further functional studies in woody plant embryogenesis.

Recent advances in iPSC technologies involving cardiovascular and neurodegenerative disease modeling.

Cardiovascular and neurodegenerative diseases are the most common health threats in developed countries. Limited cell derivation and cell number in cardiac tissue makes it difficult to study the cardiovascular disease using the existing cardiac cell model. Regarding the neurodegenerative disorders, the most potential sources of cell therapeutics such as fetal-derived primary neurons and human embryonic stem cells (ESCs) are associated with ethical or technical limitations. The successful derivation of human-induced pluripotent stem cells (iPSCs) by de-differentiation of somatic cells offers significant potential to overcome hurdles in the field of the replacement therapy. Human iPSCs are functionally similar to human embryonic stem cells, and can be derived autologously without the ethical challenges associated with human ESCs. The iPSCs can, in turn, be differentiated into all cell types including neurons, cardiac cells, blood and liver cells, etc. Recently, target tissues derived from human iPSCs such as cardiomyocytes (CMs) or neurons have been used for new disease modeling and regenerative medicine therapies. Diseases models could be advantageous in the development of personalized medicine of various pathological conditions. This paper reviews efforts aimed at both the practical development of iPSCs, differentiation to neural/cardiac lineages, and the further use of these iPSCs-derived cells for disease modeling, as well as drug toxicity testing.

Conversion of mouse fibroblasts into cardiomyocyte-like cells using small molecule treatments

The possibility of controlling cell fates by overexpressing specific transcription factors has led to numerous studies in stem cell research. Small molecules can be used, instead of transcription factors, to induce the de-differentiation of somatic cells or to induce pluripotent cells (iPSCs). Here we reported that combinations of small molecules could convert mouse fibroblasts into cardiomyocyte-like cell without requiring transcription factor expression. Treatment with specific combinations of small molecules that are enhancer for iPSC induction converted mouse fibroblasts into spontaneously contracting, cardiac troponin T-positive, cardiomyocyte-like cells. We specifically identified five small molecules that can induce mouse fibroblasts to form these cardiomyocyte-like cells. These cells are similar to primary cardiomyocytes in terms of marker gene expression, epigenetic status of cardiac-specific genes, and subcellular structure. Our findings indicate that lineage conversion can be induced not only by transcription factors, but also by small molecules.

UNRAVELLING THE REGULATION OF SOMATIC EMBRYOGENESIS BY EXTRACELLULAR CALCIUM AND AUXIN EFFLUX BLOCKERS IN HYPOCOTYL EXPLANTS OF ALBIZZIA LEBBECK L.: SIMILARITY IN ACTION

Somatic embryogenesis involves developmental reprogramming and de-differentiation of somatic cells towards embryo development that is crucial for cellular totipotency in higher plants. It is a prototype for comprehension of the physiological, biochemical and molecular biological events transpiring during plant embryo development. In this study, we analyzed the role of exogenous calcium and auxin during somatic embryogenesis in hypocotyl explants of Albizzia lebbeck L. Light microscopic studies revealed that anterior and posterior ends of hypocotyl explants had entirely different patterns of differentiation . Scanning electron microscopic analysis of cultured hypocotyl explants exhibited globular-, heart- and torpedo-shaped somatic embryos developing from the anterior cut surface and gradually proceeding towards the other end. Surface texture of these somatic embryos showed uni-or bi-celled trichomes. Presence of exogenous calcium (1-4mM) in hormone-free B5 medium is prerequisite for induction of somatic embryos in hypocotyl explants derived from 7-d-old, light grown seedlings of Albizzia lebbeck L. A two-fold increase in calcium concentration from 2mM (B 5 medium) to 4mM led to doubling of somatic embryogenic response whereas, embryogenic callus was induced in explants raised on medium containing 10 or 20mM calcium. Depletion or high dosage in the medium (20 mM) induce non-embryogenic callus in explants. A similar response induced on hypocotyl explants upon treatments with NPA and PCIB, (auxin transport blockers). Results indicate that polar auxin transport or basipetal movement of IAA triggers acropetal transport of calcium. This combination of inverse fluxes acts as the primary signal for embryogenic response at the cut ends proximal to shoot apex and callusing at the cut ends distal to shoot apex of explants. Moreover, a correlation exists between NPA and PCIB-induced disruption of basipetal IAA transport and the inhibitory effect of NPA and PCIB on calcium translocation in the induction of similar morphogenetic responses.