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1.
Methods Mol Biol ; 2827: 1-13, 2024.
Article in English | MEDLINE | ID: mdl-38985259

ABSTRACT

Plant cell, tissue, and organ cultures (PCTOC) have been used as experimental systems in basic research, allowing gene function demonstration through gene overexpression or repression and investigating the processes involved in embryogenesis and organogenesis or those related to the potential production of secondary metabolites, among others. On the other hand, PCTOC has also been applied at the commercial level for the vegetative multiplication (micropropagation) of diverse plant species, mainly ornamentals but also horticultural crops such as potato or fruit and tree species, and to produce high-quality disease-free plants. Moreover, PCTOC protocols are important auxiliary systems in crop breeding crops to generate pure lines (homozygous) to produce hybrids for the obtention of polyploid plants with higher yields or better performance. PCTOC has been utilized to preserve and conserve the germplasm of different crops or threatened species. Plant genetic improvement through genetic engineering and genome editing has been only possible thanks to the establishment of efficient in vitro plant regeneration protocols. Different companies currently focus on commercializing plant secondary metabolites with interesting biological activities using in vitro PCTOC. The impact of omics on PCTOC is discussed.


Subject(s)
Plant Cells , Tissue Culture Techniques , Cell Culture Techniques/methods , Crops, Agricultural/genetics , Crops, Agricultural/growth & development , Plant Breeding/methods , Plant Cells/metabolism , Plant Development/genetics , Plants/genetics , Plants/metabolism , Tissue Culture Techniques/methods
2.
Plants (Basel) ; 13(14)2024 Jul 22.
Article in English | MEDLINE | ID: mdl-39065528

ABSTRACT

Plant growth regulators (PGRs) play a vital role in the induction of morphogenesis in vitro. Synthetic PGRs are commonly used to induce organogenesis and somatic embryogenesis from various explants, while natural substances are rarely utilized. This study aimed to enhance the regenerative response in Nicotiana tabacum leaf explants using Tulsi (Ocimum sanctum) leaf extract and to elucidate the biochemical interactions during modulation of endogenous plant growth regulators, including indole-3-acetic acid (IAA), abscisic acid (ABA), zeatin, and 6-(γ, γ-dimethylallylamino) purine (2iP). Tulsi leaf extract significantly improved shoot production through interactions between endogenous hormones and those present in the extract, which enhanced stress mitigation. The 20% Tulsi leaf extract treatment produced significantly more shoots than the control, coinciding with increased endogenous IAA and zeatin levels starting on day 10 in culture. Furthermore, ABA and zeatin concentrations increased on days 15 and 25, respectively, in the 20% Tulsi extract treatment, suggesting their role in the induction of somatic embryo-like structures. ABA likely acts as an activator of stress responses, encouraging the development of these structures. Additionally, 2iP was involved in the induction of both forms of regeneration in the 10% and 20% extract treatments, especially in combination with ABA. These results suggest that Tulsi leaf extract holds promising potential as a natural supplement for increasing plant regeneration in vitro and advancing our understanding of how natural extracts of plant origin can be harnessed to optimize plant regeneration processes in vitro.

3.
Methods Mol Biol ; 2827: 35-50, 2024.
Article in English | MEDLINE | ID: mdl-38985261

ABSTRACT

Temporary immersion systems (TIS) have been widely recognized as a promising technology for micropropagation of various plant species. The TIS provides a suitable environment for culture and allows intermittent contact of the explant with the culture medium at different immersion frequencies and aeration of the culture in each cycle. The frequency or immersion is one of the most critical parameters for the efficiency of these systems. The design, media volume, and container capacity substantially improve cultivation efficiency. Different TIS have been developed and successfully applied to micropropagation in various in vitro systems, such as sprout proliferation, microcuttings, and somatic embryos. TIS increases multiplication and conversion rates to plants and a better response during the ex vitro acclimatization phase. This article covers the use of different immersion systems and their applications in plant biotechnology, particularly in plant tissue culture, as well as its use in the massive propagation of plants of agroeconomic interest.


