Use Of Plant Tissue Culture Research Articles

In vitro development of green manures: phytotoxicity and remediation of 2,4-D / Desenvolvimento in vitro de adubos verdes: fitotoxicidade e remediação de 2,4-D

Evaluation of phytoremediation capacity under aseptic, controlled conditions can be achieved with the use of plant tissue cultures as a model, eliminating interference from the physical environment. Herein, we developed an in vitro culture protocol for green manures of Crotalaria juncea, C. spectabilis and Canavalia ensiformis, each of which contributes to a sustainable cropping system. In particular, we studied their behavior in the presence of the herbicide 2,4-D (2,4-dichlorophenoxyacetic acid). Disinfection of C. juncea seeds was efficient, and plantlets presented the best development of roots, stem and leaves. Thus, subsequent experiments were conducted only with C. juncea. For 2,4-D tolerance tests, C. juncea seedlings were transferred to Murashige and Skoog (MS) solid media under the following concentrations of 2,4-D: 0 (control), 0.2, 0.5 and 1.0 mg L-1 for a 30-day period. After this period, MS culture media for C. juncea was analyzed by HPLC for quantification of residual 2,4-D. C. juncea development was observed to be sensitive to 2,4-D toxicity; however it was observed 2,4-D reduction in the media. Nonetheless, field assays are still necessary to evaluate the feasibility of using it as a phytoremediator, as well as determine the influence of environmental and plant density variables on 2,4-D absorption capacity from soil.

Advances in Breeding, Bioprospecting, and In Vitro Culture of Laelia Orchid Species

Orchids (Orchidaceae) are plants that are highly appreciated by their beautiful flowers worldwide. Moreover, they represent a source of metabolites with applications in medicine and biotechnology. Within the Orchidaceae family, the Laelia genus is a group of orchid species from the Neotropics and is probably one of the most representative genera of America. Laelia orchids are cultivated by their splendid flowers and are widely used in orchid breeding. Here, we revise the use of the Laelia genus in orchid breeding and metabolite bioprospecting. We also analyze the use of plant tissue culture (PTC) as an alternative to conventional propagation and as a strategy for the recovery of those Laelia species threatened with extinction. We summarize and discuss the recent advances in the application of different PTC techniques for mass multiplication based on asymbiotic germination, organogenesis, protocorm-like bodies development, and somatic embryogenesis, and the advances of in vitro conservation by cryoconservation and the use of slow-growth promoting hormones. Finally, we suggest future directions and venues in research for Laelia species.

Plant Tissue culture and Ultra High Diluted studies: suggesting a novel model using in vitro techniques

Plant tissue culture techniques have been used to evaluate the effects of many different substances and/ or conditions in plant growth and development. It provides information of great value about problems related to basic and applied aspects of plant as well as contributed to understanding of factors responsible for growth, metabolism, synthesis of secondary compounds, stress response. Considering all this wide range of applications and as all plant tissue culture techniques are undergone under axenic and controlled conditions (culture medium composition, light and temperature, for instance), it seems to be a value model for Ultra High Diluted (UHD) studies. Lippia alba is a Brazilian plant that tissue cultures protocols and in vitro essential oil production have already been described in scientific literature. None of all scientific papers evaluated the effects of UHD substances on in vitro development or secondary metabolic production. The main goal was to evaluate the use of plant tissue culture to investigate the effects of UHD benzilaminopurine (BA) on Lippia alba shoot culture. Nodal segments obtained from plants growth in vitro was subcultured to Murashigue & Skoog semi-solid medium added with 2ml of these different solutions: BA 3µmol, BA 12CH (10-24), water 12CH and water (no dilution and succussion). Weekly 1 ml of solutions were added to cultures. The experiment was repeated twice and each one consisted in 3 culture vessel with 5 nodal segments per treatment (n=30). All plants were maintained in growth room under controlled temperature (25°C), light and photoperiod (16L/8D). The tested substances were prepared according to the method of stepwise dilution and succussion as describe in Brazilian Homeopathic Pharmacopoeia. The experiment was blinded all the time. After 60d, plantlets were evaluated for number of shoots, shoot length, rooted plants (%), callus development (%) and fresh biomass. Data were submitted to ANOVA following by Duncan’s and t-test. Plants from water 12CH and BA 12CH increased the number of new shoots and promoted the highest shoot length. By adding BA 3µmol the organogenetic response was inhibited since neither shoot nor root were developed. However, it was observed a significant basal callus development. Plant tissue culture could be adapted for UHD studies. More studies are being conducted in way to analyze other experimental conditions and biochemical/phytochemical parameters.

