Journal articles on the topic 'Protoplast proliferation'

The present paper describes a simple and reliable protocol for the successful isolation, purification, culture, and regeneration of diploid cotyledon-derived protoplasts of Brassica napus L. cv. 'Topas'. Various protoplast isolation media, nutrient media, subculture procedures, and protoplast sources were tested under two culture temperatures. Protoplast viability, cell wall regeneration, and cell division were monitored. Single cotyledon-derived protoplasts formed calli in liquid protoplast medium, and when these were subcultured on solid proliferation medium and solid regeneration medium of appropriate composition, plants regenerated either by shoot formation or embryogenesis. Continuous culture at 32°C instead of 25°C favoured the initiation of cell division and cell proliferation but prevented regeneration, although calli maintained regeneration capacity. Viable haploid protoplasts were isolated from cotyledons of heat-shock-induced, microspore-derived haploid embryos and from young leaves of secondary embryos that were formed on microspore-derived embryos. Cell divisions were triggered in the two types of haploid protoplast cultures, and microcalli were formed at high frequencies. Differences between haploid and diploid protoplast cultures are discussed.Key words: cotyledon protoplast culture, haploid culture, plant regeneration.

Despite the increasing use of protoplasts in plant biotechnology research, shoot regeneration from protoplasts remains challenging. In this study, we investigated the factors involved in protoplast isolation, callus induction, and shoot regeneration in Petunia hybrida cv. Mirage Rose. The following conditions were found to be most optimal for protoplast yield and viability: 0.6 M mannitol, 2.0% cellulase, and 6 h digestion time. A plating density of 10 × 104 protoplasts/mL under osmoticum condition (0.58 M mannitol) showed high microcolony viability in liquid culture. The Kao and Michayluk medium was found to be appropriate for callus proliferation from microcalli under a 16-h light photoperiod. Calli cultured in Murashige and Skoog medium containing 1.0 mg/L 6-benzylaminopurine and 0.2 mg/L 3-indole butyric acid showed the highest shoot regeneration frequency and number of shoots obtained per explant. Random amplification of polymorphic DNA analysis showed that the protoplast-derived shoots exhibited the same banding patterns as those of donor plants. Collectively, these findings can contribute to solving problems encountered in protoplast isolation and shoot regeneration in other petunia cultivars and related species. As the protocol developed by us is highly reproducible, it can be applied in biotechnology research on P. hybrida cv. Mirage Rose.

Phytosulfokine-α (PSK), a peptidyl plant growth factor, has been recognized as a promising intercellular signaling molecule involved in cellular proliferation and dedifferentiation. It was shown that PSK stimulated and enhanced cell divisions in protoplast cultures of several species leading to callus and proembryogenic mass formation. Since PSK had been shown to cause an increase in efficiency of somatic embryogenesis, it was reasonable to check the distribution of selected chemical components of the cell walls during the protoplast regeneration process. So far, especially for the carrot, a model species for in vitro cultures, it has not been specified what pectic, arabinogalactan protein (AGP) and extensin epitopes are involved in the reconstruction of the wall in protoplast-derived cells. Even less is known about the correlation between wall regeneration and the presence of PSK during the protoplast culture. Three Daucus taxa, including the cultivated carrot, were analyzed during protoplast regeneration. Several antibodies directed against wall components (anti-pectin: LM19, LM20, anti-AGP: JIM4, JIM8, JIM13 and anti-extensin: JIM12) were used. The obtained results indicate a diverse response of the used Daucus taxa to PSK in terms of protoplast-derived cell development, and diversity in the chemical composition of the cell walls in the control and the PSK-treated cultures.

Sugarcane somatic cell hybridization can break through the barrier of genetic incompatibility between distantly related species in traditional breeding. However, the molecular mechanisms of sugarcane protoplast regeneration and the conditions for protoplast preparation remain largely unknown. In this study, young sugarcane (ROC22) leaves were enzymatically digested, and the viability of protoplasts reached more than 90% after enzymatic digestion (Enzymatic combination: 2% cellulase + 0.5% pectinase + 0.1% dissociative enzyme + 0.3% hemicellulase, pH = 5.8). Transcriptome sequencing was performed on young sugarcane leaves and protoplasts after enzymatic digestion to analyze the differences in gene expression in somatic cells before and after enzymatic digestion. A total of 117,411 unigenes and 43,460 differentially expressed genes were obtained, of which 21,123 were up-regulated and 22,337 down-regulated. The GO terms for the 43,460 differentially expressed genes (DEGs) were classified into three main categories: biological process, cellular component and molecular function, which related to developmental process, growth, cell proliferation, transcription regulator activity, signal transducer activity, antioxidant activity, oxidative stress, kinase activity, cell cycle, cell differentiation, plant hormone signal transduction, and so on. After enzymatic digestion of young sugarcane leaves, the expressions of GAUT, CESA, PSK, CyclinB, CyclinA, CyclinD3 and cdc2 genes associated with plant regeneration were significantly down-regulated to 65%, 47%, 2%, 18.60%, 21.32%, 52% and 45% of young leaves, respectively. After enzymatic digestion, Aux/IAA expression was up-regulated compared with young leaves, and Aux/IAA expression was 3.53 times higher than that of young leaves. Compared with young leaves, these key genes were significantly changed after enzymatic digestion. These results indicate that the process of somatic enzymatic digestion process may affect the regeneration of heterozygous cells to a certain extent.

Naturally induced secretions from infective juveniles of the potato cyst nematode Globodera rostochiensis co-stimulate the proliferation of tobacco leaf protoplasts in the presence of the synthetic phytohormones α-naphtha-leneacetic acid (NAA) and 6-benzylaminopurine (BAP). With the use of a protoplast-based bioassay, a low-molecular-weight peptide(s) (<3 kDa) was shown to be responsible for the observed effect. This mitogenic oligopeptide(s) is functionally dissimilar to auxin and cytokinin and, in addition, it does not change the sensitivity of the protoplasts toward these phytohormones. In combination with the mitogen phytohemagglutinin (PHA), cyst nematode secretions also co-stimulated mitogenesis in human peripheral blood mononuclear cells (PBMC). The stimulation of plant cells isolated from nontarget tissue—these nematodes normally invade the roots of potato plants—suggests the activation of a general signal transduction mechanism(s) by an oligopeptide(s) secreted by the nematode. Whether a similar oligopeptide-induced mechanism underlies human PBMC activation remains to be investigated. Reactivation of the cell cycle is a crucial event in feeding cell formation by cyst nematodes. The secretion of a mitogenic low-molecular-weight peptide(s) by infective juveniles of the potato cyst nematode could contribute to the redifferentiation of plant cells into such a feeding cell.

This study was designed to assess the general limitations of somatic hybridization as one of the key technologies for genetic manipulation in plants. The limits of somatic hybridization against different taxonomic backgrounds, intraspecific to interfamilial, were also assessed. Protoplast culture studies provided essential information relating to the species cultural and morphogenetic capacity. several #elect Ion strategies for the recovery of somatic hybrid colonies/plants were developed and assessed using various combinations of protoplast sources and species in the genera Petunia, Nicotiana, Salpiglossis and Chrysanthemum. Morphological, cytological and biochemical analyses were performed to confirm the hybridity of plants or cell lines recovered following protoplasm fusion (using 4-5 methods) and selection.The somatic hybrid callus/plants were obtained at intraspecific to interfamilial levels by complementation to chlorophyll proficiency, together with media selection or complementation of nitrate reductase deficient mutants as follows; P. Hybrida var. Monsanto (+) P. hybrida cv. Blue Lace (intraspecific), P. hybrida var. Monsanto (+) P. inflata and P. parviflora (interspecific), P. parviflora (+) N. tabacum (intergeneric), S. sinuata (+) P. hybrida var. Monsanto, P. parodii and N. tabacum (intertribal), and C. morifolium (+) S. sinuata.From this study, it appeared that there were no taxonomic limits to the production and proliferation of somatic hybrid cell lines. However, obtaining morphologically normal hybrid plants met with limited success as the taxonomic relationships became more distant. The regeneration capacity of somatic hybrids seemed to be controlled by both parental species. Somatic incompatibility mechanism was also shown to operate on chromosome elimination. Such chromosome elimination may well be advantageous in plant improvement.