Subject(s)
Acclimatization , Plant Development , Culture Media/chemistry , Tissue Culture Techniques/methods , Tissue Culture Techniques/instrumentation , Plant Shoots/growth & development , Plant Shoots/physiology , Plants , Immersion , Plant Somatic Embryogenesis Techniques/methods
4.
Methods Mol Biol ; 2827: 207-222, 2024.
Article in English | MEDLINE | ID: mdl-38985273

ABSTRACT

In this chapter, we report advances in tissue culture applied to Passiflora. We present reproducible protocols for somatic embryogenesis, endosperm-derived triploid production, and genetic transformation for such species knowledge generated by our research team and collaborators in the last 20 years. Our research group has pioneered the work on passion fruit somatic embryogenesis, and we directed efforts to characterize several aspects of this morphogenic pathway. Furthermore, we expanded the possibilities of understanding the molecular mechanism related to developmental phase transitions of Passiflora edulis Sims. and P. cincinnata Mast., and a transformation protocol is presented for the overexpression of microRNA156.


Subject(s)
Passiflora , Plant Somatic Embryogenesis Techniques , Tissue Culture Techniques , Passiflora/genetics , Passiflora/growth & development , Plant Somatic Embryogenesis Techniques/methods , Tissue Culture Techniques/methods , Transformation, Genetic , MicroRNAs/genetics , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development , Endosperm/genetics , Endosperm/growth & development , Gene Expression Regulation, Plant
5.
Methods Mol Biol ; 2827: 197-206, 2024.
Article in English | MEDLINE | ID: mdl-38985272

ABSTRACT

The coconut tree is a crop widely distributed in more than 90 countries worldwide. It has a high economic value derived from the large number of products obtained from the plant, with fast-growing global markets for some of them. Unfortunately, coconut production is decreasing mainly due to the old age of the plants and devastating pests and diseases, such as phytoplasma disease lethal yellowing (LY). Massive replanting is required with phytoplasma-resistant and high-yielding selected coconut plants to keep up with the market demand for fruit. For this purpose, an efficient micropropagation technology via somatic embryogenesis has been established at CICY, yielding fully developed vitro-plants grown within an in vitro environment. Hence, the last stage of the micropropagation process is the acclimatization of the vitro-plants, which are gradually adapted to live in external conditions outside the glass container and the growth room. A protocol has been developed at CICY to acclimate the coconut vitro-plants, and close to 80% survival can be obtained. This protocol is described here.


Subject(s)
Acclimatization , Cocos , Plant Somatic Embryogenesis Techniques/methods , Phytoplasma
6.
Methods Mol Biol ; 2827: 291-301, 2024.
Article in English | MEDLINE | ID: mdl-38985278

ABSTRACT

Somatic embryogenesis (SE) is a clear example of cellular totipotency. The SE of the genus Coffea has become a model for in vitro propagation for woody species and for the large-scale production of disease-free plants that provide an advantage for modern agriculture. Temporary immersion systems (TIS) are in high demand for the propagation of plants. The success of this type of bioreactor is based on the alternating cycles of immersion of the plant material in the culture medium, usually a few minutes, and the permanence outside the medium of the tissues for several hours. Some bioreactors are very efficient for propagating one species but not another. The efficiency of bioreactors depends on the species, the tissue used to propagate, the species' nutritional needs, the amount of ethylene produced by the tissue, and many more. In this protocol, we show how we produce C. canephora plants that are being taken to the field.


Subject(s)
Coffea , Plant Somatic Embryogenesis Techniques , Plant Somatic Embryogenesis Techniques/methods , Coffea/growth & development , Coffea/genetics , Bioreactors , Seeds/growth & development , Culture Media/chemistry
7.
Methods Mol Biol ; 2827: 223-241, 2024.
Article in English | MEDLINE | ID: mdl-38985274

ABSTRACT

Over the years, our team has dedicated significant efforts to studying a unique natural dye-producing species, annatto (Bixa orellana L.). We have amassed knowledge and established foundations that support the applications of gene expression analysis in comprehending in vitro morphogenic regeneration processes, phase transition aspects, and bixin biosynthesis. Additionally, we have conducted gene editing associated with these processes. The advancements in this field are expected to enhance breeding practices and contribute to the overall improvement of this significant woody species. Here, we present a step-by-step protocol based on somatic embryogenesis and an optimized transformation protocol utilizing Agrobacterium tumefaciens.