Exploring the Use of Bryophyllum as Natural Source of Bioactive Compounds with Antioxidant Activity to Prevent Lipid Oxidation of Fish Oil-In-Water Emulsions.

The current industrial requirements for food naturalness are forcing the development of new strategies to achieve the production of healthier foods by replacing the use of synthetic additives with bioactive compounds from natural sources. Here, we investigate the use of plant tissue culture as a biotechnological solution to produce plant-derived bioactive compounds with antioxidant activity and their application to protect fish oil-in-water emulsions against lipid peroxidation. The total phenolic content of Bryophyllum plant extracts ranges from 3.4 to 5.9 mM, expressed as gallic acid equivalents (GAE). The addition of Bryophyllum extracts to 4:6 fish oil-in-water emulsions results in a sharp (eight-fold) increase in the antioxidant efficiency due to the incorporation of polyphenols to the interfacial region. In the emulsions, the antioxidant efficiency of extracts increased linearly with concentration and levelled off at 500 μM GAE, reaching a plateau region. The antioxidant efficiency increases modestly (12%) upon increasing the pH from 3.0 to 5.0, while an increase in temperature from 10 to 30 °C causes a six-fold decrease in the antioxidant efficiency. Overall, results show that Bryophyllum plant-derived extracts are promising sources of bioactive compounds with antioxidant activity that can be eventually be used to control lipid oxidation in food emulsions containing (poly)unsaturated fatty acids.

Controlling the barriers of cloning mature Picea abies (L.) H. Karst. via tissue culture and co-cultivation with Agrobacterium rhizogenes

Providing an improved protocol for cloning of Picea abies (L.) H. Karst. mature trees via Agrobacterium rhizogenes. In reforestation programs, rejuvenation of superior genotypes is of great importance in helping juvenile rootstock production. In this regard, the use of plant tissue culture to produce axillary and/or adventitious shoots from explants of mature trees has been the objective of many researchers. There are a few reports about the successful in vitro rooting of the regenerated shoots from explants of mature trees. There is a very low rate of natural rooting in conifers, between 1 and 5%. Furthermore, they are still hard to root even when auxins are applied. In this study, in vitro propagation of Norway spruce explants and its rooting improvement by A. rhizogenes with the aim of receiving an efficient clonal propagation method was investigated. The effects of BA (0.5, 1, 1.5, and 2 μM) and TDZ (0.0025, 0.025, 0.25, and 2.5 μM) were examined on axillary shoot development from shoot-tip explants of the Norway spruce. The effects of three strains of Agrobacterium rhizogenes (K599, AR15834 and GMI9534) were also examined on enhancement of the root induction. According to the results, after the treatment of 1.5 μM BA, 87.5% of shoot-tip explants produced axillary shoots (the highest percentage among treatments), and also the maximum mean number of regenerated shoots per explant (6.5) was achieved in this treatment. Furthermore, the induction of rooting was performed using IBA (7.5 µM) and IAA (0.5 or 1 μM) in the WPM medium along with keeping the explants in the darkness at 21 °C, which yielded 37.5% and 12.5% rooting, respectively. Moreover, the application of coumarin along with IAA caused an increase of 22% in rooting, compared to the use of IAA alone. Two strains, K599 and AR15834, succeeded in improving the percentage of rooting, with the highest being 54.2% as a result of using the K599 strain alongside 7.5 μM IBA. The proposed treatment can be used efficiently for the propagation of Norway spruce in landscaping and/or reforestation programs.