In this paper, the roles of type and concentration of enzymes on protoplast isolation from in vitro leaves, multi-scalps (highly proliferating meristem culture), and young male flower of banana cv. cau man were studied. Respiration rate and content of plant hormones of these materials were analysed. Different techniques were used to culture these protoplasts. The development of protoplasts was observed under fluorescence microscope. The highest yield of protoplast (69.5 x 106 protoplasts / g fresh weight) was obtained from young male flowers after 16 hours treatment with 1.5 % cellulase, 0.25 % pectinase and 0.25 % hemicellulase. The combination of hanging drop cell technique (in 6 days), and carrot feeder layer cells in N6PKM medium supplemented with 0.2 mg/L 2,4- dichlorophenoxyacetic acid (2,4-D), 1 mg/L α- naphthalene acetic acid (NAA), and 0.5 mg/L zeatin are suitable for protoplast development. Protoplasts that were isolated from multi-scalps and young male flowers created the wall and divided when cultured by this method. The development of protoplasts from young male flower began with cell walls creation after 4 days, the cell division was after 6 days, and small colonies formation was after 28 days of culture. The differentiation and physiological activity of cells play an important role on quantity and quality of protoplasts, as on the well as protoplast development.

The structure of syncytia induced by <i>Heterodera schachtii</i> Schmidt in roots of susceptible <i>Raphanus sativus</i> L. cv. "Siletina" and resistant radish cv. "Pegletta" was investigated. In the radish cultivar "Siletina" the syncytia most often appeared in the elongation zone of lateral roots. They were initiated in the procambium and pericycle but also included the parenchyma cells of vascular cylinder. In the susceptible cultivar "Siletina" the cells forming the female's syncytia were subject to hypertrophy. Their cytoplasmic density increased. The cytoplasm contained numerous organella. The proliferation of the smooth endoplasmic reticulum took place. Branched cell wall ingrowths were formed next to the vessels. In the male's syncytia the cells were only slightly increased. Their protoplasts contained few organelles. The cell wall ingrowths were poorly developed. In the syncytia of the resistant cultivar "Pegletta" there was only a slight increase of the cell volume. A well developed system of rough endoplasmic reticulum was observed in the protoplast. Distended ER cisterns contained fine fibrillar material. Material of similar structure also appeared in numerous small vacuoles. In resistant plants only some, not numerous, syncytia spreading in procambium fully developed and functioned long enough for the parasite females to mature. At an advanced stage of infection a well developed system of a rough ER was observed also in those syncytia and numerous vacuoles appeared.

As a valuable ornamental plant, Sinningia hybrida ‘Isa’s Murmur’ (S. hybrida) has genetic flower diversity, which has great potential to develop different flower characters in the horticultural market. The present study focuses on establishing a practical approach for the sustainable propagation of S. hybrida. Compared with aseptic seeding leaves explants, field-grown leaves explants are more suitable for adventitious shoot regeneration. Adding 0.1 mg L−1 NAA and 2.0 mg L−1 TDZ could obtain the highest adventitious shoot proliferation coefficient (24.5), and the induction rate was 91.7%. The shoot proliferation coefficient (20.7) and the greatest shoot length and induction rate (95.3%) were achieved in 0.1 mg L−1 NAA and 2.0 mg L−1 BA medium, accompanied by rooting formation. Adding 0.5 mg L−1 GA3, 1.0 mg L−1 BA, and 0.2 mg L−1 IBA to MS medium can effectively prolong the regenerated buds for rooting. The best for rooting was 1/2 MS medium containing 0.3 mg L−1 IBA, with the maximum number of roots (13.4 per shoot) and survival rate for transplanting (100%). This work aims to build an efficient, definitive, and scalable protocol for S. hybrida regeneration useful for large-scale cultivation and even more protoplast fusion and genetic transformation to develop more colorful or fragrant flowers.

Several experiments were conducted to evaluate the influence of explant type, sucrose level, and callus development time on sweetpotato [Ipomoea batatas (L.) Lam] in vitro culture. Shoot tip, petiole, and leaf of Selection 75-96-1 was used as explants in Murashige and Skoog (MS) media with different plant growth regulators. Calli derived from shoot tip and petiole produced 42.1% and 10.3% somatic embryos, respectively, but the leaf failed to produce somatic embryos. The effect of sucrose level was determined using shoot tip as explants. Compared with 3% sucrose in the same plant growth regulators level medium during callus initiation and callus proliferation periods, 5% sucrose level suppressed root growth and improved shoot regeneration. The callus development time was measured by using shoot tips on callus initiation medium containing 1.5 mg/L alpha-Naphthaleneacetic acid (NAA) and 0.25 mg/L Kinetin (KIN) plus 5% sucrose. When explants were cultured for less than 6 weeks during callus initiation, then transferred onto plant regeneration medium, plant regeneration via organogenesis occurred; whereas, maintaining cultures for more than 12 weeks on the same callus initiation/proliferation medium, plant regeneration was favored via embryogenesis. Explant type and other factors affecting plant regeneration noted here could be applied to protoplast culture, somatic hybridization, and transformation in sweetpotato.

The TEOSINTE BRANCHED1, CYCLOIDEA, and PROLIFERATION CELL FACTOR (TCP) transcription factor is a plant-specific gene family and acts on multiple functional genes in controlling growth, development, stress response, and the circadian clock. In this study, a class I member of the TCP family from Fraxinus mandshurica Rupr. was isolated and named FmTCP15, which encoded a protein of 362 amino acids. Protein structures were analyzed and five ligand binding sites were predicted. The phylogenetic relationship showed that FmTCP15 was most closely related to Solanaceae and Plantaginaceae. FmTCP15 was localized in the nuclei of F. mandshurica protoplast cells and highly expressed in cotyledons. The expression pattern revealed the FmTCP15 response to multiple abiotic stresses and hormone signals. Downstream genes for transient overexpression of FmTCP15 in seedlings were also investigated. A yeast two-hybrid assay confirmed that FmTCP15 could interact with DELLA proteins. FmTCP15 participated in the GA-signaling pathway, responded to abiotic stresses and hormone signals, and regulated multiple genes in these biological processes. Our study revealed the potential value of FmTCP15 for understanding the molecular mechanisms of stress and hormone signal responses.

Macrophage migration inhibitory factor (MIF), named for its role in inhibiting macrophage/monocyte migration, has multiple functions in modulation of inflammation, cell proliferation, angiogenesis, and tumorigenesis in vertebrates. Although homologs of this gene can be found in plants, the function of MIF in plants remains obscure. Here, we characterized TaMIF1 in Triticum aestivum resembling the MIF secreted from Homo sapiens. Transcript analysis revealed that TaMIF1 responded to stripe rust infection of wheat and was upregulated during the infection stage. TaMIF1 was localized to both the cytosol and nuclei in wheat mesophyll protoplast. Additionally, TaMIF1 possessed significant tautomerase activity, indicating conservation of MIFs across kingdoms. Agrobacterium tumefaciens infiltration assay demonstrated that TaMIF1 was capable of suppressing programmed cell death hinting its role in plant immunity. Heterologous expression of TaMIF1 increased fission yeast sensitivity to oxidative stress. Silencing TaMIF1 decreased the susceptibility of wheat to Pst seemingly through increasing reactive oxygen species accumulation. In conclusion, functions of the TaMIF1 were investigated in this study, which provides significant insight into understanding the role of MIFs across kingdoms.

Torenia fournieri (T. fournieri) is one of the most widely used horticultural flowers and is considered a potential model plant for the genetic investigation of ornamental traits. In this study, we optimized an efficient protocol for high efficiency preparation and transformation of T. fournieri protoplast. The transformation rate reached ~75% when a 35S:GFP construct was used for the transformation. Using this system, we characterized the subcellular localization of several TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR (TCP) transcription factors (TFs), and found a distinct localization pattern between the CIN and CYC classes of TCP TFs. Furthermore, we also demonstrated the feasibility of the expression of dual luciferase assay system in T. fournieri protoplasts for the measurement of the activity of cis-regulatory elements. Taken together, a well-optimized transient expression system in T. fournieri protoplasts would be crucial for rapid exploration of the gene function or cis-regulatory elements.