Subject(s)
Agrobacterium tumefaciens , Bixaceae , Transformation, Genetic , Agrobacterium tumefaciens/genetics , Bixaceae/genetics , Bixaceae/metabolism , Tissue Culture Techniques/methods , Plant Somatic Embryogenesis Techniques/methods , Gene Editing/methods , Plants, Genetically Modified/genetics , Plants, Genetically Modified/growth & development
8.
Methods Mol Biol ; 2827: 279-290, 2024.
Article in English | MEDLINE | ID: mdl-38985277

ABSTRACT

This chapter presents an efficient protocol for regenerating Carica papaya plants via somatic embryogenesis from immature zygotic embryos from economically important papaya genotypes. To achieve regenerated plants from somatic embryos, in the present protocol, four induction cycles are required, followed by one multiplication cycle and one regeneration cycle. With this optimized protocol, 80% of somatic embryos can be obtained in only 3.5 months. At this stage, calli containing more than 50% globular structures can be used for transformation (via agrobacterium, biobalistics, or any other transformation method). Once transformed, calli can be transferred to the following steps (multiplication, elongation, maturation, rooting, and ex vitro acclimatization) to regenerate a transformed somatic embryo-derived full plant.


Subject(s)
Carica , Genotype , Plant Somatic Embryogenesis Techniques , Carica/genetics , Carica/embryology , Plant Somatic Embryogenesis Techniques/methods , Transformation, Genetic , Plants, Genetically Modified/genetics , Regeneration/genetics , Seeds/genetics , Seeds/growth & development
9.
Methods Mol Biol ; 2827: 363-376, 2024.
Article in English | MEDLINE | ID: mdl-38985282

ABSTRACT

Omic tools have changed the way of doing research in experimental biology. The somatic embryogenesis (SE) study has not been immune to this benefit. The transcriptomic tools have been used to compare the genes expressed during the induction of SE with the genes expressed in zygotic embryogenesis or to compare the development of the different stages embryos go through. It has also been used to compare the expression of genes during the development of calli from which SE is induced, as well as many other applications. The protocol described here is employed in our laboratory to extract RNA and generate several transcriptomes for the study of SE on Coffea canephora.


Subject(s)
Coffea , Gene Expression Profiling , Gene Expression Regulation, Plant , Plant Somatic Embryogenesis Techniques , Transcriptome , Coffea/genetics , Coffea/embryology , Coffea/growth & development , Plant Somatic Embryogenesis Techniques/methods , Gene Expression Profiling/methods , Transcriptome/genetics , Seeds/genetics , Seeds/growth & development , Gene Expression Regulation, Developmental
10.
Biol Futur ; 2024 Jul 31.
Article in English | MEDLINE | ID: mdl-39085591

ABSTRACT

This study leads with the primed seeds of rice (var. Swarna) with distilled water (D.W.) and various concentrations of Mg(NO3)2 (0-8 mM)/Kinetin (0-5 ppm) alone or in combination with screen out the regeneration medium induced tolerance level of NaCl. To fulfill the objective, the primed and non-primed rice seeds were inoculated in MS medium supplemented with 30 gL-1 maltose + 1 gL-1 casein hydrolysate and 2 mgL-1 of 2,4-D for callus induction and cultured up to 45 days in two sets: one set for regeneration purpose in NaCl-induced regeneration medium and another set was used to study the physiological potentiality of the callus. The 45-day-old calli were transferred into regeneration medium MSR (MS medium for regeneration) (BAP: NAA: Kinetin = 4:1:1) containing NaCl with a concentration range of 0 to 300 mM. The number of regenerating calli and shoot regeneration percentage, number of plantlets obtained from one callus, recovery of plantlets from each concentration of NaCl and proline estimation from the leaf of the regenerated plantlets were determined from one set obtained after 45 days. The calli obtained from another set after 45 days, the frequencies of total and embryogenic calli induction percentage, fresh and dry weights, proline content, nitrate reductase and superoxide dismutase activities were measured. The calli obtained from 2.5 ppm kinetin + 4 mM Mg(NO3)2 primed seeds were showed best result as compared to the other treatments for the above-mentioned parameters in different concentrations of NaCl-induced medium and survive up to 200 mM concentrations of NaCl.