CHARACTERISTICS OF HORNY ALTAI PEATS AND THEIR BIOLOGICAL ACTIVITY IN PLANT TISSUE CULTURE

The search for new raw materials of biologically active substances of natural origin is an urgent task for the modern period. Peat in this respect is a relatively cheap and almost unlimited raw material base. Peat of swamps can be used widely in agriculture for receipt of biologically active substances are of great interest in the territory of the Gorny Altai. The purpose of this work is to study the composition of organic matter of peats of the Gorny Altai, to choose peat raw materials for biologically active production and to study their biological activity. The object of the study was 46 swamps of the Gorny Altai. The following analyses were carried out in peats: botanical composition, degree of decomposition, ash content, group composition of peat organic matter. The composition of humic acids (ha) was analyzed by IR spectroscopy. For determine of the biological activity of humic acids was used plant tissue cultured . The results of the research allowed to distinguish peats by the content of HA: buckbean peat (47.0% of HA), wood peat (50.0% of HA), fern peat (55.0% of HA), grass peat (30.0–45.0% of HA), sedge peat (5.6–58.0% of HA), grass-buckbean peat (43.0–56.5% of HA) and to outline the raw material base for the production of BAS – peatland Turochak. According to the optimal characteristics of HAs, a sample was taken from a depth of 325-375 cm, HA was isolated. The biological activity In humic acid was determined with the use of plant tissue culture. High biological activity are proven of the preparations of HA from the peatland Turochak, which resulted in the acceleration of microclonal propagation of plants in vitro.

Plant Tissue Culture: A Battle Horse in the Genome Editing Using CRISPR/Cas9.

Plant tissue culture (PTC) is a set of techniques for culturing cells, tissues, or organs in an aseptic medium with a defined chemical composition, in a controlled environment. Tissue culture, when combined with molecular biology techniques, becomes a powerful tool for the study of metabolic pathways, elucidation of cellular processes, genetic improvement and, through genetic engineering, the generation of cell lines resistant to biotic and abiotic stress, obtaining improved plants of agronomic interest, or studying the complex cellular genome. In this chapter, we analyze in general the use of plant tissue culture, in particular protoplasts and calli, in the implementation of CRISPR/Cas9 technology.

Effect of Plant Preservative Mixture Ppmtm on the Shoot Regeneration of Watercress (Nasturtium Officinale)

Plant Preservative Mixture™ (PPM), a relatively new broad-spectrum preservative and biocide for use in plant tissue culture, was evaluated as an alternative to the use of conventional antibiotics and fungicides in plant tissue culture. Culture inoculated in MS media supplemented with PPM (1.5 ml/l) was the effective concentration which gave the best values. The top values were recorded for all studied characters using apical buds compared with lateral buds. The combination between apical buds and (1.5 ml/l ) PPM concentration showed the superior values of all studied parameters( 33.25%,19.40%, 14.46%,20.53% and 79.46%)(60.55%,39.80%,20.97%,45.33% and 54.44%) and (31.20%, 20. 06%, 12.33%, 35.13% and 81.06 %) for contamination, %bacterial contamination, % offungi contamination, dead explants% and survival explants% respectively. Different concentrations of PPM (0, 0.5, 1.0, 1.5, 2.0 and 2.5 ml/l) were tested using single node and apex explants of watercress (Nasturtium officinale). PPM at (1.5ml/l) had significant effect on the studied characters; shoots height, shoots number, leaf pairs number, fresh and dry weight which they reaches (4.16, 4.62, 42.00,0.524 and 0.063 g, respectively ). Apex bud explants showed the greatest effect on shoots height shoots number, leaf pairs number, fresh and dry weight and their values were 3.52, 32.02, 3.38, 0.405 and 0.036 g, respectively. The best parameter were recorded on MS media supplemented with PPM at (1.5ml/l) with apex buds explant (4.55, 5.34, 46.28, 0.570 and 0.085, respectively) for shoots height, shoots number, leaf pair number, fresh and dry weight. Current study aimed to determine the best concentration of PPM for limiting the contamination of watercress and micro shoot regeneration.