ABSTRACT Cowpea mosaic virus (CPMV) replication induces an extensive proliferation of endoplasmic reticulum (ER) membranes, leading to the formation of small membranous vesicles where viral RNA replication takes place. Using fluorescent in situ hybridization, we found that early in the infection of cowpea protoplasts, CPMV plus-strand RNA accumulates at numerous distinct subcellular sites distributed randomly throughout the cytoplasm which rapidly coalesce into a large body located in the center of the cell, often near the nucleus. The combined use of immunostaining and a green fluorescent protein ER marker revealed that during the course of an infection, CPMV RNA colocalizes with the 110-kDa viral polymerase and other replication proteins and is always found in close association with proliferated ER membranes, indicating that these sites correspond to the membranous site of viral replication. Experiments with the cytoskeleton inhibitors oryzalin and latrunculin B point to a role of actin and not tubulin in establishing the large central structure. The induction of ER membrane proliferations in CPMV-infected protoplasts did not coincide with increased levels of BiP mRNA, indicating that the unfolded-protein response is not involved in this process.

To investigate the ecological role of caffeine, theobromine, theophylline and paraxanthine, which are released from purine alkaloid forming plants, the effects of these purine alkaloids on the division and colony formation of lettuce cells were assessed at concentrations up to 1 mM. Five days after treatment with 500 μM caffeine, theophylline and paraxanthine, division of isolated protoplasts was significantly inhibited. Thirteen days treatment with >250 μM caffeine had a marked inhibitory effect on the colony formation of cells derived from the protoplasts. Other purine alkaloids also acted as inhibitors. The order of the inhibition was caffeine > theophylline > paraxanthine > theobromine. These observations suggest that a relatively low concentration of caffeine is toxic for proliferation of plant cells. In contrast, theobromine is a weak inhibitor of proliferation. Possible allelopathic roles of purine alkaloids in natural ecosystems are discussed.

Effector proteins play an important role in the virulence of plant pathogens such as phytoplasma, which are the causative agents of hundreds of different plant diseases. The plant hosts comprise economically relevant crops such as apples (Malus × domestica), which can be infected by ‘Candidatus Phytoplasma mali’ (P. mali), a highly genetically dynamic plant pathogen. As the result of the genetic and functional analyses in this study, a new putative P. mali effector protein was revealed. The so-called “Protein in Malus Expressed 2” (PME2), which is expressed in apples during P. mali infection but not in the insect vector, shows regional genetic differences. In a heterologous expression assay using Nicotiana benthamiana and Nicotiana occidentalis mesophyll protoplasts, translocation of both PME2 variants in the cell nucleus was observed. Overexpression of the effector protein affected cell integrity in Nicotiana spp. protoplasts, indicating a potential role of this protein in pathogenic virulence. Interestingly, the two genetic variants of PME2 differ regarding their potential to manipulate cell integrity. However, the exact function of PME2 during disease manifestation and symptom development remains to be further elucidated. Aside from the first description of the function of a novel effector of P. mali, the results of this study underline the necessity for a more comprehensive description and understanding of the genetic diversity of P. mali as an indispensable basis for a functional understanding of apple proliferation disease.

Ctn[15–34], the C-terminal fragment of crotalicidin, an antimicrobial peptide from the South American rattlesnake Crotalus durissus terrificus venom, displays remarkable anti-infective and anti-proliferative activities. Herein, its activity on Candida albicans biofilms and its interaction with the cytoplasmic membrane of the fungal cell and with a biomembrane model in vitro was investigated. A standard C. albicans strain and a fluconazole-resistant clinical isolate were exposed to the peptide at its minimum inhibitory concentration (MIC) (10 µM) and up to 100 × MIC to inhibit biofilm formation and its eradication. A viability test using XTT and fluorescent dyes, confocal laser scanning microscopy, and atomic force microscopy (AFM) were used to observe the antibiofilm effect. To evaluate the importance of membrane composition on Ctn[15–34] activity, C. albicans protoplasts were also tested. Fluorescence assays using di-8-ANEPPS, dynamic light scattering, and zeta potential measurements using liposomes, protoplasts, and C. albicans cells indicated a direct mechanism of action that was dependent on membrane interaction and disruption. Overall, Ctn[15–34] showed to be an effective antifungal peptide, displaying antibiofilm activity and, importantly, interacting with and disrupting fungal plasma membrane.

Plants are sessile organisms that have a remarkable developmental plasticity, which ensures their optimal adaptation to environmental stresses. Plant cell totipotency is an extreme example of such plasticity, whereby somatic cells have the potential to form plants via direct shoot organogenesis or somatic embryogenesis in response to various exogenous and/or endogenous signals. Protoplasts provide one of the most suitable systems for investigating molecular mechanisms of totipotency, because they are effectively single cell populations. In this review, we consider the current state of knowledge of the mechanisms that induce cell proliferation from individual, differentiated somatic plant cells. We highlight initial explant metabolic status, ploidy level and isolation procedure as determinants of successful cell reprogramming. We also discuss the importance of auxin signalling and its interaction with stress-regulated pathways in governing cell cycle induction and further stages of plant cell totipotency.

Cell-cell fusion is an important natural and engineered process for in-depth studies into hybridomas, developmental biology, immunology and various cellular therapies. It is also a powerful tool for analysis of gene expression, chromosomal mapping, antibody production, cloning mammals, and cancer immunotherapy. However, research so far has primarily focused on cell models such asC.elegans, drosophila, myoblasts, spleen-myeloma cell hybrids and various plant protoplasts. Rhabdomyosarcoma cells are a rare form of musculoskeletal cancer cells found in the head, neck, and other less skeletal areas of the human cancer patient’s body. As these cells do not normally undergo fusion naturally, they are an interesting model for studying cell fusion. Among all the techniques for fusion, electrofusion (or electroporation) can be applied to a wide range of cell types with high efficiency and high post-fusion viability. By coupling these cells with this technique, the effectson cell proliferation, growth pattern, and hybridoma count wereinvestigated. Overall, the experimental results showed that an adequate electrical stimulation helped to facilitate the fusion and proliferation of the RD cells. Furthermore,a DC current produced the highest number of hybridomas, while maintaining the highest proliferation rate.After subtracting for the control samples, an average fusion yield of 24% was obtained under this DC setting, which is comparable to the fusion yield of 20% obtained using the same technique by other researchers. This is a promising result for its application in the production of monoclonal antibodies for cancer research and treatment.

Callus growth and development, a crucial process in plant propagation, is involved in hormonal balance and abundant gene regulation. MiRNAs are key regulators in the process of cell differentiation and development. MiR397 was identified as participating in plant growth, development, and response to stress, and it was regulated by targeting the LAC gene. The regulatory function of miR397 during callus growth and development was not clear in Liriodendron. In this study, LhmiR397a and its targets were identified, and its regulatory function between LhmiR397a and LhLAC11 was shown using qRT-PCR and transient expression in protoplasts. Furthermore, to clarify the regulatory function of LhmiR397a-LhLAC11, transgenic calli overexpressing LhMIR397a, LhLAC11, and mLhLAC11 were separately obtained by Agrobacterium-mediated transfer. The results showed that overexpressing LhMIR397a might retard callus proliferation, while overexpressing LhLAC11 or mLhLAC11 could promote callus proliferation. Genes associated with the cell cycle had decreased expression when LhMIR397a was overexpressed, while increased expression was observed when LhLAC11 or mLhLAC11 was overexpressed. Additionally, the calli overexpressed with LhMIR397a could generate early cotyledons 21 days after induction, and the somatic embryo induction time was short compared with other genotypes. This study identified LhmiR397a and its targets and provided a functional characterization of LhmiR397a in callus growth and development by regulating its target in Liriodendron.