11.
Plant Physiol Biochem ; 214: 108969, 2024 Sep.
Article in English | MEDLINE | ID: mdl-39068877

ABSTRACT

Quercus aliena, a native Chinese tree species, is significant in industry and landscaping. However, it is traditionally propagated by seeds with many limitations, such as pest infestations, seed yield and quality. Thus, this study firstly introduces a somatic embryogenesis (SE) system for Q. aliena, enhancing its cultivation prospects. Thereinto, the development stage of zygotic embryo had a significant effect on SE, only immature embryos in 10-11 weeks after full bloom (WAF), rich in endogenous abscisic acid (ABA), could induce SE. Exogenous application ABA had positive roles in the early development process of both primary and secondary SE, while its antagonist had opposite roles. Transcriptome analysis showed that transcription regulation occupied the major position. Mfuzz cluster and WGCNA co-expression analysis showed that 24 candidate genes were involved in the SE process. The expression of the 24 genes were also affected by exogenous ABA signals, among which QaLEC2, QaCALS11 and QaSSRP1 occupied the important roles. Additionally, the callose content were also affected by exogenous ABA signals, which had significantly positive correlations with the expression of QaLEC2 and QaCALS11. This study not only established an efficient reproduction system for Q. aliena, but also revealed the difference in embryogenic ability of zygotic embryos from the aspects of transcriptome and endogenous hormone content, and lay a foundation for clarifying the molecular mechanism of SE, and provided a reference for exploring the vital roles of ABA in SE.


Subject(s)
Abscisic Acid , Gene Expression Regulation, Plant , Plant Somatic Embryogenesis Techniques , Quercus , Quercus/genetics , Quercus/metabolism , Quercus/embryology , Abscisic Acid/metabolism , Abscisic Acid/pharmacology , Gene Expression Regulation, Plant/drug effects , Seeds/genetics , Seeds/drug effects , Seeds/metabolism , Plant Growth Regulators/metabolism , Plant Proteins/metabolism , Plant Proteins/genetics , Gene Expression Profiling , Transcriptome
12.
BMC Plant Biol ; 24(1): 527, 2024 Jun 11.
Article in English | MEDLINE | ID: mdl-38858674

ABSTRACT

BACKGROUND: Angelica Gigas (Purple parsnip) is an important medicinal plant that is cultivated and utilized in Korea, Japan, and China. It contains bioactive substances especially coumarins with anti-inflammatory, anti-platelet aggregation, anti-cancer, anti-diabetic, antimicrobial, anti-obesity, anti-oxidant, immunomodulatory, and neuroprotective properties. This medicinal crop can be genetically improved, and the metabolites can be obtained by embryonic stem cells. In this context, we established the protoplast-to-plant regeneration methodology in Angelica gigas. RESULTS: In the present investigation, we isolated the protoplast from the embryogenic callus by applying methods that we have developed earlier and established protoplast cultures using Murashige and Skoog (MS) liquid medium and by embedding the protoplast in thin alginate layer (TAL) methods. We supplemented the culture medium with growth regulators namely 2,4-dichlorophenoxyaceticacid (2,4-D, 0, 0.75, 1.5 mg L- 1), kinetin (KN, 0, 0.5, and 1.0 mg L- 1) and phytosulfokine (PSK, 0, 50, 100 nM) to induce protoplast division, microcolony formation, and embryogenic callus regeneration. We applied central composite design (CCD) and response surface methodology (RSM) for the optimization of 2,4-D, KN, and PSK levels during protoplast division, micro-callus formation, and induction of embryogenic callus stages. The results revealed that 0.04 mg L- 1 2,4-D + 0.5 mg L- 1 KN + 2 nM PSK, 0.5 mg L- 1 2,4-D + 0.9 mg L- 1 KN and 90 nM PSK, and 1.5 mg L- 1 2,4-D and 1 mg L- 1 KN were optimum for protoplast division, micro-callus formation and induction embryogenic callus. MS basal semi-solid medium without growth regulators was good for the development of embryos and plant regeneration. CONCLUSIONS: This study demonstrated successful protoplast culture, protoplast division, micro-callus formation, induction embryogenic callus, somatic embryogenesis, and plant regeneration in A. gigas. The methodologies developed here are quite useful for the genetic improvement of this important medicinal plant.