Plant regeneration of ‘Amethyst’ purple raspberry (Rubus occidentalis×R. idaeus‘Amethyst’) from in vitro leaf tissues

Genotype-specific limitations in plant regeneration can greatly inhibit use of plant tissue culture and biotechnology in plant improvement. An experiment was conducted to discover the optimal plant growth regulator combinations for plant regeneration of a cold-hardy purple raspberry cultivar (Rubus occidentalis × R. idaeus 'Amethyst'). Regeneration of adventitious shoots was analyzed when in vitro leaf tissues were cultured on woody plant medium (WPM) with varying thidiazuron (TDZ) and indole-3-butyric acid (IBA) concentrations. The maximum regeneration rate occurred on WPM medium containing 1.0 μM TDZ and 0 μM IBA with an average of 93.3% of the explants forming shoots. The number of shoots produced from each leaf explant varied with the TDZ concentrations. An average of 3.9 shoots per explant was produced on 0.5 TDZ and 2.3 shoots on 1 µM TDZ medium. Higher TDZ or presence of IBA in the medium decreased the shoot number. Regenerated shoots were transferred to Murashige and Skoog (MS) medium with 1 to 10 μM IBA with or without polyvinylpyrrolidone (PVP) for in vitro rooting. More roots were induced in 10 μM IBA medium than lower amounts. Addition of PVP significantly enhanced rooting. Rooted explants were transferred to potting medium and grown in the greenhouse. This research will assist future raspberry improvement programs that use biotechnology techniques such as in vitro mutation and genetic transformation.

Evaluation of aqueous chlorine dioxide for disinfecting plant explants

Sterilization of explants while maintaining viability is a major task for those working in plant tissue culture. This task is usually accomplished through the use of mercuric chloride or sodium hypochlorite. Due to environmental and health concerns, however, new disinfectants are desired. Chlorine dioxide is an environmentally friendly disinfectant that has been used for many purposes, though its use in plant tissue culture has not been thoroughly investigated. This report describes the application of aqueous chlorine dioxide for the disinfection of yacon (Smallanthus sonchifolius) and pomegranate (Punica granatum) explants. The threshold concentration for obtaining high antimicrobial efficiency with aqueous chlorine dioxide was determined to be 1.25–2.5 mM; however, the viability of the explants from the two species differed with treatment. While the viability of yacon explants remained high at 1.25–1.67 mM aqueous chlorine dioxide, pomegranate explant viability was severely impaired at all tested concentrations. The level of explant viability was negatively correlated with high levels of endogenous polyphenol and subsequent free radical accumulation following treatment. Aqueous chlorine dioxide at concentrations of 1.25–2.5 mM was also suitable for explant disinfection in other low-polyphenol-containing plant species such as rice (Oryza sativa), Pinellia ternata, and Isodon amethystoides. This work shows that aqueous chlorine dioxide can be effectively utilized as an alternative disinfectant for low-polyphenol-containing explants in general.

Dissecting the Concept of the Thin Cell Layer: Theoretical Basis and Practical Application of the Plant Growth Correction Factor to Apple, Cymbidium and Chrysanthemum

A thin cell layer (TCL) is a thin layer of plant cells. TCLs have served as a simple, but important biotechnological tool in plant science, with several dozen crop species having had tissue culture regeneration protocols developed using TCLs generated from multiple explant sources. There are two types of TCLs, transverse TCLs, or tTCLs and longitudinal TCLs, or lTCLs. The former is the most common, ranging from 100 μm to 1–2 mm in thickness, usually cutting through several tissue types. In contrast, the latter usually targets a very specific layer of cells or tissues, and may vary in length but is as thick as a tTCL. The developmental question that needs to be addressed will determine the choice between one or the other and its use in plant tissue culture. The often unappreciated beauty of the TCL is not so much in its actual regeneration capacity, but rather in its potential regeneration capacity. Herein, we use data from three model species, a woody temperate fruit tree, Malus sp. (apple; Rosaceae), and two herbaceous ornamentals, Cymbidium (orchid; Orchidaceae) and Dendranthema (chrysanthemum; Asteraceae), to demonstrate the theory and functionality of TCLs. Moreover, using a new concept, the plant growth correction factor, or GCF, the ability to theoretically predicts the organogenic outcome in vitro is presented through mathematical models based on the geometric analysis of explant size and shape. A new factor, the geometric factor, or GF, was also determined for all three plants to compare regeneration from different explant types with different shapes. The GF, which is calculated, is independent of plant species or any in vitro conditions, but depends only on the size and shape of the explant and on tissue that is capable of regeneration. The GF and GCF would, in theory, allow for the direct comparison of plant in vitro studies in different laboratories provided that explant size is known, and to predict the theoretical outcome of a regeneration protocol if different explants were to be used.