Biocontrol activity of Candida saitoana and its interaction with Botrytis cinerea in apple wounds were investigated. When cultured together, yeast attached to Botrytis sp. hyphal walls. In wounded apple tissue, C. saitoana restricted the proliferation of B. cinerea, multiplied, and suppressed disease caused by either B. cinerea or Penicillium expansum. In inoculated apple tissue without the yeast, fungal colonization caused an extensive degradation of host walls and altered cellulose labeling patterns. Hyphae in close proximity to the antagonistic yeast exhibited severe cytological injury, such as cell wall swelling and protoplasm degeneration. Colonization of the wound site by C. saitoana did not cause degradation of host cell walls. Host cell walls in close contact with C. saitoana cells and B. cinerea hyphae were well preserved and displayed an intense and regular cellulose labeling pattern. In addition to restricting fungal colonization, C. saitoana induced the formation of structural defense responses in apple tissue. The ability of C. saitoana to prevent the necrotrophic growth of the pathogen and stimulate structural defense responses may be the basis of its biocontrol activity.

Abstract In China, research on tissue culture applications to fruit crop improvement has expanded greatly in the past 10 years. Tissue culture is considered to be an effective method for a) virus elimination from vegetatively propagated plants; b) rapid clonal multiplication of superior cultivars; c) the isolation and avoidance of chimeras that appear in mutation breeding, d) the establishment of haploid and polyploid lines; e) overcoming sterility in breeding through embryo rescue; and f) germplasm maintenance and storage. Tissue culture technology has been used for most of the important fruit crops in China, including a few fruit crops introduced from foreign countries (Table 1). Plants can be regenerated from various explants such as a) shoot-tips and meristems, b) cotyledons, c) anthers, d) immature and mature embryos, and e) endosperm. Plants can also be regenerated from protoplasts via organogenesis, somatic embryogenesis, or axillary bud proliferation. Procedures for the mass production in vitro of plants for commercial purposes have been established in grape (Vitis vinifera L.), hawthorn (Crataegus pinnatifida Bunge), strawberry (Fragaria spp.), and currant (Ribes nigrum L.). An overview of the various tissue culture technologies that are being used to improve fruit crops in China is presented here.

Cyclin-dependent kinase inhibitors known as KRPs (kip-related proteins) control the progression of plant cell cycles and modulate various plant developmental processes. However, the function of KRPs in rice remains largely unknown. In this study, two rice KRPs members, KRP1 and KRP2, were found to be predominantly expressed in developing seeds and were significantly induced by exogenous abscisic acid (ABA) and Brassinosteroid (BR) applications. Sub-cellular localization experiments showed that KRP1 was mainly localized in the nucleus of rice protoplasts. KRP1 overexpression transgenic lines (OxKRP1), krp2 single mutant (crkrp2), and krp1/krp2 double mutant (crkrp1/krp2) all exhibited significantly smaller seed width, seed length, and reduced grain weight, with impaired seed germination and retarded early seedling growth, suggesting that disturbing the normal steady state of KRP1 or KRP2 blocks seed development partly through inhibiting cell proliferation and enlargement during grain filling and seed germination. Furthermore, two cyclin-dependent protein kinases, CDKC;2 and CDKF;3, could interact with KRP1 in a yeast-two-hybrid system, indicating that KRP1 might regulate the mitosis cell cycle and endoreduplication through the two targets. In a word, this study shed novel insights into the regulatory roles of KRPs in rice seed maturation and germination.

ABSTRACT Positive-strand RNA viruses use diverse mechanisms to regulate viral and host gene expression for ensuring their efficient proliferation or persistence in the host. We found that a small viral noncoding RNA (0.4 kb), named SR1f, accumulated in Red clover necrotic mosaic virus (RCNMV)-infected plants and protoplasts and was packaged into virions. The genome of RCNMV consists of two positive-strand RNAs, RNA1 and RNA2. SR1f was generated from the 3′ untranslated region (UTR) of RNA1, which contains RNA elements essential for both cap-independent translation and negative-strand RNA synthesis. A 58-nucleotide sequence in the 3′ UTR of RNA1 (Seq1f58) was necessary and sufficient for the generation of SR1f. SR1f was neither a subgenomic RNA nor a defective RNA replicon but a stable degradation product generated by Seq1f58-mediated protection against 5′→3′ decay. SR1f efficiently suppressed both cap-independent and cap-dependent translation both in vitro and in vivo. SR1f trans inhibited negative-strand RNA synthesis of RCNMV genomic RNAs via repression of replicase protein production but not via competition of replicase proteins in vitro. RCNMV seems to use cellular enzymes to generate SR1f that might play a regulatory role in RCNMV infection. Our results also suggest that Seq1f58 is an RNA element that protects the 3′-side RNA sequences against 5′→3′ decay in plant cells as reported for the poly(G) tract and stable stem-loop structure in Saccharomyces cerevisiae.

ABSTRACT Most dicot-infecting geminiviruses encode a replication enhancer protein (C3, AL3, or REn) that is required for optimal replication of their small, single-stranded DNA genomes. C3 interacts with C1, the essential viral replication protein that initiates rolling circle replication. C3 also homo-oligomerizes and interacts with at least two host-encoded proteins, proliferating cell nuclear antigen (PCNA) and the retinoblastoma-related protein (pRBR). It has been proposed that protein interactions contribute to C3 function. Using the C3 protein of Tomato yellow leaf curl virus, we examined the impact of mutations to amino acids that are conserved across the C3 protein family on replication enhancement and protein interactions. Surprisingly, many of the mutations did not affect replication enhancement activity of C3 in tobacco protoplasts. Other mutations either enhanced or were detrimental to C3 replication activity. Analysis of mutated proteins in yeast two-hybrid assays indicated that mutations that inactivate C3 replication enhancement activity also reduce or inactivate C3 oligomerization and interaction with C1 and PCNA. In contrast, mutated C3 proteins impaired for pRBR binding are fully functional in replication assays. Hydrophobic residues in the middle of the C3 protein were implicated in C3 interaction with itself, C1, and PCNA, while polar resides at both the N and C termini of the protein are important for C3-pRBR interaction. These experiments established the importance of C3-C3, C3-C1, and C3-PCNA interactions in geminivirus replication. While C3-pRBR interaction is not required for viral replication in cycling cells, it may play a role during infection of differentiated cells in intact plants.

The establishment of an efficient in vitro propagation system for the conservation of the Mediterranean Mandragora autumnalis is highly desirable due to its scarcity, besides its potential medicinal and pharmacological properties. In a separate unpublished study, this species has proved to be resistant to laboratory plant regeneration from vegetative tissue cultures; therefore, an alternative decoated seed (i.e., endosperm enclosed the zygotic embryo) germination approach was conducted in this study. Pre-cold treatment of M. autumnalis seeds, removal of seed coats, and exogenous application of gibberellic acid (GA3) promoted in vitro seed germination and seedling emergence. In two separate experiments, approximately 10–27% of the germinated decoated seeds developed healthy seedlings within two weeks, compared to the non-germinated intact seeds of the potting soil controls. After 72 days, the highest rates of healthy seedlings development (67.4 and 69.4%) achieved in the in vitro decoated seed cultures supplemented with 60 and 100 mg/L GA3, respectively, compared to only 25% seedlings emergence rate of the in vitro cultures devoid of GA3, and 44.2% of the soil controls. The in vitro developed plants were healthy, survived transplantation conditions, and, significantly, grew faster, formed on average more than the double number of true leaves and shoot fresh weight (p ≤ 0.05), 90% more fresh weight of root system (p ≤ 0.05), and ultimately more than the double gross fresh weight (p ≤ 0.05) than that of the in vivo developed plants of the soil controls. Such in vitro seed germination approaches would be favorable due to the higher capacity of uniform seedling establishment year-round under lab-controlled conditions, facilitating proliferation and conservation of rare and threatened species, and providing fresh and axenic plant materials required for downstream studies such as those associated with leaf-derived protoplasts and genetic transformations.