Subject(s)
Angelica , Plant Growth Regulators , Plant Somatic Embryogenesis Techniques , Protoplasts , Angelica/embryology , Plant Growth Regulators/pharmacology , Plant Somatic Embryogenesis Techniques/methods , Protoplasts/drug effects , Cell Division/drug effects
13.
Foods ; 13(11)2024 May 27.
Article in English | MEDLINE | ID: mdl-38890899

ABSTRACT

As a significant crop growing all across the world, coffee is mostly produced in the bean belt of our global atlas. Worldwide variations in environmental conditions are causing a decline in the yield and quality of coffee varieties. Coffee production is the main emphasis of several traditional breeding techniques. But conventional breeding methods are not sufficient to tackle the problems related to coffee. The field of genomics, which includes transcriptomics, proteomics, and metabolomics, has made great paces in the last ten years. Proteomics is a well-known technique used to enhance the growth, yield, breeding, and quality of different plants under stable and shifting environments. The regulation of specific enzymes, genes, protein expression, modification, translation, and other features played an important role in the enhancement of important plants. However, relatively less research on the proteomics approach for coffee has been published in the last few years. For this reason, some of the most important aspects of proteome profiling for coffee plants have been covered in this review, including growth, the somatic embryo technique, altitude, environmental adoption, drought, and the role that proteins and important enzymes play in the flavor and taste of coffee. This review can aid in the breeding of new cultivars and improve coffee attributes. Furthermore, the present literature can pave the way for proteomics research on coffee.

14.
Plants (Basel) ; 13(11)2024 May 29.
Article in English | MEDLINE | ID: mdl-38891306

ABSTRACT

The Lanzhou lily (Lilium davidii var. unicolor) is a variant of the Sichuan lily of the lily family and is a unique Chinese 'medicinal and food' sweet lily. Somatic cell embryogenesis of Lilium has played an important role in providing technical support for germplasm conservation, bulb propagation and improvement of genetic traits. Somatic embryogenesis receptor-like kinases (SERKs) are widely distributed in plants and have been shown to play multiple roles in plant life, including growth and development, somatic embryogenesis and hormone induction. Integrating the results of KEGG enrichment, GO annotation and gene expression analysis, a lily LdSERK1 gene was cloned. The full-length open reading frame of LdSERK1 was 1875 bp, encoding 624 amino acids. The results of the phylogenetic tree analysis showed that LdSERK1 was highly similar to rice, maize and other plant SERKs. The results of the subcellular localisation in the onion epidermis suggested that the LdSERK1 protein was localised at the cell membrane. Secondly, we established the virus-induced gene-silencing (VIGS) system in lily scales, and the results of LdSERK1 silencing by Tobacco rattle virus (TRV) showed that, with the down-regulation of LdSERK1 expression, the occurrence of somatic embryogenesis and callus tissue induction in scales was significantly reduced. Finally, molecular assays from overexpression of the LdSERK1 gene in Arabidopsis showed that LdSERK1 expression was significantly enhanced in the three transgenic lines compared to the wild type, and that the probability of inducing callus tissue in seed was significantly higher than that of the wild type at a concentration of 2 mg/L 2,4-D, which was manifested by an increase in the granularity of the callus tissue.

15.
J Exp Bot ; 75(14): 4373-4393, 2024 Jul 23.
Article in English | MEDLINE | ID: mdl-38869461

ABSTRACT

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.


Subject(s)
Cellular Reprogramming , Induced Pluripotent Stem Cells , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/physiology , Animals , Plant Cells/physiology , Plants/genetics , Plants/metabolism , Gene Editing
16.
Sci China Life Sci ; 67(7): 1338-1367, 2024 Jul.
Article in English | MEDLINE | ID: mdl-38833085

ABSTRACT

Plants or tissues can be regenerated through various pathways. Like animal regeneration, cell totipotency and pluripotency are the molecular basis of plant regeneration. Detailed systematic studies on Arabidopsis thaliana gradually unravel the fundamental mechanisms and principles underlying plant regeneration. Specifically, plant hormones, cell division, epigenetic remodeling, and transcription factors play crucial roles in reprogramming somatic cells and reestablishing meristematic cells. Recent research on basal non-vascular plants and monocot crops has revealed that plant regeneration differs among species, with various plant species using distinct mechanisms and displaying significant differences in regenerative capacity. Conducting multi-omics studies at the single-cell level, tracking plant regeneration processes in real-time, and deciphering the natural variation in regenerative capacity will ultimately help understand the essence of plant regeneration, improve crop regeneration efficiency, and contribute to future crop design.