Effects of Environmental Temperature on the Regeneration Frequency of the Immature Embryos of Wheat (Triticum aestivum L.)

The immature embryos (IEs) of wheat are the most widely used tissues for in vitro culture and genetic transformation due to its high regeneration competency. However, this explant can only be maintained in 4°C daily cooler for a short period time for its use in plant tissue culture or transformation experiments. This study aimed to investigate the effects of environmental temperature, cryopreservation storage temperature, and heat shock culture (HSC) temperature on the regeneration frequency of wheat IEs. Results indicated that environmental temperature significantly affected the induction of embryonic calli. The optimum total accumulated temperature (TAT) during the time of anthesis and sampling for regeneration of these tissues was around 280°C for spring wheat type cv. CB037 and approximately 300°C for winter wheat type cv. Kenong 199. Regeneration ability obviously declined when the highest environmental temperature was over 35°C for 1 d or a high temperature between 30 and 33°C lasted for 5 d during anthesis and sampling. This finding was verified by culturing the freshly isolated IEs under different temperatures from 29 to 37°C in different controlled growth incubators for 5 d; the IEs almost completely lost regeneration ability when the temperature rose to 37°C. Cryopreservation of −20°C caused the wheat samples lost ability of producing callus or embryonic callus in a few days, and cryopreservation of −10°C more than 10 d made the regeneration potential of the tissues dramatically declined. Comparatively, the temperature that best maintained high regeneration ability was −5°C, at which the materials can be maintained for around 1 mon. In addition, the preservation of the immature samples at −5 or −10°C inhibited the direct germination of the IEs, avoiding the embryo axis removing process. Our results are useful for ensuring that field collection and cryopreservation of the wheat IEs are done correctly to enable tissue culture and genetic transformation.

Micropropagation of Cacti—a Review

In the past, members of the Cactaceae family were mostly propagated by seeds or vegetatively by cuttings and grafting. Seeds, however, do not guarantee genetic stability and, in the case of some cacti, seeds are difficult to obtain, their germination rates are low, or they need to be stored for a long time in specific light and temperature conditions. Traditional vegetative reproduction in vivo, on the other hand, may be less efficient insofar as a limited number of plants can be obtained. Major issues associated with traditional propagation methods may be solved by the use of plant tissue culture. Today micropropagation techniques are applied in order to produce large numbers of new high-quality plants in a relatively short time and space. This is especially important for endangered and desirable species such as cacti. This paper discusses the achievements, current state and future prospects of cactus micropropagation methods. We also provide an overview of cactus multiplication by areole activation, direct and indirect organogenesis (caulogenesis and rhizogenesis) and somatic embryogenesis, as well as consider acclimatization. Micropropagation of cacti is still sufficiently idiosyncratic that different protocols must be used for different species, even closely related ones.

“Genetically Modified Lite” placates public but not activists

“Genetically Modified Lite” placates public but not activists

Lignin Biosynthesis Studies in Plant Tissue Cultures

Lignin, a phenolic polymer abundant in cell walls of certain cell types, has given challenges to scientists studying its structure or biosynthesis. In plants lignified tissues are distributed between other, non-lignified tissues. Characterization of native lignin in the cell wall has been difficult due to the highly cross-linked nature of the wall components. Model systems, like plant tissue cultures with tracheary element differentiation or extracellular lignin formation, have provided useful information related to lignin structure and several aspects of lignin formation. For example, many enzyme activities in the phenylpropanoid pathway have been first identified in tissue cultures. This review focuses on studies where the use of plant tissue cultures has been advantageous in structural and biosynthesis studies of lignin, and discusses the validity of tissue cultures as models for lignin biosynthesis.