In the animal life cycle, the earliest manifestations of programmed cell death (PCD) can already be seen during embryogenesis. The aim of this work was to determine if PCD is also involved in the elimination of certain cells during plant embryogenesis. We used a model system of Norway spruce somatic embryogenesis, which represents a multistep developmental pathway with two broad phases. The first phase is represented by proliferating proembryogenic masses (PEMs). The second phase encompasses development of somatic embryos, which arise from PEMs and proceed through the same sequence of stages as described for their zygotic counterparts. Here we demonstrate two successive waves of PCD, which are implicated in the transition from PEMs to somatic embryos and in correct embryonic pattern formation, respectively. The first wave of PCD is responsible for the degradation of PEMs when they give rise to somatic embryos. We show that PCD in PEM cells and embryo formation are closely interlinked processes, both stimulated upon withdrawal or partial depletion of auxins and cytokinins. The second wave of PCD eliminates terminally differentiated embryo-suspensor cells during early embryogeny. During the dismantling phase of PCD, PEM and embryo-suspensor cells exhibit progressive autolysis, resulting in the formation of a large central vacuole. Autolytic degradation of the cytoplasm is accompanied by lobing and budding-like segmentation of the nucleus. Nuclear DNA undergoes fragmentation into both large fragments of about 50 kb and multiples of approximately 180 bp. The tonoplast rupture is delayed until lysis of the cytoplasm and organelles, including the nucleus, is almost complete. The protoplasm then disappears, leaving a cellular corpse represented by only the cell wall. This pathway of cell dismantling suggests overlapping of apoptotic and autophagic types of PCD during somatic embryogenesis in Norway spruce.

Abstract Background Although quantitative single-cell analysis is frequently applied in animal systems, e.g. to identify novel drugs, similar applications on plant single cells are largely missing. We have exploited the applicability of high-throughput microscopic image analysis on plant single cells using tobacco leaf protoplasts, cell-wall free single cells isolated by lytic digestion. Protoplasts regenerate their cell wall within several days after isolation and have the potential to expand and proliferate, generating microcalli and finally whole plants after the application of suitable regeneration conditions. Results High-throughput automated microscopy coupled with the development of image processing pipelines allowed to quantify various developmental properties of thousands of protoplasts during the initial days following cultivation by immobilization in multi-well-plates. The focus on early protoplast responses allowed to study cell expansion prior to the initiation of proliferation and without the effects of shape-compromising cell walls. We compared growth parameters of wild-type tobacco cells with cells expressing the antiapoptotic protein Bcl2-associated athanogene 4 from Arabidopsis (AtBAG4). Conclusions AtBAG4-expressing protoplasts showed a higher proportion of cells responding with positive area increases than the wild type and showed increased growth rates as well as increased proliferation rates upon continued cultivation. These features are associated with reported observations on a BAG4-mediated increased resilience to various stress responses and improved cellular survival rates following transformation approaches. Moreover, our single-cell expansion results suggest a BAG4-mediated, cell-independent increase of potassium channel abundance which was hitherto reported for guard cells only. The possibility to explain plant phenotypes with single-cell properties, extracted with the single-cell processing and analysis pipeline developed, allows to envision novel biotechnological screening strategies able to determine improved plant properties via single-cell analysis.

<span>Phytosulfokines (PSK) are peptidyl growth factors with the potential of inducing cell proliferation. We examined the effect of supplementation of liquid culture medium with 0.1 µM phytosulfokine-α (PSK-α) on protoplast viability and division frequencies in seven accessions of <em>Brassica oleracea</em> var. <em>capitata</em> L., including cultivars and breeding lines. Protoplasts were isolated from leaves and hypocotyls of in vitro grown plants and immobilized in calcium-alginate layers. Cabbage protoplast-derived cells cultured in medium supplemented with 0.1 µM of PSK-α had higher viability and division frequencies compared to cells cultured in PSK-α-free control medium. The effect of PSK-α was more pronounced in low-responding accessions (‘Sława z Gołębiewa’, ‘Ramkila F1’, LM, and LM98); however, in two cultivars with very low response (‘Badger Shipper’ and ‘Oregon 123’), although the division frequencies in the media supplemented with PSK-α were increased over the control, the differences were not significant. Obtained callus colonies were subjected to regeneration. PSK-α supplemented into the liquid culture medium had an indirect effect on shoot regeneration by inducing sustained cell divisions leading to an increase in shoot regeneration in Sława z Gołębiewa and both breeding lines.</span>

ABSTRACT Bamboo mosaic virus (BaMV), a member of the Potexvirus genus, has a monopartite positive-strand RNA genome on which five open reading frames (ORFs) are organized. ORF1 encodes a 155-kDa nonstructural protein (REPBaMV) that plays a core function in replication/transcription of the viral genome. To find out cellular factors modulating the replication efficiency of BaMV, a putative REPBaMV-associated protein complex from Nicotiana benthamiana leaf was isolated on an SDS-PAGE gel, and a few proteins preferentially associated with REPBaMV were identified by tandem mass spectrometry. Among them, proliferating cell nuclear antigen (PCNA) was particularly noted. Overexpression of PCNA strongly suppressed the accumulation of BaMV coat protein and RNAs in leaf protoplasts. In addition, PCNA exhibited an inhibitory effect on BaMV polymerase activity. A pulldown assay confirmed a binding capability of PCNA toward BaMV genomic RNA. Mutations at D41 or F114 residues, which are critical for PCNA to function in nuclear DNA replication and repair, disabled PCNA from binding BaMV genomic RNA as well as suppressing BaMV replication. This suggests that PCNA bound to the viral RNA may interfere with the formation of a potent replication complex or block the replication process. Interestingly, BaMV is almost invisible in the newly emerging leaves where PCNA is actively expressed. Accordingly, PCNA is probably one of the factors restricting the proliferation of BaMV in young leaves. Foxtail mosaic virus and Potato virus X were also suppressed by PCNA in the protoplast experiment, suggesting a general inhibitory effect of PCNA on the replication of potexviruses. IMPORTANCE Knowing the dynamic interplay between plant RNA viruses and their host is a basic step toward first understanding how the viruses survive the plant defense mechanisms and second gaining knowledge of pathogenic control in the field. This study found that plant proliferating cell nuclear antigen (PCNA) imposes a strong inhibition on the replication of several potexviruses, including Bamboo mosaic virus, Foxtail mosaic virus, and Potato virus X. Based on the tests on Bamboo mosaic virus, PCNA is able to bind the viral genomic RNA, and this binding is a prerequisite for the protein to suppress the virus replication. This study also suggests that PCNA plays an important role in restricting the proliferation of potexviruses in the rapidly dividing tissues of plants.

In accordance with the recommendations of The International Council for Standardization in Haematology (ICSH, https://icsh.org/), this article describes the morphophysiological characteristics of the precursors and mature erythroid cells in the early period of postnatal development of birds (Gallus gallus L.) including calculation of the surface area of these cells (S, μm2 , X±SEM). Depending on cell shape, cytoplasm color, and chromatin organization in nucleus, the following types are distinguished: basophilic erythroblasts (69.60±4.01 μm2 , p≤0.05), polychromatophilic erythroblasts (65.42±2.49 μm2 , p≤0.05), and oxyphilic erythroblasts (71.10±4.43 μm2). Formation of cell pool is characteristic for erythropoiesis in birds due to mitotic proliferation of basophilic erythroblasts. There are often proerythroblasts and polychromatophilic erythroblasts. The nucleus of a polychromatophilic proerythroblast contains a large number of histone proteins; therefore, it has an intensely basophilic color with a pronounced oxyphilic hue (proteinrelated oxyphilia). The accumulation of hemoglobin in the protoplasm of these cells contributes to the gradual transition of the basophilic staining of cytoplasm to the oxyphilic one which is typical for mature red blood cells (73.95±2.10 μm2 , p≤0.05). Cell shape and the structure of erythroblast nucleus approaches to these of mature red blood cells.

abstract Shoot regeneration involves reprogramming of somatic cells and de novo organization of shoot apical meristems (SAMs). In the best-studied model system of shoot regeneration using Arabidopsis, regeneration is mediated by the auxin-responsive pluripotent callus formation from pericycle or pericycle-like tissues according to the lateral root development pathway. In contrast, shoot regeneration can be induced directly from fully differentiated epidermal cells of stem explants of Torenia fournieri (Torenia), without intervening the callus mass formation in culture with cytokinin; yet, its molecular mechanisms remain unaddressed. Here, we characterized this direct shoot regeneration by cytological observation and transcriptome analyses. The results showed that the gene expression profile rapidly changes upon culture to acquire a mixed signature of multiple organs/tissues, possibly associated with epidermal reprogramming. Comparison of transcriptomes between three different callus-inducing cultures (callus induction by auxin, callus induction by wounding and protoplast culture) of Arabidopsis and the Torenia stem culture identified genes upregulated in all the four culture systems as candidates of common factors of cell reprogramming. These initial changes proceeded independently of cytokinin, followed by cytokinin-dependent, transcriptional activations of nucleolar development and cell cycle. Later, SAM regulatory genes became highly expressed, leading to SAM organization in the foci of proliferating cells in the epidermal layer. Our findings revealed three distinct phases with different transcriptomic and regulatory features during direct shoot regeneration from the epidermis in Torenia, which provides a basis for further investigation of shoot regeneration in this unique culture system.