Subject(s)
Arabidopsis , Biotechnology , Regeneration , Regeneration/genetics , Regeneration/physiology , Biotechnology/methods , Arabidopsis/genetics , Arabidopsis/physiology , Crops, Agricultural/genetics , Crops, Agricultural/physiology , Plant Growth Regulators/metabolism , Transcription Factors/metabolism , Transcription Factors/genetics , Gene Expression Regulation, Plant , Epigenesis, Genetic , Plant Development/genetics , Plants/genetics , Plants/metabolism
17.
BMC Plant Biol ; 24(1): 561, 2024 Jun 15.
Article in English | MEDLINE | ID: mdl-38877454

ABSTRACT

BACKGROUND: Somatic embryogenesis (SE) is recognized as a promising technology for plant vegetative propagation. Although previous studies have identified some key regulators involved in the SE process in plant, our knowledge about the molecular changes in the SE process and key regulators associated with high embryogenic potential is still poor, especially in the important fiber and energy source tree - eucalyptus. RESULTS: In this study, we analyzed the transcriptome and proteome profiles of E. camaldulensis (with high embryogenic potential) and E. grandis x urophylla (with low embryogenic potential) in SE process: callus induction and development. A total of 12,121 differentially expressed genes (DEGs) and 3,922 differentially expressed proteins (DEPs) were identified in the SE of the two eucalyptus species. Integration analysis identified 1,353 (131 to 546) DEGs/DEPs shared by the two eucalyptus species in the SE process, including 142, 13 and 186 DEGs/DEPs commonly upregulated in the callus induction, maturation and development, respectively. Further, we found that the trihelix transcription factor ASR3 isoform X2 was commonly upregulated in the callus induction of the two eucalyptus species. The SOX30 and WRKY40 TFs were specifically upregulated in the callus induction of E. camaldulensis. Three TFs (bHLH62, bHLH35 isoform X2, RAP2-1) were specifically downregulated in the callus induction of E. grandis x urophylla. WGCNA identified 125 and 26 genes/proteins with high correlation (Pearson correlation > 0.8 or < -0.8) with ASR3 TF in the SE of E. camaldulensis and E. grandis x urophylla, respectively. The potential target gene expression patterns of ASR3 TF were then validated using qRT-PCR in the material. CONCLUSIONS: This is the first time to integrate multiple omics technologies to study the SE of eucalyptus. The findings will enhance our understanding of molecular regulation mechanisms of SE in eucalyptus. The output will also benefit the eucalyptus breeding program.


Subject(s)
Eucalyptus , Plant Somatic Embryogenesis Techniques , Proteome , Transcriptome , Eucalyptus/genetics , Eucalyptus/metabolism , Eucalyptus/growth & development , Proteome/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Gene Expression Regulation, Plant , Gene Expression Profiling
18.
Cryobiology ; 116: 104915, 2024 Jun 12.
Article in English | MEDLINE | ID: mdl-38830567

ABSTRACT

A cryopreservation protocol has been developed for embryogenic cultures (ECs) of Castanea mollissima, an important economic species of the Castanea genus in China. We achieved 100 % regrowth when ECs were treated with Plant Vitrification Solution 2 (PVS2) for 30, 60 and 90 min on ice. Optimal PVS2 treatment for cryopreservation was determined to be 30 min on ice based on the highest biomass regrowth after thawing. Fluorescein diacetate (FDA) staining could rapidly and reliably determine post-thaw cell viability and its use facilitated the optimization of the cryopreservation protocols. Although the proliferation rate of the re-established ECs remained largely unchanged compared to non-cryopreserved ECs, the capacity of the re-established ECs to differentiate (on two media) into somatic embryos nearly doubled to approximately 2200-2300 globular somatic embryos per 1 g of re-established ECs. Based on cell cluster size analysis, this enhanced growth is primarily attributed to the presence of significantly greater cell clusters with a diameter of 100-200 µm, which have the highest level of differentiation ability. In order to understand the increased embryogenic potential following cryopreservation, we analyzed the expression of key genes related to somatic embryogenesis. Genes CmWUS and CmABP1 were downregulated while CmLEC1, CmAGL15, CmGRF2, and CmFUS3 were upregulated in re-established ECs when compared to non-cryopreserved ECs.