Molecular Identification of Twelve Medicinal Plants of Huperziaceae

Twelve species of Huperziaceae have been identified for resolving the resource depletion of Huperziaceae plants. The chloroplast genes rbcL, rpL16, and psbA-trnH intergenic spacers were used in this study. Identification of different species of Huperziaceae is possible by analyzing the information obtained from rbcL, rpL16, and psbA-trnH intergenic spacer sequences. The result also can provide information with regard to the use of plant tissue culture and cuttage propagation to solve the problem of the resource depletion of these plants.

Assessment of somaclonal variability in hop (Humulus lupulus L.) in vitro meristem cultures and clones by molecular methods

In vitro meristem tissue cultures are used for production of virus-free rootstocks of hop (Humulus lupulus L.). Because use of plant tissue cultures is associated with occurrence of somaclonal variability, we assessed somaclonal variability in hop meristem in vitro cultures before and after thermotherapy by different molecular methods (RFLP, RAPD, STS, ISSR and AFLP) and compared it with existing clonal variability of Osvald's clones 31, 72 and 114. No molecular differences were observed between mother plants and in vitro mericlones by RFLP and STS analyses. Amplified molecular differences were found in RAPD and ISSR products of one from five in vitromericlones cvs. Eroica (E5) and Southern Brewer (SB2), respectively. Similarities with mother plants were 0.965 and 0.913 (JSC), respectively. Specific amplified polymorphic products were found for every mericlone and mother plant in AFLP reactions and variability of DNA sequence ranged from 0.824 to 0.993 (JSC). This variability was very similar to determined intra-clonal variability within Osvald's clones 31, 72 and 114 by AFLP analysis. Inter-clonal variability of DNA sequence was exactly higher than intra-clonal variability of DNA sequence in these clones. The molecular differences between Osvald's clone 72 normal and meristem derived were not verifiable. Thermotherapy increased frequency of molecular changes, since amplified differences were found in 14 from 20 in vitro mericlones of cv. Eroica, in 6 from 11 in vitro mericlones of cv. Yeoman and in 15 from 23 in vitro mericlones of cv. Southern Brewer by RAPD and ISSR analyses.

Plant Preservative Mixture™ Can Affect Shoot Regeneration from Leaf Explants of Chrysanthemum, European Birch, and Rhododendron

Plant Preservative Mixture™ (PPM), a relatively new, broad-spectrum preservative and biocide for use in plant tissue culture, was evaluated as an alternative to the use of conventional antibiotics and fungicides in plant tissue culture. Concentrations of 0.5 to 4.0 mL·L-1 were tested with leaf explants of chrysanthemum (Dendranthem×grandiflora Kitam), European birch (Betula pendula Roth), and rhododendron (Rhododendron catawbiense Michx.). PPM had little effect on the percentage of explants forming shoots and the number of shoots formed per explant in birch and rhododendron, but dramatically reduced both responses in chrysanthemum. Therefore, the effects of PPM must be evaluated for each species of interest prior to use.

Plant Biotechnology: Promises and Challenges .

Development of procedures in cell biology to regenerate plants from single cells in any desired quantity provides the prerequisite for the practical use of plant tissue culture and genetic engineering in crop improvement. Such regenerating cell cultures are used for selection of mutants and for DNA transformation experiments. DNA transfer by means of engineered Ti and Ri plasmids has become an established technique for the rapidly growing list of dicotyledonous plants. Considerable success has also been achieved in making gene transfer techniques independent of cell culture methods. These techniques have given the opportunity to create, characterise and select plant cultivars which cannot be obtained by traditional breeding methods. The exploitation of plant cell cultures for production of pharmaceuticals, natural products of commercial importance and mass propagation of high-value crops by automation, have developed into an important industry with considerable potential for future. This paper discusses the recent advances and applications of plant biotechnology in agriculture and industry and the challenges the still exist.

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