PENDAHULUAN Stem cell sering disebut sebagai sel punca atau sel induk, bertanggung jawab atas regenerasi dan pemeliharaan jaringan serta memiliki karakteristik yang unik yaitu menghasilkan salinan dirinya dan keturunan sel yang berbeda ketika membelah.1 Stem cell yang berasal dari tumbuhan memiliki sifat membantu merangsang dan meregenerasi tanaman setelah cedera. Sifat unik plant stem cell telah menjadi bidang yang banyak diminati baru-baru ini, terutama dalam mengembangkan kosmetik baru dan mempelajari bagaimana ekstrak/phytohormone ini dapat mempengaruhi kulit. Usaha regeneratif yang dilakukan tumbuh-tumbuhan tidak hanya pada perbaikan jaringan akibat kerusakan, tetapi juga perkembangan tumbuhan yang baru.2,3Penuaan kulit merupakan suatu proses kompleks yang dipengaruhi faktor internal dan eksternal serta melibatkan seluruh lapisan epidermis dan dermis.4,5 Tujuan dari kosmetik antipenuaan modern adalah untuk memperbaiki tampilan kulit dengan menstimulasi dan meregenerasi proses fisiologis alami demi perbaikan kondisi kulit dan perlindungan dari berbagai faktor yang menyebabkan penuaan, terlepas berapapun usia sesungguhnya. Kandungan yang menarik perhatian adalah plant stem cell, telah dinyatakan memberikan efek proteksi terhadap stem cell manusia dengan cara menstimulasi regenerasi kulit dan mencegah proses penuaannya.4 Penuaan KulitPenuaan adalah proses biologis yang tak terhindarkan, kompleks dan dinamis yang ditandai dengan kemunduran progresif dari berbagai sistem pada tubuh dan penurunan kapasitas cadangan fisiologis.5 Kulit manusia mengalami dua jenis penuaan yaitu penuaan intrinsik dan ekstrinsik. Penuaan intrinsik mencakup serangkaian perubahan fisiologis bertahap yang merupakan konsekuensi dari waktu ke waktu dan dipengaruhi genetik dan hormonal. Penuaan ekstrinsik merupakan perubahan struktural dan fungsional yang disebabkan oleh faktor eksogen, terutama paparan sinar matahari (disebut juga photoaging), alkohol, merokok, malnutrisi dan lingkungan yang merugikan, namun dalam kondisi tertentu masih dapat dihindari.5-7Proses penuaan kulit intrinsik ditandai dengan adanya proses penuaan seluler, penurunan kapasitas proliferasi, penurunan kemampuan perbaikan deoxyribonucleic acid (DNA), stres oksidatif dan mutasi gen.8 Fungsi sawar kulit terganggu akibat perubahan struktur filamen keratin dan penurunan filagrin.Dalam hormonal, hormon seks terutama estrogen mempengaruhi sintesis kolagen, asam hialuronat, elastin dan komponen lain dari matriks ekstraseluler. Bersama-sama, ketiga komponen ini memberikan tampilan kulit yang sehat dan muda.Perubahan biokimia yang terjadi pada kolagen, elastin dan komponen dasar kulit menyebabkan penuaan kulit.5,9 Umumnya terjadi pada area kulit yang terlindungi dari paparan sinar matahari dengan klinis yang relatif lebih ringan, ditandai dengan tampilan kulit kering, pucat dan kendur dengan kerutan halus dan atau berbagai bentuk neoplasma jinak.10Sumber terbesar penuaan ekstrinsik adalah akumulasi dan paparan sinar matahari pada area yang tidak terlindungi seperti wajah, leher, dada dan lengan ekstensor. Faktor lain yang berpengaruh adalah merokok, terbukti kadar matrix metalloproteinase-1 (MMP-1) lebih tinggi pada perokok.5,6 Diet nutrisi seimbang memperlambat penuaan dengan menyediakan nutrisi, air dan oksigen yang diperlukan sel dalam pembelahan, mengirimkan informasi dan memperbaiki kerusakan.5 Photoaging atau penuaan kulit dini merupakan istilah yang digunakan untuk menggambarkan klinis dan histologi akibat paparan sinar matahari kronis.5-7,11 Peningkatan kerusakan dan penurunan produksi kolagen adalah landasan dari photoaging, ditandai dengan penampilan kulit yang kasar, kerutan, warna kulit menjadi pudar, telangiektasis, pigmentasi tidak merata, dan berbagai lesi jinak, premaligna dan neoplasma ganas.5,9-11 Stres oksidatif dan penuaanSalah satu teori penuaan melibatkan proses penuaan seluler atau apoptosis sekunder adalah stres oksidatif. Stres oksidatif merupakan kondisi ketidakseimbangan antara reactive oxygen species (ROS) dengan mekanisme antioksidan. Pertahanan antioksidan sistem enzimatik dan non-enzimatik pada kulit cenderung melemah seiring bertambahnya usia.5,10 Kerusakan oksidatif menyebabkan peningkatan pembentukan faktor-faktor yang berhubungan dengan stres, kemudian memicu proses penuaan intrinsik. Misalnya, hypoxia-inducible factor dan nuclear factor ĸB menginduksi ekspresi sitokin interleukin 1, interleukin 6, vascular endothelial growth factor (VEGF) dan tumor necrosis factor α (TNFα), yang telah terbukti merupakan regulator proinflamasi dan modulator penghancuran MMP. Selain itu, kerusakan oksidatif terhadap protein seluler bersamaan dengan penurunan aktivitas proteasom sejalan usia membentuk akumulasi kerusakan protein yang dapat mengganggu fungsi seluler normal.5Stres oksidatif juga mampu memodifikasi telomer yang awalnya bertahan terhadap degradasi, fusi atau rekombinasi abnormal.3,5 Pemendekan telomer adalah hasil dari ketidakmampuan DNA polimerase untuk mereplikasi pasangan basa akhir kromosom. Ketika mencapai ambang "sangat pendek", sel akan mengalami penuaan proliferatif atau apoptosis. Mekanisme umum penuaan intrinsik dan photoaging disebabkan oleh gangguan struktur siklus putaran normal pada akhir telomer. Pemaparan ini kemudian mengaktifkan pensinyalan p53 yang mengarah pada peristiwa penuaan proliferatif dan apoptosis.5Penuaan kulit merupakan proses kompleks yang melibatkan seluruh lapisan epidermis dan dermis, mencakup denaturasi protein dan penurunan fungsi regeneratif stem cell.3 Penurunan fungsi stem cell epidermis telah diamati berhubungan dengan telomer yang lebih pendek, yang mengurangi potensi proliferatif sebagai respon terhadap paparan UV.5 Plant Stem CellKarakteristik plant stem cellSalah satu teori Weismann mengenai penuaan menjelaskan bagaimana organisme multiselular menua melalui germ line yang imortal dengan akumulasi kerusakan lebih didistribusikan pada sel somatik. Tumbuhan tidak secara jelas memisahkan mana germ line dan sel somatik, sehingga menjadi pertanyaan apakah teori penuaan berlaku untuk tumbuh-tumbuhan. Plant stem cell memiliki tampilan seperti germ line, sementara jaringan tumbuhan yang mati pada saat musiman, seperti daun dan xilem, memiliki sifat seperti sel somatik. Bagaimana sebuah tumbuhan dapat mencapai usia yang ekstrem, plant stem cell mungkin memegang kunci dalam hal ini karena tumbuhan memiliki pasokan stem cell terus menerus.12Stem cell dipertahankan secara terus menerus melalui mekanisme pembaruan diri yang terjadi di dalam niches stem cell atau dediferensiasi struktur dewasa. Sel-sel ini memunculkan organ baru sehingga memungkinkan bertahan dalam kondisi ekstrem. Sebuah studi mengenai model embriogenesis somatik menunjukkan sistem yang menarik yaitu regenerasi tanaman dari protoplas mesofil sebagai jenis sel yang terpapar dan berkemungkinan dapat mengakumulasi kerusakan lalu berdediferensiasi menjadi sel pluripoten. Contoh model protoplas dari sel-sel mesofil yaitu Arabidopsis, dapat diinduksi dengan menambahkan phytohormones cytokinin dan auksin. Akar dan pucuknya dapat beregenerasi dari callus untuk menghasilkan tanaman dewasa dan memungkinkan untuk digunakan dalam perangkat molekuler genetik yang tersedia. 