19.
Gene ; 927: 148698, 2024 Nov 15.
Article in English | MEDLINE | ID: mdl-38908456

ABSTRACT

Glutamate decarboxylase (GAD) is involved in GABA metabolism and plays an essential regulatory role in plant growth, abiotic stresses, and hormone response. This study investigated the expression mechanism of the GAD family during longan early somatic embryogenesis (SE) and identified 6 GAD genes based on the longan genome. Homology analysis indicated that DlGAD genes had a closer relationship with dicotyledonous plants. The analysis of cis-acting elements in the promoter region suggests that the GAD genes were associated with various stress responses and hormones. RNA sequencing (RNA-Seq) and the qRT-PCR data indicated that most DlGAD genes were highly expressed in the incomplete compact pro-embryogenic cultures (ICpEC) and upregulated in longan embryogenic callus (EC) after treatments with 2,4-D, high temperature (35 °C), IAA, and ABA. Moreover, the RNA-Seq analysis also revealed that DlGADs exhibit different expression patterns in various tissues and organs. The subcellular localization results showed that DlGAD5 was localized in the cytoplasm, suggesting that it played a role in the cytoplasm. Transient overexpression of DlGAD5 enhanced the expression levels of DlGADs and increased the activity of glutamate decarboxylase in longan embryogenic callus (EC), while the content of glutamic acid decreased. Thus, the DlGAD gene can play an important role in the early somatic embryogenesis of longan by responding to hormones such as IAA and ABA. DlGAD5 can affect the growth and development of longan by stimulating the expression of the DlGAD gene family, thereby increasing the GAD activity in the early SE of longan, participating in hormone synthesis and signaling pathways.


Subject(s)
Gene Expression Regulation, Plant , Glutamate Decarboxylase , Plant Growth Regulators , Plant Proteins , Sapindaceae , Glutamate Decarboxylase/genetics , Glutamate Decarboxylase/metabolism , Plant Growth Regulators/pharmacology , Plant Growth Regulators/metabolism , Sapindaceae/genetics , Sapindaceae/metabolism , Plant Proteins/genetics , Plant Proteins/metabolism , Stress, Physiological/genetics , Phylogeny , Plant Somatic Embryogenesis Techniques , Genome, Plant , Seeds/genetics , Seeds/metabolism , Seeds/growth & development , Multigene Family , Abscisic Acid/metabolism , Abscisic Acid/pharmacology
20.
Plants (Basel) ; 13(10)2024 May 15.
Article in English | MEDLINE | ID: mdl-38794439

ABSTRACT

The medicinal plant tulsi (Ocimum sanctum L.) is acknowledged for its invigorating and healing properties that enhance resilience to stress in various human and animal models by modulating antioxidant compounds. While extensive research has documented these effects in humans, the adaptogenic potential of tulsi in stressful in vitro plant systems has not been explored. This study aimed to elucidate the adaptogenic properties of tulsi leaf extract on the in vitro regeneration of tobacco leaf explants through an investigation of the indoleamines at different developmental stages. Shoot regeneration from leaf explants on the medium supplemented with tulsi extract (20%) was compared to the control, and the differences in indoleamine compounds were analyzed using ultra-performance liquid chromatography. Treatment of the explants with the extract resulted in an almost two-fold increase in the number of regenerants after four weeks of culture, and 9% of the regenerants resembled somatic embryo-like structures. The occurrence of browning in the extract-treated explants stopped on day 10, shoots began to develop, and a significant concentration of tryptamine and N-acetyl-serotonin accumulated. A comparative analysis of indoleamine compounds in intact and cut tobacco leaves also revealed the pivotal role of melatonin and 2-hydroxymelatonin functioning as antioxidants during stress adaptation. This study demonstrates that tulsi is a potent adaptogen that is capable of modulating plant morphogenesis in vitro, paving the way for further investigations into the role of adaptogens in plant stress biology.

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