12Plant stem cell dikelompokkan menjadi niches yang disebut meristem, terdiri atas primer dan sekunder. Meristem primer adalah meristem apikal (pucuk pertumbuhan batang dan akar), meristem interkalari (sisipan) dan meristem germ. Meristem sekunder adalah bagian lateral yaitu kambium (smear) dan phellogen serta bagian traumatik (callus). Pada meristem apikal pucuk batang, proliferasi dan diferensiasi plant stem cell dikendalikan oleh banyak faktor, termasuk proses siklus putaran reversibel negatif antara produk ekspresi gen, yaitu protein WUSCHEL (WUS) dan CLAVATA3 (CLV3). Protein WUS disekresi oleh sel-sel pusat organisasi dan merupakan sinyal untuk proliferasi stem cell, sedangkan protein CLV3 disekresikan oleh stem cell dan terbatas pada area  WUS. Kelebihan stem cell menyebabkan CLV3 berlebihan dan merangsang pengurangan sekresi WUS sehingga sinyal proliferasi menurun. Namun, jika jumlah stem cell terlalu rendah (defisit CLV3), maka WUS akan meningkatkan jumlah stem cell.2,13Meristem traumatik (callus) muncul pada bagian tumbuhan yang terlukai, paling sering berdiferensiasi menjadi kambium. Fenomena penciptaan callus pertama kali dijelaskan oleh ahli botani Austria, Gottlieb Heberlandt pada tahun 1902. Dia menyatakan bahwa sel tumbuhan mampu meregenerasi seluruh tanaman dan percobaan pada tahun 1958, wortel berhasil dikloning dari sel wortel yang dibudidayakan secara in vitro. Proses pembuatan callus adalah satu tahap embriogenesis sel-sel somatik (pembentukan zigot tanpa pembuahan) atau disebut juga tumbuhan mengalami dediferensiasi menjadi stem cell yang mampu menghasilkan jaringan baru atau bahkan seluruh organ. Penelitian menunjukkan bahwa sitokin bertanggung jawab dalam memproduksi batang dari callus, sedangkan auksin bertanggung jawab memproduksi akar.2,13 Contoh lain adalah pada pucuk akar Arabidopsis sp., bagian pusatnya tidak aktif bermitosis namun dikelilingi oleh stem cell yang membentuk bagian distal, lateral serta bagian sel akar proksimal.12Kemampuan diferensiasi plant stem cell untuk dediferensiasi kembali menjadi status pluripotensial saat ini banyak dimanfaatkan untuk menghilangkan gejala penuaan kulit pada manusia dalam bentuk sediaan perawatan kulit atau prosedur kosmetik. Teknologi kultur plant stem cellTeknologi kultur sel tumbuhan memastikan pertumbuhan sel tumbuhan, jaringan atau organ dalam lingkungan dengan nutrisi yang bebas mikroba dan memungkinkan untuk sintesis zat aktif biologis. Kultur ini memungkinkan akses dengan material bebas polusi, mikroorganisme atau toksin, mampu di setiap musim, dengan kandungan zat aktif yang hampir sama di setiap bagian.4 Teknik kultur sel tumbuhan dengan metode perbanyakan plant stem cell bertujuan mendapatkan metabolit tumbuhan.3 Dasar biologis di dalam semua tumbuhan adalah reservoir stem cell yang pluripoten dan sel-sel dengan kemampuan berdediferensiasi (pluripoten). Dengan nutrisi yang tepat, callus dapat tumbuh dalam kultur dan dengan menggunakan stimulasi hormon yang tepat, mungkin dapat menstimulasi regenerasi tumbuhan dewasa (disebut sebagai teknik reproduksi mikro).4,13Langkah pertama adalah memilih bahan tumbuhan yang tepat (buah, daun atau akar). Selanjutnya jaringan tumbuhan disterilisasi dan direduksi menjadi fragmen mikroskopis (disebut "eksplan"). Eksplan ditempatkan pada cawan petri beserta nutrisi solid. Kultur dibuat dalam kondisi gelap (tidak berfotosintesis) dan disuplai oleh zat sumber karbon dan energi organik (seperti sakarosa), phytohormone (auksin dan sitokin), vitamin serta unsur mikro dan makro. Kemudian dipilih turunan sel dengan karakteristik biokimia dan metabolisme terbaik (produktif dalam waktu pembelahan terpendek), stabil dan seragam.Selanjutnya pembesaran volume biomassa yang disebut "scaling". Kultur suspense beradaptasi secara bertahap dimulai dari tabung (volume 200 mL) hingga dalam bioreaktor (volume 100 L). Proses fermentasi dipantau dengan pengukuran kadar gula, konduktivitas, tingkat pH, densitas optik, vitalitas sel dan isi metabolit sekunder seperti, asam ursolat.3,4,14Tahap akhir dari fermentasi adalah pengolahan biomassa yang diawali dengan pencampuran konten kultur dalam suspense, yang terdiri atas liposom, phenoxyethanol (pengawet) dan antioksidan (asam askorbat atau tokoferol). Selanjutnya, dilakukan homogenisasi tekanan tinggi, di mana dinding stem cell dihancurkan, komponen yang dimasukkan dilepaskan secara bersamaan komponen lipofilik ditutup di dalam liposom (lecithin), sedangkan komponen hidrofilik dilarutkan dalam fase air. Produk yang diperoleh adalah likuid berwarna kekuningan dan amber. Solusio ini berasal dari perusahaan Swiss Mibelle AG Biochemistry dan menamakan teknologi mereka PhytoCellTecTM (PCT).3,4,14,15   Plant stem cell versus ekstrak plant stem cellTerminologi merupakan hal yang sangat penting dalam hal penegasan cosmeceutical, yaitu pemahaman bahwa ketika istilah “plant stem cell” digunakan sebagai komposisi, sebenarnya mengacu pada ekstrak dari plant stem cell. Banyak perusahaan perawatan kulit mempromosikan produk mereka dengan mengklaim memanfaatkan teknologi stem cell.Ekstrak dari plant stem cell tidak dapat bertindak dengan cara yang sama seperti stem cell yang hidup.3 Menggabungkan ekstrak plant stem cell di dalam zat pembawa yang dapat membantu sel menembus hingga ke dalam kulit untuk memberikan manfaat kosmetik yang sebenarnya memerlukan teknologi yang tepat untuk mendapatkan potensi yang melekat dalam sediaan perawatan kulit.3,14Dengan pengaturan sedemikian rupa, kultur sel tumbuhan terdediferensiasi dapat mewarisi beberapa modifikasi epigenetik yaitu karakteristik biosintesis dan pertumbuhan jaringan yang sangat heterogen. Fakta ini memungkinkan untuk menghasilkan turunan sel tumbuhan dengan jumlah hampir tak terbatas dengan sifat phytochemical yang unik dan karakteristik tumbuh yang hampir sama dengan yang digunakan untuk inisiasi. Istilah “plant stem cell” dalam komposisi kosmetik sebenarnya mengacu pada kultur callus atau suspensi sel yang didapatkan dari ekstrak kultur sel tumbuhan yang berdediferensiasi ditambah solusio berteknologi tinggi yang mampu mempertahankan potensinya.14 1. 2. Ekstrak Plant Stem Cell sebagai Antipenuaan KulitEkstrak plant stem cell sebagai terapi regeneratif kulit Ekstrak terbaik dapat diperoleh dari plant stem cell yang biji atau buahnya mampu mempertahankan kesegaran dan reproduksibilitas dalam jangka waktu yang lama. Faktor penting yang harus dipertimbangkan selama pemilihan tanaman adalah habitatnya dalam kondisi lingkungan yang sulit atau kemampuannya untuk "menyembuhkan" tanaman lainnya.5 Pelopor dalam memproduksi plant stem cell untuk industri kosmetik adalah Mibelle AG Biochemistry company (Swiss), yang mengimplementasikan stem cell buah apel (PhytoCellTecTM Malus Domestica) dan dipublikasi pada tahun 2008. Pada studi klinis menggunakan krim PhytoCellTecTM Malus Domestica 2% selama 4 minggu, kerutan pada wajah berkurang.3 Sejak saat itu, Mibelle AG Biochemistry telah memperkenalkan ekstrak dari plant stem cell Vitis vinifera (PhytoCellTecTM Solar Vitis), Saponaria pumila (PhytoCellTecTM nunatak®) atau Argania spinosa (PhytoCellTecTM Argan) di pasar dalam bentuk suspensi yang memberikan efek antikerut serta meningkatkan aktivitas stem cell epidermis.21 Selain itu, terdapat bukti yang menunjukkan auksin tanaman memiliki efek regulasi terhadap panjang telomer.4Penting diketahui bahwa plant stem cell sangat sensitif terhadap faktor eksternal, seperti cahaya atau suhu. Dalam produk kosmetik, agar mereka dapat bertahan maka digunakan dalam bentuk ekstrak yang larut dalam lipid (diekstraksi dengan minyak) dan larut dalam air (diekstraksi dengan gliserol), ekstrak bubuk (dengan maltodekstrin), liposom, nanoemulsi atau suspensi.4,14 Aktivitas antioksidan dari ekstrak plant stem cellSeperti yang telah disebutkan di atas, radikal bebas dianggap sebagai senyawa aktif yang paling berperan dalam proses penuaan kulit. Ia merusak DNA dan membantu dehidrogenasi, hidroksilasi dan glikasi protein selain merusak lipid di stratum korneum. Akibatnya, kulit kehilangan elastisitas dan kapasitas dalam mengatur transepidermal water loss (TEWL) serta replikasi sel kurang efisien. Oleh karena itu, antioksidan adalah bahan baku penting dalam cosmeceutical.4 Ekstrak dari plant stem cell merupakan sumber senyawa antioksidan yang sangat baik, seperti polifenol, asam phenolic, flavonoid, triterpenes, karotenoid, stilbenes, steroidal saponin dan peptide.14Goutzourelas et al. mempelajari efek antioksidan senyawa phenolic dari ekstrak stem cell anggur (Vitis vinifera) pada sel endotel dan otot.Terapi ekstrak stem cell anggur konsentrasi rendah mampu meningkatkan status redoks sel. Trans-resveratrol, asam gallic, (+)-catechin, asam ferullic, asam caffeic, quercetin, asam coumaric dan kaempferol merupakan bahan utama aktivitas antioksidan dari ekstrak stem cell anggur. Sumber lain yang kaya senyawa phenylpropanoid, terutama isoverbascoside adalah ekstrak kultur sel daun Syringa Vulgaris.4,14 Tito juga melaporkan bahwa ekstrak stem cell tomat memiliki kandungan asam rutin, coumaric, protocatechuic dan chlorogenic yang lebih tinggi dari pada buah tomat dan memiliki kemampuan antioksidan lebih tinggi. Aktivitas antioksidan yang tinggi dari ekstrak stem cell raspberry juga telah dilaporkan oleh Barbulova dan rekannya. Selain senyawa polyphenolic, ditemukan juga banyak kandungan asam ferulic dan quercitin ramnoside.4Studi Bazylak dan Gryn membandingkan beberapa ekstrak plant stem cell dalam total konten polyphenolic dan scavenging radikal diphenyl picrylhydrazin (DPPH) pada ekstrak stem cell paper mulberry (Brussonetia kazinoki), anggur (Vitis vinifer), magnolsi (Magnolia sieboldii), teh hijau (Camelia sinensis), ginseng putih (Panax ginsgen) dan ginseng hidroponik. Hasilnya menunjukkan semua ekstrak memiliki aktivitas antioksidan serta yang paling tinggi dan efektif adalah teh hijau dan ginseng putih.3,17 KinetinSalah satu agen terpenting yang memberikan sifat antipenuaan pada ekstrak plant stem cell adalah kinetin (N6-furfuryladenine), suatu phytohormone cytokinin. Kinetin merupakan salah satu basa purin asam nukleat turunan adenin.18,19 Kinetin terbentuk secara alami dalam plant stem cell, misalnya, pohon pinus Australia (Casuarina equisetifolia) atau gingergrass (Cymbopogon martinii var. Motia). Konsentrasi kinetin yang sangat tinggi ditemukan dalam stem cell lemon (Citrus limon) dan raspberry (Rubus chamaemorus).4Kinetin dianggap sebagai antioksidan alami terkuat. Ia terlibat dalam induksi sintesis enzim regeneratif dan membentuk senyawa kompleks dengan ion tembaga (II) dan mengaktifkan superoksida dismutase. Kinetin juga melindungi DNA sel dengan menghambat pembentukan 8-oxo-dG, penanda kerusakan oksidatif yang terbentuk dari reaksi Fenton.18-21Faktor pertumbuhan alami ini adalah agen yang tepat untuk merangsang stem cell kulit. Menurut penelitian, ia memperbaiki fungsi sawar pada lapisan spinosum epidermis, merangsang keratinosit, menurunkan TEWL, yang memperbaiki pigmentasi dan mengurangi kerutan permukaan. Efek antipenuaan dari kinetin juga telah terbukti bermanfaat pada sel-sel endotel kulit yaitu dengan cara mengaktifkan proliferasi sel, menghambat penuaan sel dan menstimulasi zat-zat substansi proliferasi dan metaboliknya.4,20,21 SIMPULANTumbuhan memiliki umur panjang karena stem cell-nya mampu berdediferensiasi menjadi pluripoten dan dapat dimanfaatkan guna menghambat penuaan kulit manusia. Penggunaan plant stem cell memerlukan teknologi yang tepat agar mendapatkan potensi yang diharapkan dalam sediaan perawatan kulit. Ekstrak dari plant stem cell merupakan sumber senyawa antioksidan yang sangat baik seperti polifenol, asam phenolic, flavonoid, triterpenes, karotenoid, stilbenes, steroidal saponin dan peptida. Efek yang ditimbulkan adalah stimulasi fibroblas, perbaikan epidermis, perbaikan DNA sel serta melindungi kulit dari paparan sinar UV dan stres oksidatif sebagai antioksidan. Dibutuhkan penelitian lanjutan untuk menilai efektifitas dan keamanan ekstrak plant stem cell sebagai antipenuaan kulit. DAFTAR PUSTAKASlack JMW. What is a Stem Cell?. Dalam: The Science of Stem Cells. United States: John Wiley & Sons;2018.h.1-11.Fehér A. Somatic embryogenesis—stress-induced remodeling of plant cell fate. BBA-Gene Regulatory Mechanisms. 2015;1849(4):385-402.Trehan S, Michniak-Kohn B, Beri K. Plant stem cells in cosmetics: Current trends and future directions. FSOA. 2017;3(4):FS0026.Miastkowska M, Sikora E. Anti-Aging Properties Of Plant Stem Cell Extracts. 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